Dissertations / Theses: 'Antimicrobial peptide resistance'

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Vila, Farrés Xavier. "Development of new antimicrobial peptides and peptidomimetics and mechanism of resistance to peptide antibiotics." Doctoral thesis, Universitat de Barcelona, 2014. http://hdl.handle.net/10803/285375.

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Nowadays in the world there is a very big problem associated with two factors related to each other. The first factor is the increase in the resistant of certain bacteria, especially the bacteria from the ESKPAE group. The second factor is the dramatically decrease of new antibiotics approved by the FDA. These two problems show that there is an urgent need to find new antibiotics active against these resistant bacteria. In this thesis, we have tackled two different topics closely related in the race to find new antimicrobials. The first topic tackled was the knowledge of the mechanism of resistance of Gram-negative (A. nosocomialis) and Gram-positive (S. mitis) bacteria. The two antibiotics studied were peptides, colistin and daptomicin, these two peptides are resistant to A. nosocomialis and S. mitis, respectively. Both peptides had a similar mechanism of action related to the membrane of bacteria, therefore we are going to focus just in the modifications in the membrane of the strains resistant to the antibiotic peptides. In S. mitis it was observed, using proteomic techniques, that two proteins related with the membrane were observed. These two proteins has some homologue domains to several proteins involved in daptomycin resistant in S. aureus and Enterococci. In A. nosocomialis, the bacteria showed a high tolerance to colistin, and at 8 mg/L an inflexion point is observed. In this inflexion point, the MIC of colistin, against bacteria increase from <0.1 mg/L to 128 mg/L. These bacteria with high resistance to colistin showed no production of LPS due to the fact that mutations and a stop codon in lpxD gene were observed. This gene is involved in the synthetic pathway of the LPS. Apart from the understanding of the mechanism of action of peptide antibiotics, we have proposed several peptides and peptidomimetics against Acinetobacter species. We have used two different approaches. The first approach is the normal approach, testing several peptides or peptidomimetics against the desired bacteria. The first peptides tested were commercially available, and we found mastoparan that was active against both colistin-susceptible and colistin-resistant A. baumannii. This peptide was optimized specially in terms of stability in human serum. After several in vivo trials we did not observe any activity of the peptides tested, however we found a very strong bindoing with some proteins present in the human serum. Frog skin secretions peptides were also tested against colistin-susceptible and colistin-resistant Acinetobacter species, the results obtained were really interesting specially in two peptides. The last peptides tested were peptidomimetics. These peptidomimetics act as an antimicrobial peptide, with two different faces, one face with a cation charge and the other very amphipathic. These peptidomimetics are analogues from the original structure of cholic acid, the structure was modified in order to have antibacterial activity that was found in colistin-susceptible and colistin-resistant A. baumannii, K. pneumonia and P. aeruginosa. The second approach was completely different, in this case the idea was to block the virulence of bacteria caused by OmpA. This protein is involved in the adherence between bacteria and host cells, therefore several hexacylcic peptides were synthesized in order to inhibit the action of this protein. The results obtained were satisfactory, obtaining good activity in both in vitro and in vivo.
Actualment al mon hi ha un greu problema derivat de dos factors relacionats, el primer factor es el increment de la resistència, especialment del bacteris del grup ESKAPE. El segon factor es la disminució dràstica en el nombre d’antibiòtics aprovats per la FDA. Aquests dos problemes fan que hi hagi una urgència per trobar nous antimicrobians efectius en front d’aquestes soques resistents. En aquesta tesi hem abordat dos temes diferents però que estan relacionats a la hora de trobar nous antibiòtics. El primer tema abordat es el de conèixer a fons els mecanismes de resistència de certs antibiòtics, en aquest cas peptídics, en front diferents tipus de soques tant Gram-positives (S. mitis) com Gram-negatives (A. nosocomialis). Els dos antibiòtics peptídics pels que s’ha estudiat la resistència son daptomicina i colistina, en front de S. mitis i A. nosocomialis respectivament. Ambdós pèptids actuen a nivell de membrana, per tant ens centrarem en veure les modificacions produïdes en els soques resistents. Per part de S. mitis resistent a daptomicina, es pot veure una sobreexpressió de dues proteïnes que tenen dominis homòlegs amb altres proteïnes involucrades en la resistència a daptomicina en altres bacteris. En la resistència a colistina es pot apreciar com les soques resistents d’A. nosocomialis presenten una deficiència del LPS. També hem proposat diferents alternatives com a nous antibiòtics, en aquest cas en front de soques A. baumannii. Dos tipus d’aproximacions van ser utilitzades, la primera, i mes clàssica es la de trobar nous antimicrobians, vàrem trobar mastoparan i va diferents paràmetres van ser optimitzat però sense obtindré bons resultats in vivo. També es van provar diferents pèptids provinents de les secrecions de les granotes, presentant bona activitat en front soques d’Acinetobacter, i per últim, les ceragenines, anàlegs del àcid cólic, que tenen bona activitat en front de totes les soques tant colistina sensibles com colistina resistents en Gram-negatius. La segona aproximació es buscant pèptids capaços d’inhibir l’adherència entre el bacteri i la cèl•lula del hoste bloquejant l’acció de la proteïna OmpA. S’ha trobat un pèptid amb bona activitat fins i tot in vivo.

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Gooderham, William James. "Regulation of virulence and antimicrobial peptide resistance in Pseudomonas aeruginosa." Thesis, University of British Columbia, 2008. http://hdl.handle.net/2429/1014.

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Pseudomonas aeruginosa is a ubiquitous environmental Gram-negative bacterium that is also a major opportunistic human pathogen in nosocomial infections and cystic fibrosis chronic lung infections. These P. aeruginosa infections can be extremely difficult to treat due to the high intrinsic antibiotic resistance and broad repertoire of virulence factors, both of which are highly regulated. It was demonstrated here that the psrA gene, encoding a transcriptional regulator, was up-regulated in response to sub-inhibitory concentrations of antimicrobial peptides. Compared to wild-type and the complemented mutant, a P. aeruginosa PAO1 psrA::Tn5 mutant displayed intrinsic super-susceptibility to polymyxin B, a last resort antimicrobial used against multi-drug resistant infections, and indolicidin, a bovine neutrophil antimicrobial peptide; this super-susceptibility phenotype correlated with increased outer membrane permeability. The psrA mutant was also defective in simple biofilm formation, rapid attachment, and normal swarming motility, phenotypes that could be complemented by the cloned psrA gene. The role of PsrA in global gene regulation was studied by comparing the psrA mutant to wild-type by microarray analysis, demonstrating that 178 genes were up or down-regulated by greater than 2-fold (P ≤0.05). Dysregulated genes included those encoding known PsrA targets, the type III secretion apparatus and effectors, adhesion and motility genes and a variety of metabolic, energy metabolism and outer membrane permeability genes. This indicates that PsrA is a central regulator of antimicrobial peptide resistance and virulence. P. aeruginosa containing a mutation in the PhoQ sensor kinase-encoding gene was highly attenuated for persistence in a rat chronic lung infection model. In addition, the polymyxin B hyper-resistant phoQ mutant displayed reduced type IV pili-dependent twitching motility and was less cytotoxic towards human bronchial epithelial cells, indicating that the virulence defect observed could be due at least in part to these phenotypes. Using microarrays it was further demonstrated that PhoQ regulates a large number of genes that are PhoP-independent and that the phoQ mutation leads to up-regulation of PhoP- and PmrA regulated genes as well as other genes consistent with its virulence phenotypes.

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thew, Francis Matthew Francis. "Investigating antimicrobial resistance mechanisms in Neisseria gonorrhoeae using peptide probes." Thesis, Durham University, 2009. http://etheses.dur.ac.uk/185/.

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The continuing evolution of antibiotic resistance strains of Neisseria gonorrhoeae coupled with the paucity of new antimicrobial agents makes the treatment of gonococcal infections challenging. A major cause of resistance is the expression of a multidrug efflux pump termed MtrCDE, which exports a wide range of antimicrobial agents. Efflux pumps are membrane-bound systems and consequently challenging to study and target with drugs. The transcriptional regulator (MtrR) of the efflux pump, however, is a soluble protein and therefore more amenable to study and drug target validation investigations. This thesis serves to investigate the hypothesis that substrates for the MtrCDE efflux pump are also ligands for the regulator MtrR. Isothermal titration calorimetry (ITC) was used to show that MtrR binds commercial antibiotics and antimicrobial peptides. -lactam antibiotics not only bind MtrR but are hydrolysed by the multidrug protein. Evidence for this novel enzymatic activity is provided by ITC, mass spectrometric and microbiological techniques. A series of peptides derived from LL-37 were synthesised and screened for binding to MtrR. A key region of LL-37 with a higher affinity to MtrR than the natural product was then identified. The peptide binding site in MtrR was elucidated via a photoactivated peptide binding study. Electrophoresis mobility shift assays indicated that the peptides do not induce derepression of the genes controlled by MtrR, although the peptide derivatives of LL-37 were shown to be substrates for the MtrCDE efflux pump.

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4

Dintner, Sebastian. "Characterization of a sensory complex involved in antimicrobial peptide resistance." Diss., Ludwig-Maximilians-Universität München, 2014. http://nbn-resolving.de/urn:nbn:de:bvb:19-176173.

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In their habitats, microorganisms are often in competition for limited nutrients. In order to succeed, many Gram-positive bacteria resort to production of peptide antibiotics. Therefore, resistance mechanisms against these compounds are essential. The first step of ensuring survival is the perception of the harmful drugs and mediation of resistance against it. In recent years, a group of ABC-transporters have been recognized as important resistance determinate against antimicrobial peptides. The expression of these transporters is generally regulated by a two-component system, which in most cases is encoded next to the transporter. Together they are described as detoxification modules. The permeases of the transporters are characterized by a large extracellular domain, while the histidine kinases lack an obvious input domain. One of the best understood examples is the BceRS-BceAB system of Bacillus subtilis, which mediates resistance against bacitracin, mersacidin and actagardine. For this system it was shown that the histidine kinase is not able to detect the substrate directly and instead has an absolute requirement for the transporter in stimulus perception. This describes a novel mode of signal transduction in which the transporter is the actual sensor and therefore regulates its own expression. To date, mechanistic details for this unique mode of signal transduction remain unknown. Several other examples have been described for transport proteins that have acquired additional sensing or regulatory functions beyond solute transport, and these have been designated trigger transporters. For these bifunctional transporters a direct protein-protein interaction with membrane-integrated or soluble components of signal transduction relays has been postulated. However, for most sensor/co-sensor pairs, conclusive proof of such an interaction is lacking, and so far little is known about the sites that might mediate contacts between the putative protein interfaces and how communication is achieved. Based on sequence and architectural similarities, we identified over 250 BceAB-like transporters in the protein database, which occurred almost exclusively in Firmicutes bacteria. To whether the regulatory interplay between the ABC transporter and the two-component system was a common theme in these antimicrobial peptide resistance modules, we carried out a phylogenetic study of these identified systems. We identified a clear coevolutionary relationship between transport permeases and histidine kinases. Furthermore, we identified conserved putative response regulator binding sites in the promoter regions of the transporter operons. Additionally, we were able to provide a tool to identify TCSs for transporters lacking a regulatory system in their genomic neighbourhood, which was based on the coclustering of histidine kinases and transporter permeases. These findings also suggested the existence of a sensory complex between BceAB-like transporters and BceS-like histidine kinases. To further investigate the signaling mechanism, we performed a random mutagenesis of the transport permease BceB with the aim to identify regions or residues within the transporter that are involved in signaling and/or resistance. With this approach we were able to identify mutations that affected either the ability for signaling or mediation of resistance. This showed a partial genetic separation of the two qualities, which could be achieved by single amino acid replacements. These results provide first insights into the signaling mechanism of the Bce system. In order to analyse the proposed communication between two-component system and ABC transporter, we further characterized their interactions by in vivo and in vitro approaches. We could demonstrate that the transporter BceAB is indeed able to interact directly with the histidine kinase. Because it was unknown how the signal perception by BceAB-type transporters occurs, we next analyzed substrate binding by the transporter permease BceB and could show direct binding of bacitracin by BceB. Finally, in vitro signal transduction assays indicated that complex formation with the transporter influenced the activity of the histidine kinase. In summary this thesis clearly shows the existence of a sensory complex comprised of BceRS-like two-component systems and BceAB-like ABC transporters and provides first functional insights into the mechanism of stimulus perception, signal transduction and antimicrobial resistance mechanism employed by these wide spread detoxification systems against antimicrobial peptides.
Um sich in solch hart umkämpften Habitaten wie dem Boden zu behaupten sind Bakterien dazu übergegangen Antibiotika zu produzieren, um das Wachstum der Konkurrenz einzudämmen. Eine Gruppe solcher Substanzen sind antimikrobielle Peptide, die von Gram-positiven Bakterien produziert werden. Zum Schutz vor Peptidantibiotika haben Gram-positive Bakterien eine Vielzahl verschiedener Resistenzmechanismen entwickelt. Den effizientesten Resistenzmechanismus gegen Peptidantibiotika stellt eine Gruppe ATP-abhängiger ABC-Transporter dar. Diese Transporter weisen einen besonderen Transmembranaufbau auf. Sie bestehen aus zehn Transmembranhelices und einer großen extrazellulären Domäne. Die Expression dieser Transportergruppe wird durch ein Zweikomponentensystem reguliert. Die Histidinkinase besitzt ebenfalls einen ungewöhnlichen Transmembranaufbau, da sie keine offensichtliche Bindedomäne besitzt. Zusammen bilden der Transporter und die Histidinkinase ein Resistenzmodul gegen Peptidantibiotika, das in Firmicutes weit verbreitet ist. Eines der am besten verstandenen Systeme ist das BceRS-BceAB System in Bacillus subtilis. Dieses System vermittelt Resistenz gegen Bacitracin, Actagardin und Mersacidin. Für dieses System konnte gezeigt werden, dass die Histidinkinase BceS alleine nicht in der Lage ist, auf Bacitracin zu reagieren, sondern stattdessen für die Reizwahrnehmung und die Vermittlung der Resistenz auf den Transporter BceAB angewiesen ist. Der Transporter reguliert somit eine eigene Produktion. Wie der Resistenzmechanismus in diesem System genau funktioniert konnte bisher aber noch nicht hinreichend geklärt werden. Dass Transporter neben ihrer Funktion Substrate über eine Zellmembran zu transportieren auch an der Reizwahrnehmung und der Antwortregulation beteiligt sein können, ist in unterschiedlichsten Beispielen beschrieben worden. Um die Signalweiterleitung an membranständige oder zytoplasmatische Komponenten des Signalwegs gewährleisten zu können, müssen diese miteinander interagieren, zum Beispiel durch direkte Protein-Protein Interaktionen. Bisher konnte jedoch für viele solcher Sensorkomplexe keine endgültige Erklärung für solch eine Interaktion dargestellt werden. Basierend auf einer Datenbankanalyse konnten über 250 BceAB-artige Transporter identifiziert und ein Großteil davon einer BceS-artigen Histidinkinase zugeordnet werden. Durch eine phylogenetische Studie konnte weiterhin gezeigte werden, dass BceRS-artige Zweikomponentensysteme und BceAB-artige Transporter in Firmicutes Bakterien weit verbreitet sind und sich über Ko-Evolution gemeinsam zu Resistenzmodulen gegen Peptidantibiotika entwickelt haben. Dazu konnte eine konservierte Antwortregulator-Bindestelle in den Promoter Regionen der Transporteroperons bestimmt werden. Zudem war es möglich aufgrund dieser Klassifizierung für diejenigen Permeasen ohne ein benachbartes Zweikomponentensystem anhand der Genomsequenz ein mögliches Regulationssystem zuzuordnen. Diese Erkenntnisse unterstützten die Vermutung über einen sensorischen Komplex zwischen BceS-ähnlichen Histidinkinasen und BceAB-ähnlichen ABC Transportern. In einer weiteren Studie konnten mittels zufälliger Mutagenese der Transporterpermease BceB Aminosäurereste identifizierte werden, die an der Signalweiterleitung und/oder Resistenzvermittlung beteiligt waren. Durch einige der eingefügten Mutationen wurde nur die Signalweiterleitung bzw. nur die Resistenz beeinträchtigt. Dies spricht dafür, dass eine partielle genetische Trennung der Aufgaben des Transporters möglich ist. Hierdurch konnten erste wichtige Einblicke in den Signalweiterleitungsmechanismus des Bce-Systems gewonnen werden. Um die vorgeschlagene Kommunikation zwischen Zweikomponentensystem und ABCTransporter weiterführend zu untersuchen, wurden Interaktionsstudien durchgeführt. Die auf in vitro und in vivo Studien basierenden Ergebnisse konnten eine direkte Interaktion zwischen BceS und BceAB darstellen. Darüber hinaus konnten wir in dieser Arbeit durch eine Oberflächenresonanz- Spektroskopie zum ersten Mal zeigen, dass die Transporterpermease Bacitracin direkt und spezifisch bindet. Außerdem konnte durch eine in vitro Rekonstruktion des Signalwegs im Bce-System gezeigt werden, dass die Aktivität der Histidinkinase durch die Anwesenheit des Transporters beeinflusst wird. Zusammenfassend zeigt die vorliegende Arbeit direkte Hinweise, dass BceRS-artige Zweikomponentensysteme und BceAB-artige ABC-Transporter zusammen einen sensorischen Komplex für Peptidantibiotika bilden. Dies wird unterstützt durch erste funktionelle Einblicke in die Mechanismen der Reizwahrnehmung und Signalweiterleitung in diesen in Firmicutes Bakterien weit verbreiteten Resistenzsystemen.

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Thomassin, Jenny-Lee. "Antimicrobial peptide resistance mechanisms used by Enteropathogenic and Enterohemorrhagic «Escherichia coli»." Thesis, McGill University, 2014. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=121462.

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Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) are Gram-negative pathogens that cause diarrheal disease in the developed and developing world. To cause infection, these pathogens must overcome innate host defenses, such as secreted cationic antimicrobial peptides (AMPs). There are two groups of human AMPs: cathelicidins (LL-37) and defensins (α-defensin 5). AMPs are expressed in specific locations of the human body. In the small intestine, the infectious niche for EPEC, human α-defensins 5 and 6 (HD-5 and HD-6) are abundant and there are low levels of LL-37. Conversely in the colon, the infectious niche for EHEC, HD-5 and HD-6 are not expressed and LL-37 is abundant. Pathogens can overcome AMP-killing using several mechanisms, including proteolytic inactivation, producing shielding structures and modifying their lipopolysaccharide (LPS). We hypothesized that EPEC and EHEC use AMP-resistance mechanisms to resist killing by secreted AMPs during infection. Previously, CroP the omptin protease in Citrobacter rodentium, a murine pathogen used to model EPEC and EHEC infections, was shown to degrade murine cathelicidin. Both EPEC and EHEC have a CroP-homologue: OmpT. The contribution of OmpT to LL-37 resistance was analyzed in both pathogens. Peptide cleavage assays showed that EHEC OmpT cleaves and inactivates LL-37 more rapidly than EPEC OmpT. Higher ompT-expression and protein levels in EHEC than EPEC are responsible for the differences observed in LL-37 inactivation rates. Additional studies showed that OmpT was unable to cleave folded α-defensins. These data suggest that EPEC uses other mechanisms to resist killing by the AMPs in its infectious niche. To assess this possibility, surface structures that may shield the bacterial membrane from AMPs were identified. High transcript levels of gfcA, a gene required for group 4 capsule (G4C) secretion, were observed in EPEC but not EHEC. The unencapsulated EPEC ΔgfcA and EHEC wild-type strains were more susceptible to HD-5 killing than EPEC wild-type. Since the G4C is composed of the same sugar repeats as the LPS O-antigen, an O-antigen ligase (waaL) deletion mutant was generated to assess the role of the O-antigen in HD-5 resistance. The EPEC ΔwaaL strain was more susceptible to HD-5 than both the wild-type and ΔgfcA strains. Addition of exogenous polysaccharide increased survival of the ΔgfcAΔwaaL strain in the presence of HD-5, suggesting that HD-5 binds the polysaccharides present on the surface of EPEC. These data show that EPEC relies on both the G4C and O-antigen to resist the bactericidal activity of HD-5. Altogether, these data indicate that EHEC and EPEC differentially regulate AMP-specific resistance mechanisms as an adaptation to their specific infectious niches.
Les Escherichia coli entéropathogènes et entérohémorrhagiques (EPEC et EHEC) sont des bactéries à coloration Gram-négative qui causent des diarrhées dans les pays développés et en développement. Pour causer une infection, ces pathogènes doivent surmonter les défenses de l'immunité innée de l'hôte, tel que les peptides antimicrobiens sécrétés (PAMs). Chez l'humain, les PAMs sont divisés en deux groupes, les cathélicidines (ex. LL-37) et les défensines (ex. α-défensine humaine 5). L'expression des PAMs varie selon les tissus. Dans l'intestin grêle, la niche infectieuse des EPEC, les α-défensines humaines 5 et 6 (HD-5 et HD-6) sont abondantes et le niveau de LL-37 est bas. Inversement, HD-5 et HD-6 ne sont pas exprimées dans le côlon, la niche infectieuse des EHEC, et LL-37 est très abondant. Les pathogènes peuvent résister aux PAMs en utilisant différent mécanismes comme l'inactivation protéolytique, la production de structures recouvrant la cellule bactérienne et la modification du lipopolysaccharide (LPS). Notre hypothèse est que les EPEC et EHEC utilisent des mécanismes de résistance aux PAMs pour établir une infection. Précédemment, il a été démontré que la protéase de type omptin, CroP, de Citrobacter rodentium, un pathogène murin utilisé comme modèle pour les infections des EPEC et EHEC, dégrade la cathélicidine murine. Les EPEC et EHEC possèdent un homologue de CroP, OmpT. La contribution de OmpT à la résistance au LL-37 a été examinée chez ces deux pathogènes. Nos tests de clivage de peptide ont démontré que EHEC OmpT clive et inactive LL-37 plus rapidement que EPEC OmpT. La différence observée a été associée à une plus forte expression et production de OmpT chez les EHEC que chez les EPEC. Des tests supplémentaires ont démontré que OmpT ne peut pas cliver les α-défensines repliées. Ces données suggèrent qu'EPEC utilise d'autres mécanismes de résistance pour surmonter l'activité des PAMs présents dans sa niche infectieuse. Pour tester cette possibilité, les structures recouvrant la cellule ont été identifiées. Un haut niveau de transcription de gfcA, un gène requit pour la sécrétion de la capsule du groupe 4 (G4C), a été observé chez EPEC mais pas chez EHEC. Le mutant EPEC non-encapsulé ΔgfcA et la souche sauvage EHEC sont plus susceptible à l'effet du HD-5 que la souche sauvage EPEC. Étant donné que la G4C est composée des mêmes sucres que l'antigène O, la ligase de l'antigène O, waaL, a été délétée pour déterminer le rôle de l'antigène O dans la résistance au HD-5. La souche EPEC ΔwaaL est plus susceptible au HD-5 que la souche sauvage EPEC et le mutant EPEC ΔgfcA. L'addition de polysaccharide exogène augmente la survie du mutant ΔwaaLΔgfcA en présence de HD-5. Ceci indique que HD-5 se lie aux polysaccharides présents à la surface des EPEC. Ces données démontrent que la résistance à HD-5 chez EPEC repose sur la présence de la G4C et de l'antigène O. Toutes ces données indiquent que EHEC et EPEC utilisent des mécanismes de résistance différents aux PAMs, ce qui démontre une adaptation à leurs niches infectieuses respectives.

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Smith, Ryan Douglas. "Investigating the antimicrobial peptide resistance and mechanism of RosB and the Sap system." Thesis, University of Aberdeen, 2016. http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=230775.

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Bacteria have adapted to CAMP insult in many ways, this study has focused on two resistance mechanism for CAMPs, firstly the putatative potassium proton antiporter RosB has been shown not to efflux potassium with treatment of polymyxin B. The mechanism of resistance of RosB in Yersinia enterococcus was proposed to be a lowering of internal pH from a potassium efflux and a proton influx. The RosB homologue from Vibrio paraheamolyticus is able to efflux potassium with electrophile treatment, but unlike the potassium proton antiporter KefKC this potassium transport is not associated with CAMP resistance. The lack of resistance is likely to be due to an absence of intracellular pH regulation seen with the Vibrio RosB. The RosB homologue from E. coli YbaL has been shown to increase resistance to the electrophile NEM in high potassium media, but YbaL does not transport potassium when NEM is present. This suggests that YbaL is transporting another ion. The mechanism of ion transport for RosB is based on the sodium antiporter NhaA and NapA, this is due to similarities in the ion selection motif. There are differences between the Vibrio and Yersina homologue which would suggest that the Yersinia homologue is not transporting potassium. This second CAMP resistance mechanism studied was the ABC transporter Sap, with the focus of work done on the periplasmic substrate binding protein SapA. SapA binds the peptide within the periplasm and delivers it to the membrane domain for uptake in to the cell. It was not possible to detect any peptide binding to the SapA homologues from Klebsiella pneumonia or E. coli. Finally CAMPs purification was attempted under taken through Ni-NTA and chitin chromatography to produce human antimicrobial peptides. It was possible to 12 produce the human antimicrobial peptide HBD2 with thioredoxin fusion complex via Ni-NTA chromatography.

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Zhang, P. "Identification of staphylococcal genes involved in resistance to the human antimicrobial peptide LL-37." Thesis, University College London (University of London), 2012. http://discovery.ucl.ac.uk/1380282/.

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Staphylococcus aureus is well-known for its ability to acquire resistance to a broad range of antimicrobial agents and a limited number of commercially available antibiotics exist that are active against multidrug resistant strains. Antimicrobial peptides have been suggested as promising alternatives to current antimicrobials due to their potent antimicrobial activity against a broad range of microorganisms including multidrug resistant bacteria, and a membrane-lytic mode of action that is thought to have low possibility of inducing bacterial resistance. This study describes the identification of S. aureus genes involved in resistance to the human cationic antimicrobial peptide LL-37, with a particular interest in the effects of a physiological concentration of bicarbonate on the resistance mechanism. Transposon mutagenesis and recombinase-based in vivo expression technology systems were designed to enable genome-wide screening. A S. aureus transposon mutant library was screened for increased resistance to LL-37 in the presence of bicarbonate. Mutants with insertions in yycH and yycI, demonstrated bicarbonate-dependent resistance to LL-37. Both yycH and yycI form part of a predicted operon yycFGHI in S. aureus, and have been shown to be suppressors of an essential two component system YycFG in B. subtilis that regulates cell wall metabolism. The resistance of S. aureus small colony variants (SCVs) to LL-37 was also investigated. SCVs defective in hemB, menD or aroD, demonstrated bicarbonate-dependent resistance to LL-37. Furthermore, SigB (a global regulator) and TcaR (an activator of protein A) were found to exert opposite effects on resistance to LL-37 in the presence of bicarbonate. Strains defective in TcaR showed bicarbonate-dependent resistance to LL-37, interestingly, this resistance was abolished by either deleting sigB or repairing tcaR in these strains. These data suggest that YycFG, SigB, TcaR and the SCV phenotype may play important roles in resistance to LL-37 under in vivo conditions where bicarbonate is present.

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Ackroyd, Bryony Kate. "Structural and biochemical analysis of E. coli ABC transporters implicated in antimicrobial peptide resistance." Thesis, University of York, 2018. http://etheses.whiterose.ac.uk/22751/.

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Cationic antimicrobial peptides (CAMPs) are a key component of the innate immune system of many organisms, including humans. They target invading pathogens in a variety of ways often integrating into, and permeabilising, bacterial cell membranes and causing cell death. In response, bacteria have developed a variety of CAMP resistance mechanisms, including those based on ATP-binding cassette (ABC) transporters such as Sap and Yej, which are the subject of studies described herein. ABC importers use an extracellular substrate binding protein (SBP) to recognise substrates and deliver them to a cognate membrane complex for uptake into the cell. A primary aim of this study was to unravel the structural basis of CAMP binding by the SBPs, SapA and YejA. CAMPs are larger than conventional peptides handled by ABC transporters and usually contain secondary structure.

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Linde, Charlotte M. A. "Defense peptides against Mycobacteria /." Stockholm, 2005. http://diss.kib.ki.se/2005/91-7140-480-5/.

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Jacob, Rebecca. "Lipid bilayers and their interactions with the antimicrobial peptide LL37: a TIR Raman study." Thesis, KTH, Skolan för kemivetenskap (CHE), 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-207018.

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As a direct consequence of the misuse of antibiotics since its first discovery, bacteria have developed extensive resistance mechanisms making them once again potential lethal threats. This eventuality has triggered a vast research effort from scientists worldwide to find solutions to mitigate antimicrobial resistance. One such option is the identification of new potential antimicrobial agents, like for example antimicrobial peptides (AMPs). Various methods have been applied to evaluate the properties and determine the complex mechanism of AMPs. However, the details of the mechanism remain unknown and hence the work in this project seeks to examine the suitability of using TIR Raman, a vibrational spectroscopy technique which is sufficiently surface sensitive to study the interaction of AMPs in contact with lipid bilayers, which are just a few nanometres thick. In order to evaluate the information that could be extracted from TIR Raman, measurement of solid supported lipid bilayers in the absence of peptides were first carried out. In particular, the lipid DMPC with a phase transition close to room temperature, was measured at various temperatures to determine spectral changes associated with the transition. For the peptide-membrane interactions, the AMP LL37 was put into contact with solid supported lipid bilayers modelling the cell membranes of bacteria (DOPE, DOPG) or humans (DOPC) respectively. The data clearly indicates that the membrane composition, and specifically the lipid head group charge, play an important role in the peptide-membrane interactions. In the bilayers mimicking bacteria cell membranes, the peptide either absorbed onto or inserted into the bilayer. In contrast, for the bilayer modelling a human cell membrane, no significant variations were detected, indicating no interaction with LL37. The findings presented in this work generally coincide with similar research of LL37 using other techniques. At hand of the herein presented data, TIR Raman has proven suitable and effective in detecting effects of antimicrobial peptides in contact with model lipid bilayers, and hence can be recommended for further studies.
Som en direkt följd av missbruket av antibiotika sedan det först upptäcktes, har bakterier utvecklat omfattande resistensmekanismer vilket föranlett dem att återigen utgöra potentiellt dödlig hot. Denna situation har manat fram en väsentlig forskningsinsats från forskare världen över att hitta lösningar för att minska antimikrobiell resistens. Ett sådant alternativ har varit identifieringen av nya potentiella antimikrobiella substanser, så som till exempel antimikrobiella peptider. Ett flertal metoder har använts för att både evaluera peptiders egenskaper och fastställa deras komplexa mekanism. Detta till trots förblir de exakta detaljerna i mekanismen okända, vilket föranlett arbetet i detta projekt att undersöka lämpligheten i att använda TIR Raman, en vibrational-spektroskopisk metod som är tillräckligt ytkänslig för att studera interaktionen hos antimikrobiella peptider i kontakt med lipidmembran, vilka endast är några få nanometer tjocka. För att evaluera informationen som kan utvinnas med TIR Raman, utfördes först mätningar av lipidmembran, skapade på ett solitt underlag, utan tillägg av peptider. Mer noggrant, har lipiden DMPC med en fasövergång nära vid rumstemperatur, mätts vid olika temperaturer för att fastställa spektrala variationer associerade till övergången. För peptid-membran interaktionerna, sattes den antimikrobiella peptiden LL37 i kontakt med lipidmembran som modellerar cellmembranet hos bakterier (DOPE, DOPG) respektive människor (DOPC). Mätdatan indikerar tydligt att membrankompositionen, och där specifikt laddningen av lipidens huvudgrupp, spelar en viktig roll i membran-peptid interaktionerna. För lipidmembranen som imiterar bakteriella cellmembran, adsorberade peptiden till membranet eller integrerades in i det. Till skillnad från detta, kunde inga signifikanta variationer detekteras för lipidmembranet som modellerade ett mänskligt membran vilket indikerar att det inte interagerar med peptiden LL37. Upptäckterna som presenteras i detta arbete sammanfaller generellt med andra, liknande studier där andra instrument använts för att undersöka LL37. Det kan ur materialet som presenterats här utläsas att TIR Raman visat sig lämpligt och effektivt i detekteringen av antimikrobiella peptider i kontakt med modeller av lipidmembran, och kan därav rekommenderas för fortsatta studier.

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El, Shazely Baydaa Mohamed Ahmed [Verfasser]. "Antimicrobial peptide resistance: infection dynamics, crosssensitivity to insect immune effectors, and evolution of pharmacodynamic / Baydaa Mohamed Ahmed El Shazely." Berlin : Freie Universität Berlin, 2021. http://d-nb.info/1230797637/34.

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12

Chen, Heng-Chang. "Identification and characterization of a novel Salmonella gene product, STM0029, which contributes to the resistance to host antimicrobial peptide killing." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2013. http://dx.doi.org/10.18452/16658.

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Salmonella spp. sind fakultative intrazelluläre Pathogene, die gastrointestinale und systemische Erkrankungen in einem umfassenden Wirtsbereich, einschließlich Tier und Mensch, hervorrufen. Salmonella benötigt verschiedene Virulenzgene für die Infektion welche auf sogenannten Salmonella Pathogenitäts-Inseln (SPI) kodiert sind. Hinzu kommt, dass auch zahlreiche im Salmonella Genom verstreuten Gene an verschiedenen Aspekten von Virulenz und Pathogenese beteiligt sind. In der vorliegenden Studie wurde die Funktion eines zuvor nicht beschriebenen putativen transkriptionellen Regulators (STM0029) charakterisiert und definiert. Dieser scheint für die Abwehr von zellulären bakterizid wirkenden Verbindungen und das Überleben des Bakteriums innerhalb einer intrazellulären Nische von entscheidender Bedeutung zu sein. Die STM0029-deletierte Mutante wies eine gesteigerte Sensitivität gegenüber antimikrobiellen Peptiden und bakteriziden Verbindungen auf. Dazu zählten α-Defensin-1, β- Defensin-1, β-Defensin-2, LL-37 und Polymyxin B sowie Komponenten des Komplementsystems. Unerwartet war die Beobachtung, dass die Expression von STM0029 durch das PmrA/B Zwei Komponenten System reprimiert vorlag, während das PhoP/Q Zwei Komponenten System keinen Einfluss auf die Expression von STM0029 zu scheinen hat. Beide Komponent Systeme spielen bekanntlich eine entscheidende Rolle bei der Expressionsregulation von Genen die für das intrazelluläre Überleben von Salmonella wichtig sind. Bemerkenswert ist, dass ein Set von Genen welche an der Biosynthese und/oder der Modifikation für das LPS O-Antigen sowie des Peptidoglykans in der bakteriellen Zellwand beteiligt ist, im STM0029 Deletionshintergrund herab reguliert vorlag. Dieses Ergebnis deutet darauf hin, dass das STM0029 Genprodukt die Persistenz des Pathogen in Wirtszellen beeinflusst. Möglicherweise geschieht dies durch das Umgehen von wirtseigenen Abwehrmechanismen.
Salmonella spp. are facultative intracellular pathogens, which cause gastrointestinal and systemic diseases in a broad range of hosts including animals and humans. In addition to virulence genes clustered within pathogenicity islands, numerous additional genes scattered throughout the genome are also involved in various aspects of Salmoenlla virulence and pathogenesis. In this study, I identified a Salmonella putative transcriptional regulator encoded by a previously uncharacterized open reading frame designated STM0029. Deletion of STM0029 altered the expression of genes involved in both the resistance to host bactericidal challenges, and bacterial cell wall biosynthesis in S. Tyhpimurium. The ΔSTM0029 strain showed a defect in the resistance to host antimicrobial peptides, including α-defensin-1, β-defensin-1, β-defensin-2, LL-37, and polymyxin B as well as serum challenges compared to the wildtype. Unexpectedly, expression of STM0029 was found to be repressed by the PmrA/B two component system, but appeared to be independent of the PhoP/Q two component system, both of which are well-known regulatory systems involved in the regulation of expression of genes involved in Salmonella intracellular survival. Notably, the expression of a set of genes involved in bacterial LPS O-antigen and peptidoglycan biosyntheses and modifications showed decreases in the absence of STM0029. These experimental results indicate that the STM0029 gene product in S. Typhimurium contributes to resistance against host cell defense mechanisms, likely through regulation of genes involved in LPS O-antigen and peptidoglycan biosynthesis and modifications.

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Richards, Susan Michelle. "PhoPQ- and PmrAB-mediated Lipopolysaccharide Modification and Cationic Antimicrobial Peptide Resistance in Salmonella enterica Serovars Typhimurium and Typhi." The Ohio State University, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=osu1290981691.

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Katvars, Laura K. "Novel cationic peptides and polymers in the treatment of methicillin-resistant Staphylococcus aureus and multi-drug resistant Acinetobacter spp. skin infection isolates." Thesis, University of Aberdeen, 2015. http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=228984.

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Huang, En. "Naturally Occurring Antimicrobial Peptides for Enhancing Food Safety and Protecting the Public against Emerging Antibiotic-resistant Pathogens." The Ohio State University, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=osu1366298199.

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Dintner, Sebastian [Verfasser], and Susanne [Akademischer Betreuer] Gebhard. "Characterization of a sensory complex involved in antimicrobial peptide resistance : communication between a histidine kinase and an ABC transporter in Bacillus subtilis / Sebastian Dintner. Betreuer: Susanne Gebhard." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2014. http://d-nb.info/1062492692/34.

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Chen, Heng-Chang [Verfasser], Richard Akademischer Betreuer] Lucius, Lothar H. [Akademischer Betreuer] [Wieler, Thomas [Akademischer Betreuer] Eitinger, and Klaus [Akademischer Betreuer] Heuner. "Identification and characterization of a novel Salmonella gene product, STM0029, which contributes to the resistance to host antimicrobial peptide killing / Heng-Chang Chen. Gutachter: Richard Lucius ; Lothar H. Wieler ; Thomas Eitinger ; Klaus Heuner." Berlin : Humboldt Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2013. http://d-nb.info/1030313628/34.

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Chen, Heng-Chang [Verfasser], Richard [Akademischer Betreuer] Lucius, Lothar H. [Akademischer Betreuer] Wieler, Thomas [Akademischer Betreuer] Eitinger, and Klaus [Akademischer Betreuer] Heuner. "Identification and characterization of a novel Salmonella gene product, STM0029, which contributes to the resistance to host antimicrobial peptide killing / Heng-Chang Chen. Gutachter: Richard Lucius ; Lothar H. Wieler ; Thomas Eitinger ; Klaus Heuner." Berlin : Humboldt Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2013. http://nbn-resolving.de/urn:nbn:de:kobv:11-100206853.

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19

Vargues, Thomas. "Antimicrobial peptides : structure, function and resistance." Thesis, University of Edinburgh, 2009. http://hdl.handle.net/1842/4076.

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Higher eukaryotes produce a vast range of antimicrobial peptides (AMPs) that play important roles in their defence against microbial infection. Beta defensins are small (3-5 kDa), cationic peptides that display broad, potent antimicrobial activity against a range of microbes and also act as chemoattractants of important immunomodulatory cells. To generate highly pure peptides for structural and functional studies, we developed a method to prepare recombinant human beta defensin-2 (HBD2). The HBD2 gene was synthesised by recursive PCR with codons optimised for expression in Escherichia coli. HBD2 was expressed as an insoluble fusion to a His-tagged ketosteroid isomerase. After cleavage from the fusion with cyanogen bromide, 1H NMR spectroscopy and mass spectrometry confirmed that the oxidised HBD2 was folded and possessed the correct b-defensin disulfide bond topology. The recombinant HBD2 was active against E. coli, P. aeruginosa, S. aureus and C. albicans and was also a chemoattractant against HEK293 cells expressing the chemokine receptor CCR6. 15N-labelled HBD2 was also prepared and was highly suitable for future structural studies. Since defensins are thought to interact with bacterial membranes we also tested the recombinant HBD2 in biophysical studies (surface plasmon resonance, SPR, Biacore). We observed different binding to artificial model membranes containing either E. coli Kdo2-lipid A or phospholipids. Bacterial resistance to AMPs has been linked to the covalent modification of the outer membrane lipid A by 4-amino-4-deoxy-L-arabinose (L-Ara4N). This neutralises the charge of the LPS, thereby decreasing the electrostatic attraction of cationic peptides to the bacterial membrane. The pathogen Burkholderia cenocepacia displays extremely high resistance to AMPs and other antibiotics and the Ara4N pathway appears to be essential. To explore this further we expressed recombinant forms of two enzymes (ArnB and ArnG) from the B. cenocepacia Ara4N pathway. Purified ArnB is a pyridoxal 5’-phosphate (PLP)-dependent transaminase and we tested its ability to bind amino acid substrates. We investigated the binding of inhibitors L- and D-cycloserine to ArnB and tested their antibiotic activity against Burkholderia strains. We also studied the B. cenocepacia ArnG – a proposed membrane protein undecaprenyl-L-Ara4N flippase – and showed that the protein behaved as a dimer by non-denaturing gel analysis. The B. cenocepacia ArnG failed to complement E. coli knock-out strains encoding the equivalent flippase proteins ArnE and ArnF, suggesting that ArnG is a Burkholderia-specific protein.

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Malik, Sohaib Zafar. "Interaction of cyclotides and bacteria : A study of the cyclotide action and the bacterial reaction." Doctoral thesis, Uppsala universitet, Avdelningen för farmakognosi, 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-318656.

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The growing problem of antibiotic resistance and the lack of promising prospective antibiotics have forced us to search for new classes of antibiotics. Among the candidates to develop into future antibacterials are antimicrobial peptides (AMPs). These potent, broad spectrum compounds are important components of innate immunity of organism from all kingdoms of life. One such family of mini-proteins from plants is called cyclotides, whose members are defines by cyclic backbone and a cystine knot (CCK), which confers to them extreme stability in the face of biological, chemical and physical insults. Some cyclotides possess Gram-negative specific antibacterial activity; the purpose of this thesis was to characterize how these molecules kill bacteria, and how bacteria would respond to treatment with cyclotides. For this purpose, Salmonella enterica and Escherichia coli mutants resistant to the cyclotides cycloviolacin O2 and cycloviolacin O19, respectively, were selected. These mutants were characterized by whole genome sequencing, genetic reconstitution, fitness measurements, and cross-resistance studies. These studies identified a number of genetic pathways for resistance development to cyclotides. These mutants displayed variable fitness profiles in laboratory growth media and in mice competition experiments, with some mutants possessing a fitness advantage in mice. Cross-resistance studies resulted in the identification of several cases of cross-resistance and collateral sensitivity between cyclotides and other AMPs/antibiotics. Antimicrobial effects of cyclotides were assayed in different conditions and in bacterial organisms with different surface characteristics. In addition, immunolocalization experiments were performed to explore the biological distribution of cyclotides in plants and to determine the mechanism of action of cyclotides in bacteria, respectively. Antibodies raised against cyO2 were used for this purpose. Immunohistochemical techniques applied to plant cells, tissues and organs provided the information that cyclotides were distributed in all plant organs, and were found in tissues vulnerable to pathogen attack, and that cyclotides were stored in the vacuoles of plant cells. Immunogold staining of cyclotide treated cells of S. typhimurium, showed effects of cyclotide treatment on the cell envelope components as well as cytoplasm. A higher number of cyclotide molecules was associated with the cell envelope, but a considerable fraction of them penetrated into the cytoplasm.

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Pränting, Maria. "Bacterial Resistance to Antimicrobial Peptides : Rates, Mechanisms and Fitness Effects." Doctoral thesis, Uppsala universitet, Institutionen för medicinsk biokemi och mikrobiologi, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-130168.

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The rapid emergence of bacterial resistance to antibiotics has necessitated the development of alternative treatment strategies. Antimicrobial peptides (AMPs) are important immune system components that kill microbes rapidly and have broad activity-spectra, making them promising leads for new pharmaceuticals. Although the need for novel antimicrobials is great, we also need a better understanding of the mechanisms underlying resistance development to enable design of more efficient drugs and reduce the rate of resistance development. The focus of this thesis has been to examine development of bacterial resistance to AMPs and the resulting effects on bacterial physiology. The major model organism used was Salmonella enterica variant Typhimurium LT2. In Paper I, we observed that bacteria resistant to PR-39 appeared at a high rate, and that the underlying sbmA resistance mutations were low cost or even cost-free. Such mutants are more likely to rapidly appear in a population and, most importantly, will not disappear easily once the selective pressure is removed. In paper II, we isolated protamine-resistant hem- and cydC-mutants that had reduced growth rates and were cross-resistant to several other antimicrobials. These mutants were small colony variants (SCVs), a phenotype often associated with persistent infections. One SCV with a hemC-mutation reverted to faster growth when evolved in the absence of protamine. In paper III, the mechanism behind this fitness compensation was determined, and was found to occur through hemC gene amplification and subsequent point mutations. The study provides a novel mechanism for reversion of the SCV-phenotype and further evidence that gene amplification is a common adaptive mechanism in bacteria. In Paper IV, the antibacterial properties of cyclotides, cyclic mini-proteins from plants, were evaluated. Cycloviolacin O2 from violets was found to be bactericidal against Gram-negative bacteria. Cyclotides are very stable molecules and may be potential starting points for development of peptide antibiotics.

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Yu, Guozhi [Verfasser]. "Antimicrobial peptides: pharmacodynamics, combinatorial effects and resistance evolution / Guozhi Yu." Berlin : Freie Universität Berlin, 2018. http://d-nb.info/1150238127/34.

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Lofton, Tomenius Hava. "Mechanisms and Biological Costs of Bacterial Resistance to Antimicrobial Peptides." Doctoral thesis, Uppsala universitet, Institutionen för medicinsk biokemi och mikrobiologi, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-284119.

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The global increasing problem of antibiotic resistance necessarily drives the pursuit and discovery of new antimicrobial agents. Antimicrobial peptides (AMPs) initially seemed like promising new drug candidates. Already members of the innate immune system, it was assumed that they would be bioactive and non-toxic. Their common trait for fundamental, non-specific mode of action also seemed likely to reduce resistance development. In this thesis, we demonstrate the ease with which two species of pathogenic bacteria, the gram-negative Salmonella typhimurium (S. typhimurium), and the gram-positive Staphylococcus aureus (S. aureus), can gain increased tolerance and stable resistance to various AMPs. By serially passaging each bacterial species separately under increasing AMP selection pressure we observed increasing AMP tolerance. Resulting in independent bacterial lineages exposed to four different AMPs (including a two-AMP combination) that exhibited 2 to 16-fold increases in MIC. Substantial cross-resistance between the AMPs was observed. Additionally, the S. aureus mutants were found to be cross-resistant to human beta-defensins 1, 2, 3, and 4. The LPS molecule, with mutations in the waaY, pmrB and phoP genes, was the principal target for S. typhimurium resistance development. The main target for S. aureus remained elusive. Reduced membrane potential was a common change for two of the mutants, but not for the others. All sequenced mutants had one or more mutations in various stress response pathways. Fitness of the resistant mutants was assayed by growth rate analysis and in vitro virulence factor testing (e.g. survival response to bile, superoxide, acidic pH). Furthermore an in vivo survival/virulence test involving a mouse competition experiment (S. typhimurium) and sepsis model (S. aureus) was performed. In the absence of AMPs there was often little or no fitness reduction in the mutants. Our results suggest that AMP resistance mechanisms do not irrevocably weaken either species with regard to virulence characteristics or survival within the host. In light of these findings, we suggest that the progression of therapeutic use of AMPs should proceed with great caution since otherwise we might select for AMP resistant mutants that are more resistant to our innate host defenses and thereby potentially more virulent.

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Felek, Arif. "Discovery of antimicrobial peptides active against antibiotic resistant bacterial pathogens." Thesis, University of Manchester, 2015. https://www.research.manchester.ac.uk/portal/en/theses/discovery-of-antimicrobial-peptides-active-against-antibiotic-resistant-bacterial-pathogens(cc408f16-e24a-4f49-ac8e-e7a5fe7185d0).html.

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Rapid development of antimicrobial resistance (AMR) among bacteria, combined with diminished new antibiotic discovery rates, is an increasing threat to human health. Bacterially derived antimicrobial peptides (AMP) hold excellent potential as potent novel therapeutics. This study embraces traditional natural AMP discovery methods and the newer in silico genome mining tool BAGEL 3 to facilitate identification of novel antimicrobial agents. The traditional screening efforts led to the discovery of two promising antimicrobial producer strains; Bacillus pumilus J1 producing two AMPs, peptides NI03 and NI04, and Klebsiella pneumoniae A7, which produced peptide NI05. In silico mining of the B. pumilus J1 and K. pneumoniae A7 genomes and those from under exploited anaerobic bacteria using BAGEL 3 yielded 18 putative bacteriocin structures that were associated with multiple known and relevant bacteriocin accessory genes and/or carried significant homologies to known bacteriocins. Peptide NI04 proved to be active against Gram positive species only, including meticillin resistant Staphylococcus aureus and vancomycin resistant enterococci and peptide NI03, in addition to these pathogens, showed activity against E. coli. Peptide NI05 was active against Gram-negative pathogens including extended spectrum β-lactamase producing E. coli. All isolated peptides were observed to be proteinaceous in nature and highly heat stable. Peptides were purified or partially purified using solid phase extraction followed by RP-HPLC. The mass of the peptides was determined using ESI or MALDI-TOF mass spectrometry. Tandem MS fragmentation of peptide NI04 generated several sequence tags. Draft genome sequences of the B. pumilus J1 and K. pneumoniae A7 producer strains were obtained using the Illumina MiSeq platform. This allowed identification of the genes encoding peptide NI04, which was confirmed to be novel and was named pumicin NI04. Further characterisation of pumicin NI04 demonstrated it was non-toxic to keratinocytes, Vero cells and non-haemolytic up to at least 18x the minimum inhibitory concentration. The discovery revealed that pumicin NI04 belongs to the WXG-100 peptide superfamily, having homology with the mycobacterial and staphylococcal virulence factor EsxA. This represents the first report of antimicrobial activity in a WXG-100 peptide and has intriguing evolutionary implications. Although it was not possible to fully characterise peptides NI03 and NI05, when BAGEL 3 was used to mine the B. pumilus J1 genome, a promising putative bacteriocin candidate was identified that was homologous to Enterocin AS-45, which also confers anti Gram-negative activity and may be related to the activity observed for NI03, however more evidence is required. Investigations of the K. pneumoniae A7 bacteriocin on the other hand helped establish that the K. pneumoniae microcin E492 pathway was present and highly conserved in strain A7, and is likely to be responsible for the activity observed indicating that NI05 was not a novel peptide.

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Ashby, Martin. "The development of computer-designed and naturally occurring antimicrobial peptides to target methicillin-resistant Staphylococcus aureus (MRSA) using SPOT peptide synthesis." Thesis, St George's, University of London, 2018. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.764275.

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The aim of this thesis is the identification and development of computer-designed and naturally occurring antimicrobial peptides (AMPs) which demonstrate potential as clinically viable compounds in the treatment of MRSA infections. To achieve this aim the method of SPOT peptide synthesis was employed, this allows for hundreds of unique peptide sequences to be synthesised rapidly and relatively cheaply. Using this technique, peptide libraries were constructed based upon two general strategies: 1. Synthesis of novel 9-mer peptide sequences predicted in silico to possess a high therapeutic index. 2. Synthesis of short naturally occurring AMPs sequences ranging in length from 5-17 residues along with truncated forms of longer peptides. In addition, two potent novel peptides were optimised by designing additional libraries containing hundreds of derivatives based upon their sequences. Each library was screened for both antimicrobial and hemolytic activity and several peptides were identified that demonstrate potent in vitro antistaphylococcal activity (MIC 0.5-4 pg/ml) and minimal hemolytic activity (HCso 111- >250 pg/ml). To assess the activity of the peptides in vivo the model organism Galleria mellonella was used. Neither artificial or natural peptides demonstrated substantial toxicity towards this organism, however no peptide could rescue 6. mellonella from a systemic MRSA infection. When applied topically to G. mellonella, several of the peptides could significantly reduce the bacterial burden on the skin. Mode of action studies revealed that all peptides cause rapid depolarisation of the bacterial membrane and differing degrees of membrane permeabilisation. Using small angle X-ray scattering morphological changes that take place in bacteria exposed to different concentrations of peptides for different lengths of time could be tracked. Visualisation of bacteria treated with one novel peptide revealed changes to the intracellular environment. In conclusion, the usefulness of SPOT synthesis in identifying several peptides with potent antimicrobial activity towards MRSA and minimal toxicity towards mammalian cells in vitro was demonstrated. Analysis ofthe screening data revealed peptide features which could be correlated with their level of activity, thus improving our ability to predict active peptides in the future.

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Mohammadihashemi, Marjan. "Antibacterial and Antifungal Activity of Ceragenins, Mimics of Endogenous Antimicrobial Peptides." BYU ScholarsArchive, 2019. https://scholarsarchive.byu.edu/etd/7411.

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The continuous emergence of drug-resistance pathogens is a global concern. As a result, substantial effort is being expended to develop new therapeutics and mechanisms for controlling microbial growth to avoid entering a "post-antibiotic" era in which commonly used antibiotics are no longer effective in treating infections. In this work, we investigate the efficacy and application of ceragenins as non-peptide mimics of antimicrobial peptides (AMPs). First, this work examines the susceptibility of drug-resistant Gram-negative bacteria. The susceptibility of colistin-resistant clinical isolates of Klebsiella pneumoniae to ceragenins and AMPs suggests that there is little to no cross-resistance between colistin and ceragenins/AMPs. Furthermore, Lipid A modifications are found in bacteria with modest changes in susceptibility to ceragenins and with high levels of resistance to colistin. Next, we investigated the potential for cross resistance between chlorhexidine, colistin, AMPs and ceragenins as repeated exposure of bacteria to chlorhexidine might result in cross resistance with colistin, AMPs or ceragenins. Furthermore, a proteomics study on the chlorhexidine-resistant strains showed that chlorhexidine resistance is associated with upregulation of proteins involved in the assembly of LPS for outer membrane biogenesis and virulence factors in Pseudomonas aeruginosa.Second, this dissertation describes the antifungal activity of ceragenins against an emerging multidrug-resistant fungus, Candida auris. We found that lead ceragenins displayed activities comparable to known antifungal agents against C. auris isolates. We also found that fungal cell morphology was altered in response to ceragenin treatment, that ceragenins exhibited activity against sessile organisms in biofilms, and that gel and cream formulations including CSA-44 and CSA-131 resulted in a significant log reduction against established fungal infections in ex vivo mucosal tissues. Finally, a hydrogel film containing CSA-131 was generated on endotracheal tubes (ETTs). ETTs provide an abiotic surface on which bacteria and fungi form biofilms that cause serious infections. In this study, the eluting ceragenin prevented fungal and bacterial colonization of coated ETTs for extended periods while uncoated tubes were colonized by bacteria and fungi. Coated tubes were well tolerated in intubated pigs. The ability of ceragenins to eradicate established biofilms make them attractive potential therapeutics for persistent infections in the lung, including those associated with cystic fibrosis. In ex vivo studies, we initially found that this ceragenin, at concentrations necessary to eradicate established biofilms, also causes loss of cilia function. However, by formulating CSA-131 in poloxamer micelles, cilia damage was eliminated and antimicrobial activity was unaffected. These findings suggest that CSA-131, formulated in micelles, may act as a potential therapeutic for polymicrobial and biofilm-related infections in the lung and trachea.

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Visser, Marike. "An evaluation of the efficacy of antimicrobial peptides against grapevine pathogens." Thesis, Stellenbosch : University of Stellenbosch, 2011. http://hdl.handle.net/10019.1/6729.

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Thesis (MSc (Genetics))--University of Stellenbosch, 2011.
Includes bibliography
ENGLISH ABSTRACT: This study investigated the use of antimicrobial peptides (AMPs) as possible source of resistance against a range of pathogens in grapevine. Whilst the ultimate aim would be to express AMPs in grapevine, the development of transgenic grapevine is time consuming and therefore pre-screening of potential AMPs is necessary. These small molecules, of less than 50 amino acids in length, are expressed by almost all organisms as part of their non-specific defence system. In vitro pre-screening of AMP activity is valuable but is limited since the activity on artificial media may differ from the AMP activity in planta. These tests are also restricted to pathogens which can be cultured in vitro. These limitations can be overcome by using transient expression systems to determine the in planta activity of AMPs against pathogens of interest. In this study transient systems were used to express AMPs in developed plant tissue to test their efficacy against grapevine pathogens such as Agrobacterium vitis, Xylophilus ampelinus and aster yellows phytoplasma. Aster yellows phytoplasma, which was recently discovered in local vineyards, is known to cause extensive damage and therefore pose a great threat to the South African grapevine industry. To study the in planta effect of AMPs against the abovementioned pathogens, transient expression vectors were constructed expressing either of the AMPs D4E1 or Vv-AMP1. D4E1 is a synthetically designed AMP known to be active against bacteria and fungi, while Vv-AMP1, isolated from grapevine berries, has already shown activity against fungi. In a transient approach in grapevine, the expression of foreign genes from viral and non-viral vectors was confirmed by expression of the marker genes β-glucuronidase and Green Fluorescent Protein, while tissue-printing immunoassays confirmed viral replication and systemic spread in Nicotiana benthamiana. The viral vectors were based on the phloem-limited virus grapevine virus A. Only Agrobacterium-mediated 35S transient expression vectors were used for AMP in planta activity screening since the viral-mediated expression in grapevine was insufficient for screening against A. vitis and X. ampelinus as it was restricted to phloem tissues after whole-leaf infiltration. No phytoplasma-infected material could be established and as a result AMP activity screening was only performed against the A. vitis and X. ampelinus. Quantification of the bacteria was performed by qPCR. Vv-AMP1 did not show activity against either of the two bacteria in planta while D4E1 was found to be active against both. The observed in planta activity of D4E1 correlated with the in vitro activity as measured in an AMP plate bioassay. In contrast to in vitro screenings, the in planta AMP activity screening might give a more accurate representation of the potential antimicrobial activity of the peptide in a transgenic plant environment. This study proved that transient expression systems can be used as a pre-screening method of AMP activity in planta against grapevine pathogens, allowing the screening of various AMPs in a relatively short period of time before committing to transgenic grapevine development.
AFRIKAANSE OPSOMMING: Hierdie studie het die gebruik van antimikrobiese peptiede (AMPe) as 'n moontlik bron van weerstand teen 'n reeks van patogene in wingerd ondersoek. Alhoewel die uiteindelike doel sal wees om AMPe uit te druk in wingerd, is transgeniese wingerd ontwikkeling tydrowend en daarom is vooraf evaluering van potensiële AMPe nodig. Hierdie klein molekules, van minder as 50 aminosure in lengte, word uitgedruk deur amper alle organismes as deel van hul nie-spesifieke verdedigingsisteem. In vitro vooraf evaluering van AMP aktiwiteit is van waarde, maar is beperk aangesien die aktiwiteit op kunsmatige media mag verskil van die AMP-aktiwiteit in planta. Hierdie toetse is ook beperk tot patogene wat in vitro gekweek kan word. Hierdie beperkinge kan oorkom word deur gebruik te maak van tydelike uitdrukkingsisteme om die in planta aktiwiteit van AMPe te bepaal teen patogene van belang. In hierdie studie is tydelike uitdrukkingsisteme gebruik om AMPe uit te druk in ontwikkelde plantweefsel om hul effektiwiteite te toets teen wingerdpatogene soos Agrobacterium vitis, Xylophilus ampelinus en aster yellows fitoplasma. Aster yellows fitoplasmas, wat onlangs in plaaslike wingerde ontdek is, is bekend vir die uitgebreide skade wat hul aanrig en hou daarom 'n groot bedreiging in vir die Suid-Afrikaanse wingerd industrie. Om die in planta effek van AMPe teen die bogenoemde patogene te bestudeer is tydelike uitdrukkingsvektore ontwikkel wat die AMPe D4E1 of Vv-AMP1 uitdruk. D4E1 is 'n sinteties-ontwerpte AMP wat aktief is teen bakterieë en fungi, terwyl Vv-AMP1, wat uit druiwekorrels geïsoleer is, alreeds aktiwiteit teen fungi getoon het. In 'n tydelike uitdrukkingsbenadering in wingerd is die uitdrukking van transgene, vanaf virus of nie-virus gebaseerde vektore, bevestig deur die uitdrukking van die merker gene β-glukuronidase en die Groen Fluoresserende Proteïen, terwyl weefsel afdrukkings-immunotoetse virus replisering en sistemiese beweging in Nicotiana benthamiana bevestig het. Die virusvektore was gebaseer op die floëem-beperkte virus, wingerdvirus A. Slegs Agrobacterium-bemiddelde 35S tydelike uitdrukkingsvektore is gebruik om die AMP in planta aktiwiteit te bepaal aangesien die virus-bemiddelde uitdrukking in wingerd onvoldoende was vir evaluering teen A. vitis en X. ampelinus weens die beperking tot die floëem weefsel na infiltrering van die totale blaar. Geen fitoplasma geïnfekteerde materiaal kon gevestig word nie, en daarom is AMP aktiwiteitsevaluering slegs teen A. vitis en X. ampelinus uitgevoer. Kwantifisering van die bakterieë is deur middel van qPCR uitgevoer. Vv-AMP1 het geen aktiwiteit getoon teen enige van die bakterieë in planta nie, terwyl D4E1 aktief was teen beide. Die waargenome in planta aktiwiteit van D4E1 het ooreengestem met die in vitro aktiwiteit soos bepaal deur 'n AMP plaat bio-toets. In kontras tot in vitro evaluering kan die in planta AMP-aktiwiteit evaluering 'n meer akkurate voorspelling bied van die potensiële antimikrobiese aktiwiteite van die peptied in 'n transgeniese plant omgewing. Hierdie studie het bewys dat tydelike uitdrukkingsisteme gebruik kan word as 'n voorafgaande evalueringsmetode vir AMP in planta aktiwiteit teen wingerdpatogene, wat die evaluering van 'n verskeidenheid AMPe in 'n relatiewe kort tydperk toelaat voor verbintenis tot die ontwikkeling van transgeniese wingerd.

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Burkart, David. "UNDERSTANDING CHYTRIDIOMYCOSIS RESISTANCE BY INVESTIGATING THE CUTANEOUS DEFENSE MECHANISMS OF MARSUPIAL FROGS." OpenSIUC, 2015. https://opensiuc.lib.siu.edu/theses/1835.

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Anurans are declining worldwide because of the spread of Batachochytrium dendrobatidis (Bd), the fungus that causes chytridiomycosis. However, some frogs are resistant to this disease, and understanding why may be critical to saving those that are susceptible. In Peru, Gastrotheca excubitor is resistant to chytridiomycosis while Gastrotheca nebulanastes is susceptible. Two anuran skin defenses, symbiotic bacteria and antimicrobial peptides (AMPs), have demonstrated the ability to inhibit Bd in vitro when isolated from certain frogs. We tested if these defenses can explain the difference in susceptibility between the two Gastrotheca species. The cutaneous bacteria and AMPs of both species were collected, tested for their abilities to inhibit the growth of Bd, and analyzed for their compositions. Results indicate that 34%of the strains of skin bacteria from G. excubitor were able to inhibit the growth of Bd whereas only 10% isolated from G. nebulanastes were effective. Gastrotheca excubitor also has stronger anti-Bd skin bacteria. Neither frog species has peptide mixtures capable of completely inhibiting Bd, and overall species did not differ in the anti-Bd abilities of their peptides. These results suggest that the chytridiomycosis resistance experienced by G. excubitor may be attributed to its skin bacteria.

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Pinto, Natália dos Anjos. "Transformação genética de fumo visando controle de Fusarium oxysporum via Oxalato Descarboxilase e resistência a Xanthomonas fragarie via peptídeo antimicrobiano PgAMP1." Universidade Federal de Juiz de Fora, 2012. https://repositorio.ufjf.br/jspui/handle/ufjf/1719.

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O morango é produzido e consumido nas mais variadas regiões do mundo, sendo a espécie do grupo de pequenos frutos de maior apreciação e grande retorno econômico. Um dos principais problemas na cultura do morangueiro é a incidência de doenças, que podem aparecer em várias fases do ciclo da cultura, atacando desde as mudas recém plantadas até os frutos na fase final de produção. Devido a susceptibilidade às doenças e pragas, o uso de pesticidas é usual no cultivo de morangos. Nesse sentido, o uso de variedades resistentes a fungos e bactérias pode ser uma importante alternativa visando a melhoria da qualidade dos frutos e um menor custo de produção dos mesmos, uma vez que os consumidores exigem cada vez mais, frutos com menor nível residual de agrotóxicos. No presente estudo, plantas de Nicotiana tabacum foram transformadas com os genes OxDc de Flammulina velutipes e Pg-AMP1 de Psidium guajava com o objetivo de avaliar os efeitos da enzima Oxalato Descarboxilase, produzida pelo gene OxDc, na resistência das plantas a fatores que induzem a morte celular de tecidos infectados pelo fungo Fusarium oxysporum, assim como os efeitos in vitro do extrato bruto contendo o peptídeo Pg-AMP1 extraído das folhas de tabaco contra a bactéria Gram-negativa Xanthomonas fragarie. Experimentos de transformação genética mediada por Agrobacterium permitiram a obtenção de 8 linhagens transgênicas de N. tabacum OxDc e 7 linhagens transgênicas de N. tabacum Pg-AMP1, representando eventos de transformação distintos. As plantas foram caracterizadas molecular e bioquimicamente a fim de se confirmar a inserção, expressão e funcionalidade dos genes inseridos nas linhagens obtidas tanto para o gene OxDc quanto para o Pg-AMP1. Os testes de resistência ao ácido oxálico mostraram danos menos severos nas linhagens transformadas de X tabaco OxDc do que nas plantas não transformadas. No que se refere à resistência das folhas ao ataque pelo fungo Fusarium Oxysporum, o gene OxDc também se mostrou capaz de conferir resistência às linhagens transgênicas. Através da técnica de Western blot, foi possível detectar a presença do peptídeo Pg-AMP1 no extrato bruto extraído das folhas transformadas de tabaco Pg-AMP1. O bioensaio realizado in vitro utilizando-se o extrato bruto contendo o peptídeo Pg-AMP1 demonstrou a ação bactericida desse peptídeo contra a bactéria fitopatogênica Xanthomonas Fragarie. Em conjunto os resultados indicam a viabilidade da utilização dos genes OxDc e Pg-AMP1 transformação genética de morangueiro visando à redução nos danos causados por fitopatógenos que acometem suas lavouras, assim como a redução no uso de pesticidas.
The strawberry is produced and consumed in a variety of regions in the world, being the group of small fruits’ species of greater appreciation and high economic returns. A major problem in strawberry culture is the disease incidence that can appear in several stages of the cycle, attacking from the newly planted seeding to the fruit in the final stage of production. Due to this susceptibility to diseases and pests, the pesticide use is common in strawberries cultivation. Regarding this, the use of varieties resistant to fungi and bacteria may be a good alternative in order to improve fruit quality and to lower production cost, since consumers are increasingly demanding fruits with a lower level of pesticides. In the present study, Nicotiana tabacum plants were transformed with Flammulina velutipes OxDc gene and Psidium guajava Pg-AMP1 aiming to evaluate the effect of the enzyme Oxalate Decarboxylase produced by the gene OxDc in plant resistance to factors that induce cell death in tissues infected by the fungus Fusarium oxysporum, and the effect of raw extract containing the Pg-AMP1 peptide extracted from tobacco leaves against gram-negative bacterium Xanthomonas fragarie in vitro. Genetic transformation Agrobacterium-mediated experiments allowed the production of 8 transgenic lines N. tabacum OxDc and 7 transgenic lines of N. tabacum Pg-AMP1, representing different transformation events. The plants were biochemically and molecular characterized to confirm the integration, expression and function of the genes inserted in the lines obtained both for the gene OxDc and Pg-AMP1. Tests for resistance to 20 mM oxalic acid had less severe symptoms in OxDc transformed tobacco lines than the non-transformed plants. Concerning the strength of the leaves against Fusarium oxysporum attack, OxDc gene has also shown to confer resistance to the transgenic lines. It was possible to detect the presence of Pg-AMP1 peptide in the raw extract from Pg-AMP1 tobacco leaves through Western blot. Thus, the in vitro bioassay carried out using the raw extract containing the Pg-AMP1 peptide showed bactericidal activity against the phyto-pathogenic bacteria Xanthomonas Fragarie. Together these results XII determine the viability of OxDc and Pg-AMP1 genes in the use of strawberry genetic transformation aiming the reduction in the damage caused by pathogens that attack the crop.

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Adams, Shanice Raquel. "Bioactivity and genome guided isolation of a novel antimicrobial protein from Thalassomonas viridans." University of the Western Cape, 2019. http://hdl.handle.net/11394/7003.

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>Magister Scientiae - MSc
The continued emergence of bacterial resistance to the antibiotics currently employed to treat several diseases has added to the urgency to discover and develop novel antibiotics. It is well established that natural products have been the source of the most effective antibiotics that are currently being used to treat infectious diseases and they remain a major source for drug production. Natural products derived from marine microorganisms have received much attention in recent years due to their applications in human health. One of the biggest bottlenecks in the drug discovery pipeline is the rediscovery of known compounds. Hence, dereplication strategies such as genome sequencing, genome mining and LCMS/MS among others, are essential for unlocking novel chemistry as it directs compound discovery away from previously described compounds. In this study, the genome of a marine microorganism, Thalassomonas viridans XOM25T was mined and its antimicrobial activity was assessed against a range of microorganisms. Genome sequencing data revealed that T. viridans is a novel bacterium with an average nucleotide identity of 81% to its closest relative T. actiniarum. Furthermore, genome mining data revealed that 20% of the genome was committed to secondary metabolisms and that the pathways were highly novel at a sequence level. To our knowledge, this species has not previously been exploited for its antimicrobial activity. Hence, the aim of this study was to screen for bioactivity and identify the biosynthetic gene/s responsible for the observed bioactivity in T. viridans using a bioassay-and-genome- guided isolation approach to assess the bioactive agent. The bioassay-guided fractionation approach coupled to LCMS/MS led to the identification of a novel antimicrobial protein, TVP1. Bioinformatic analyses showed that TVP1 is a novel antimicrobial protein that is found in the tail region of a prophage in the T. viridans genome. Phage-derived proteins have previously been shown to induce larval settlement in some marine invertebrates. Since the mechanism of action of TVP1 remains unknown, it remains a speculation whether it may offer a similar function. More research is required to determine the biotechnological application and the role of TVP1 in its host and natural environment.

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Montesinos, Barreda Laura. "Rice seeds as biofactories of the production of antimicrobial peptides." Doctoral thesis, Universitat de Girona, 2014. http://hdl.handle.net/10803/135054.

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A series of antimicrobial peptides (AMPs) derived from the BP134 peptide (a member of the CECMEL11 library) exhibited potent bactericidal effect and low cytotoxicity, and were selected for expression in transgenic rice. Transgenic plants were generated expressing a codon-optimized AMP, under the control of a seed-specific promoter. It was confirmed that the expected peptides accumulated in the transgenic rice seeds, but not in other plant tissues, resulting in enhanced levels of protection against bacterial and fungal plant pathogens. Procedures for purification of the AMPs from seeds have been established based on solubilization/selective precipitation and Weak Cation Exchange-Reverse Phase chromatography. One of the peptides obtained, the S-Cec A, exhibited a high antibacterial and bactericidal activity against Dickeya sp. The accumulation levels of the recombinant AMP peptides in rice seeds were dependent on the strategy used, and its production in seeds offers a promising perspective for the use of rice as biofactory
Una sèrie de pèptids antimicrobians (PAMs) derivats del pèptid BP134 (provinent de la quimioteca CECMEL11), que exhibien un potent efecte bactericida i baixa citotoxicitat, es van seleccionar per a l'expressió en arròs. Es van generar plantes transgèniques que expressaven les seqüències gèniques corresponents als PAMs, i adaptades a l’ús de codons, sota el control de promotors específics de llavor. Es va confirmar que els PAMs esperats s’acumulaven en les llavors, però no en altres teixits de la planta, i conferien una protecció molt significativa a la infecció per patògens fúngics i bacterians de plantes. La purificació dels PAMs es va realitzar mitjançant solubilització/precipitació selectiva/ i cromatografia de Bescanvi Catiònic Feble i Fase Reversa. Un dels pèptids obtinguts, S-Cec A, exhibia una potent activitat antibacteriana i bactericida contra Dickeya sp. Els nivells d'acumulació dels PAMs en les llavors eren dependents de l'estratègia utilitzada. La llavor d’arròs ofereix una plataforma adequada pel seu ús com a biofactoria de PAMs

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32

Su, Ma. "Structure-based Design, Synthesis and Applications of a New Class of Peptidomimetics: 'Y-AA Peptides and Their Derivatives." Scholar Commons, 2018. https://scholarcommons.usf.edu/etd/7580.

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Peptidomimetics can mimic hierarchical structures of peptides and proteins. Thus, they are extensively studied for therapeutic applications. To break the limitation of backbones and frameworks and expand the peptidomimetics family, a new class of peptidomimetics - “γ-AApeptides” was developed. Design of γ-AApeptides is based on the chiral peptide nucleic acids (PNAs) backbone. The World Health Organization estimates that one -third of all deaths in the world are on account of infectious diseases. AMPs are important because of their high activity against broad spectrum microbes, less susceptible to grow resistance and selectivity in binding to bacterial cells over human cells. γ-AApeptides as a new class of peptidomimetics have increased stability and enhanced chemical diversity. We have developed polymyxin mimic cyclic peptides, small linear molecules and hydantoin derivatives as potent antibiotic agents with γ-AApeptides. They have good bioactivity and selectivity. Combinatorial library is key technology for accelerating the discovery of novel therapeutic agents. One-bead-two-compound γ-AApeptides-based library was developed and screened against SMYD2 protein which is essential for tumor growing.

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Noonan, Joseph Ali. "Investigation of Antimicrobial Peptide Genes in Maize ( Zea mays) Inbred Lines Resistant to Lepidoptera Larvae Feeding and Fungus Infection." Thesis, Mississippi State University, 2018. http://pqdtopen.proquest.com/#viewpdf?dispub=10841223.

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Present throughout all classes of life, antimicrobial peptides (AMPs) confer defense against bacteria, viruses, fungi, and insects. Identifying maize AMPs would provide breeders with a new defense resource. Here, the investigation of maize AMPs is reported. The distribution of AMPs within a panel of ten Mississippi maize inbred lines with varying resistance to Lepidoptera larvae feeding and Aspergillus infection is explored to characterize their observed resistances. Homology data-mining with two comprehensive AMP databases revealed 88 unique maize AMP protein sequences across 81 genes in the MaizeGDB B73 genome assembly. AMP-related polymorphic sites were identified using genomic primers. Analyses with qRT-PCR revealed 8 differentially expressed maize AMP genes. Computational 3D models of AMPs are limited, and models of these eight maize AMP genes were predicted. Two-dimensional electrophoresis gels were used to contrast protein profiles of inbred lines with varying resistance to identify regions related to AMPs and other defense-related protein.

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Alexander, Todd E. "Bringing together engineering and entrepreneurship: understanding the role of tethered C-CHY1 in the fight against antimicrobial resistance." Digital WPI, 2019. https://digitalcommons.wpi.edu/etd-dissertations/575.

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Healthcare associated infections (HAIs) cost the US healthcare system over $45 billion to treat and cause millions of deaths annually. A large subset of HAIs are associated with medical devices that are meant to improve and save lives. Infected devices are treated using traditional antibiotics, contributing to development of antibiotic resistance. Antibiotic resistance is expected to cost $100 trillion and kill more people a year than cancer by 2050; thus, new alternative antimicrobials for the treatment of device-associated HAIs are critically needed. Antimicrobial peptides (AMPs), such as 26 amino-acid (aa), marine-derived Chrysophsin-1 (CHY1), are poised to reduce HAIs due to their broad antimicrobial activity and unique mechanisms of action that do not promote bacterial resistance. AMPs are short (12-50aa), positively charged (+2-+9) proteins found in the innate immune systems of many different species. Their high separation of hydrophilic and hydrophobic residues leads to many unique mechanisms derived from many unique secondary and tertiary structures that are not yet well understood. Despite the discovery of over 2000 natural AMPs and many more synthetically designed AMPs, none have been successfully commercialized for healthcare applications due to challenges surrounding cytotoxicity, short in vivo half-life (degradation), high costs of production and effectiveness in physiological environments (such as those with high-salt). Several strategies have been investigated to overcome these challenges, for example, truncation of cytotoxic sequences or D-amino acid substitution to improve AMP toxicity and stability; however, many of these strategies can reduce antimicrobial effectiveness. A unique strategy of increasing stability, reducing cytotoxicity, and maintaining antimicrobial activity that is relevant for medical devices is the covalent tethering (binding) of AMPs via a flexible tethering molecule to the surface. However, the effect of tethering parameters on resulting AMP mechanisms and activity is still widely debated. AMP activity can vary widely by utilizing different tethering strategies, which include additional variables such as: (1) peptide choice and properties (such as native mechanism, concentration, charge, and structure), (2) tether choice and properties (such as chemical composition, length, charge, surface density, and flexibility), and (3) testing conditions (such as temperature, solvent composition and substrate type). Some studies suggest that AMP performance may be tether-dependent, for example some AMPs require longer tethers while others do not and some need a flexible tether. Thus, models for predicting successful tethering strategies for different AMP properties, which currently do not exist, must be developed. Further, complicated and often destructive techniques, such as XPS and SEM, are typically implemented to study the relationship of all these parameters vs. antimicrobial activity, which are labor-intensive and limited in scope. Predictive models guiding tether strategy need to be constructed, but also new techniques to study tethering be developed. If these technical milestones are achieved they can serve as a predicate for commercial implementation of a host of new therapies targeted at reducing device-associated HAIs. The overall goal of this thesis was to study the relationship between antimicrobial activity of tethered C-CHY1 examining both spacer length and peptide surface density and the development of a feasible clinical business case for tethered AMPs. To achieve this goal, a traditional entrepreneurial approach was taken in which a minimally-viable product was first designed and business case analyzed, followed by studies to better optimize and understand the underlying structure-mechanism relationships. CHY1 with a C-terminus cysteine to allow for surface-binding (C-CHY1) was tethered onto a silicon dioxide surface via a flexible poly(ethylene glycol) (PEG) tether, and then both surface binding behavior and antimicrobial success of C-CHY1 were examined as a function of tether properties and reaction conditions. For these studies, quartz-crystal microbalance with dissipation (QCM-D) was the primary technique, a real-time, non-destructive flow method that was then coupled with downstream characterization techniques: fluorescent microscopy and contact angle measurements. In parallel a deep dive into domestic and international business models for commercializing AMP technologies. Specifically, tether length and surface density effects on C-CHY1 mechanisms were studied, followed by the effect of temperature, type of microbe, and salt concentration on the antimicrobial mechanisms of tethered C-CHY1. QCM-D was used to measure binding of C-CHY1 via three different length tethers, PEG molecular weight (MW) 866, 2000 and 7500, followed by microscopy to measure antimicrobial effectiveness against two model microbes Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). Modeling of QCM-D data allowed for surface density and thickness to be calculated and related to C-CHY1 antimicrobial activity. PEG 7500 allowed proper C-CHY1 orientation and mobility, allowing for its native pore-forming mechanism and highest activity while PEG 866 tethers led to denser grafting and an effective, yet non-native ion displacement mechanism. The QCM-D was used to characterize the effect of salt concentration and temperature reaction conditions on the grafting density of C-CHY1 tethered via PEG 866 and PEG 7500, which was then related to antimicrobial activity. For PEG MW 866, neither temperature nor salt concentration increases significantly altered the grafting density of C-CHY1 while for PEG 7500 increasing temperature allowed for significantly increased grafting density. C-CHY1 density had no significant effect on antimicrobial activity against either microbe. Temperature of bacterial incubation did demonstrate microbe-specific changes in C-CHY1 antimicrobial activity. These results demonstrated that small changes in reaction conditions can drastically change membrane selectivity of C-CHY1. An in-depth investigation of the effects of bacterial membrane composition and temperature on soluble C-CHY1 mechanism was implemented to better understand the molecular membrane- and temperature-dependent selectivity and structure-function of C-CHY1. Supported lipid bilayers (SLBs) formed in QCM-D can be used as model membranes to elucidate AMP action mechanisms against membranes of different compositions. Two and three component SLBs representative of Gram-negative phosphatidylethanolamine (PE) and phosphatidyglycerol acid (PG) with and without charged lipopolysaccharide, LPS and Gram-positive bacteria phosphatidylcholine (PC) and PG with and without charged lipoteichoic acid, (LTA) were formed at both 23°C and 37°C. C-CHY1 at 5 µM was exposed to the different membranes and mechanistic surface action was studied. The membranes formed highly different baseline responses in QCM-D, indicative of vastly different membrane structures, thicknesses and deposition behaviors on SiO2, warranting future studies. Further, significant effects of LTA incorporation were observed in both peptide interaction and deposition. There were measurable effects of temperature on membrane formation as well as peptide interaction kinetics and even mode of interaction. Lastly, business models for the commercialization of novel medical device technologies such as surface-tethered C-CHY1 were investigated. While this technology has the potential to solve many unmet needs, there must a commercialization plan implemented in order to have an impact. There is a clear disconnect between technology development in academia and technology commercialization in industry that must be connected. Development of an entrepreneurial mindset at the graduate school level, can help bridge the gap. A thorough investigation of domestic and international business models for commercializing AMP technologies was carried out and distilled in the form of the Business Model Canvas developed by Alexander Osterwalder that can be used as a roadmap for commercialization efforts. Using the QCM-D a relationship between both spacer length and peptide surface density and the antimicrobial activity of tethered C-CHY1 was determined. A business plan was developed in order to increase the impact of this and other AMP based work. This work provides a roadmap for future researchers to quickly develop and commercial novel AMP based coating technology.

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Odendaal, Clerisa. "Structure-function studies of peptide fragments derived from a defensin of the tick Ornithodoros savignyi Audouin (1827)." Diss., University of Pretoria, 2013. http://hdl.handle.net/2263/41020.

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Overuse of conventional antibiotics has led to increased multidrug resistant micro-organisms. Therefore, development of alternative drugs with new mechanisms of action in the control of resistant micro-organisms is urgently needed. Defensins, one of the larger groups of naturally occurring antimicrobial peptides (AMPs), found in a variety of species, may serve as templates for the development of novel therapeutic agents. The work completed in this study is based on an antimicrobial peptide (AMP), Os, derived from the C-terminus of a tick Ornithodoros savignyi defensin isoform 2 (OsDef2). OsDef2 was found to be active against Gram-positive bacteria only, whereas Os, showed bactericidal activity towards both Gram-positive and Gram-negative bacteria. In this study a series of synthetic shorter peptides, based on the sequence of Os, was utilised in order to determine whether shorter peptides would retain their antibacterial activity and selectivity. Initial screening indicated that only two fragments, Os(3-12) and Os(11-22), were active towards the tested Gram-negative and Gram-positive bacteria. The minimum bactericidal concentrations (MBCs) of the two fragments were determined and ranged from 30 μg/ml to 120 μg/ml. The MBCs of the parent peptide, Os (1.88 to 15 μg/ml), was considerably lower than that of Os(3-12) and Os(11-22). As previously observed for Os, neither of the peptides showed cytotoxic effects towards eukaryotic cells. The amidated analogue of one of the active peptides, Os(11-22)NH2, was further evaluated in terms of its secondary structure, antibacterial and antioxidant activities as well as cytotoxicity. Amidation increased the activity of Os(11-22) 16 fold towards B. subtilis (MBC of 1.88 μg/ml) and 32 fold towards both Escherichia coli and Pseudomonas aeruginosa (MBC of 3.75 μg/ml), whereas a 2 fold decrease in activity was observed against Staphylococcus aureus (MBC of 60 μg/ml). Circular dichroism data showed that amidation altered the secondary structure of Os(11-22) from α-helical to mostly random coiled. In the presence of 30% serum the activity of Os(11-22)NH2 unexpectedly increased 8 fold against S. aureus (MBC of 7.5 μg/ml), but decreased 32 fold against E. coli (MBC of 120 μg/ml). The activity of Os(11-22)NH2 in 100 mM NaCl decreased 4 fold against E. coli (MBC of 15 μg/ml), but was completely lost (MBC >120 μg/ml) against S. aureus. The kinetics of bactericidal activity indicated that Os(11-22)NH2 killed B. subtilis and E. coli within 30 min and 120 min, respectively, whereas Os killed both bacteria within 5 min. Even at high concentrations Os(Os(11-22)NH2 was non-toxic towards human erythrocytes and SC-1 cells, moreover an increase in SC-1 cell number was observed at 120 μg/ml. The peptide showed strong antioxidant activity and was found to be 4 fold more active than glutathione (GSH), however Os was 3.4 fold more antioxidative than Os(11-22)NH2. Os(11-22)NH2 can be considered a dual functional peptide, since it possesses both antibacterial and antioxidant activity. The amidated peptide has the potential for use against the damaging effects of oxidative stress associated with infectious diseases and recovery of chronic wounds. Further investigation into structure-function properties of Os(11-22)NH2 is necessary.
Dissertation (MSc)--University of Pretoria, 2013.
gm2014
Biochemistry
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Olleik, Hamza. "Identification and characterization of new antibacterial molecules effective against multi-resistant bacteria including Helicobacter pylorie." Thesis, Aix-Marseille, 2019. http://www.theses.fr/2019AIXM0436.

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L’infection gastrique par Helicobacter pylori (bactérie à Gram négatif) touchant plus de 50 % des humains est la seconde cause d’infection bactérienne dans le Monde après les carries. A long terme, les complications causées par cette infection (ulcères et cancers) sont responsables de plus de 700,000 décès par an. Les antibiotiques “conventionnels” utilisés en médecine ont permis de traiter les patients infectés par H. pylori mais ont également malheureusement conduit à l’apparition de souches résistantes aux traitements responsables d’échecs thérapeutiques observés chez 20-30 % des patients. L’augmentation constante de l’incidence de souches résistantes d’H. pylori a motivée la recherche de nouvelles molecules ayant comme caractéristiques: i) d’être actives contre H. pylori, notamment les souches déjà résistantes aux antibiotiques conventionnels, ii) d’être peu ou pas toxiques et iii) de ne pas causer / sélectionner de résistance. Durant ma thèse, plusieurs molécules naturelles ou synthétiques pouvant remplir ces critères ont été évaluées : i) les Aurones et les Berbérines qui sont des molécules issues de plantes, ii) les peptides antimicrobiens (PAM) issus de champignons (les cyclodepsipeptides), de grenouilles (Temporine SHa et ses dérivés) ou d’estomac de vache (les Lynronnes). Les résultats obtenus montrent que plusieurs de ces pistes peuvent être considérées comme prometteuses dans l’éradication d’H. pylori, particulièrement celle des PAM de type Temporine SHa et Lynronnes qui sont peu toxiques et actifs même contre les souches résistantes aux antibiotiques conventionnels responsables d’échecs thérapeutiques
Helicobacter pylori, a gram-negative bacterium infecting the stomach, is the second cause of bacterial infection (after caries) in the world with 50 % of the human beings chronically infected. Long term infection by H. pylori leads to gastric ulcer and eventually to gastric cancer, with around 700,000 deaths per year. Although antibiotics were successfully used to treat patients, they are also responsible for the selection of resistant strains accounting for the 20-30 % treatment failure observed. For that reason, new molecules should be considered to treat H. pylori, ideally ones that are: i) active on H. pylori, including strains already resistant to conventional antibiotics, ii) not or poorly toxic and iii) not able to cause significant resistance. Few options were considered during my thesis that we thought could fulfill those purposes: i) Aurones and Berberines that are plant molecules and ii) antimicrobial peptides (AMP) originated from fungi (cyclodepsipeptides), from frog’s skin (Temporin-SHa and its derivatives), or present in the stomach of cow (Lynronnes). Taken together, my results demonstrated that among the different options we tested to treat H. pylori, Temporin-SHa and Lynronnes are the best candidates, particularly in the case of resistant strains responsible for treatment failure

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Hoffman, Jared Michael. "The Role of the Transcriptional Antiterminator RfaH in Lipopolysaccharide Synthesis, Resistance to Antimicrobial Peptides, and Virulence of Yersinia pseudotuberculosis and Yersinia pestis." BYU ScholarsArchive, 2016. https://scholarsarchive.byu.edu/etd/6401.

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RfaH is a unique bacterial protein that enhances transcription of a select group of long operons in many Gram-negative bacteria. Operons regulated by RfaH possess an upstream operon polarity suppressor sequence, which recruits the RfaH protein to the RNA polymerase during transcription of genes, most of which are involved in the synthesis of cell-surface features. These include synthesis of the lipopolysaccharide (LPS) core and O-antigen in Salmonella and Escherichia coli, as well as F-plasmid conjugation pilus and capsule in E. coli. LPS is an important virulence factor in many Gram-negative bacteria, and protects Y. pseudotuberculosis against host antimicrobial chemokines. Recently published high-throughput transposon mutant screens have also suggested a role for RfaH in the ability of Y. pseudotuberculosis to colonize mice. However, the role of RfaH in Y. pseudotuberculosis and its descendent Yersinia pestis has not been carefully examined. In these studies we investigated the effect RfaH has on the structure of the LPS in both species at different temperatures. We also identified LPS-synthesis related genes that are regulated by RfaH. We determined the effect of RfaH on bacterial resistance to host defense peptides, and the ability of Y. pseudotuberculosis to colonize mice. We found that the loss of the rfaH gene had different effects in Y. pseudotuberculosis and Y. pestis. Loss of rfaH caused a truncation in the core region in Y. pseudotuberculosis strain IP32953 at both 21°C and 37°C, but only at 37°C in Y. pestis strain KIM6+. Similarly, we found that transcription of individual genes that are predicted to function in core or O-antigen synthesis were downregulated in the rfaH mutant strains in both species, but the impact of rfaH deletion was greater in Y. pseudotuberculosis. When tested for their ability to survive in the presence of antimicrobial peptides, the Y. pseudotuberculosis rfaH deficient bacteria were much more susceptible than wild-type to killing by polymyxin and by the antimicrobial chemokine CCL28. However, the Y. pestis rfaH mutant strain was equally susceptible to CCL28 as the wild-type strain. Infection of mice with Y. pseudotuberculosis show that rfaH deficient bacteria were able to survive as effectively as the wild-type following oral or intravenous inoculation, with or without the pYV virulence plasmid. Overall, our results show that while RfaH controls LPS gene expression in both Y. pseudotuberculosis and Y. pestis, its impact is much greater in Y. pseudotuberculosis. Furthermore, although loss of rfaH greatly reduces the ability of Y. pseudotuberculosis to resist antimicrobial peptides, it is not required for virulence in this species.

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Furlanetto, Alessandra. "Síntese de nanoemulsão e nanopartícula de ouro (AuNPs) contendo nisina e seus efeitos sobre os fatores de virulência de Staphylococcus aureus." Botucatu, 2020. http://hdl.handle.net/11449/192336.

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Orientador: Ary Fernandes Júnior
Resumo: O aumento no número de bactérias multirresistentes aos fármacos antibacterianos é preocupação de saúde pública e tem motivado pesquisas na buscsa por antimicrobianos alternativos para minimizar este problema, e na obtenção de substâncias com capacidade de matar bactérias e/ou interferir com a sua patogenicidade. O Staphylococcus aureus é uma bactéria altamente virulenta, capaz de causar inúmeras doenças, incluindo intoxicações alimentares. Esta bactéria se tornou resistente aos diversos antimicrobianos ao longo dos anos com destaque para o S. aureus meticilina-resistente (MRSA). O peptídeo antimicrobiano (AMP) nisina, bacteriocina produzida por Lactococcus lactis é um que vem sendo estudado na forma de nanoemulsões e nanopartículas. O objetivo desse estudo foi sintetizar, caracterizar e testar nanoemulsões e nanopartículas de ouro (AuNPs) de nisina, para a verificação da ação antibacteriana, através da Concentração Inibitória Mínima (CIM), atividade antibiofilme, antienterotoxina, atividade hemolítica, ação sobre a membrana bacteriana determinada pelo extravasamento de proteínas, sobre linhagens padrões ATCC de S. aureus, e teste de viabilidade celular em linhagem HCT-116 por citometria de fluxo. Os tratamentos utilizados foram nisina, cinco nanoemulsões com nisina (Nano-Nis), AuNPs com nisina (AuNPs-Nis), AuNPs com Nano-Nis (AuNPs + Nano-Nis) e AuNPs-Nis com nanoemulsão (AuNPs-Nis + Nano). De acordo com os resultados obtidos para CIM, observou-se que para a cepa ATCC 33591 d... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The growth in the number of multiresistant bacteria resistant to traditional antimicrobial drugs is a public health concern which has been motivated researchs worldwide, seeking new antimicrobial drugs to minimize this problem besides getting new substances able to erradicate bacteria and/or interfer with their pathogenicity. Staphylococcus aureus is a highly virulent bacteria, able to cause countless diseases including food poisoning. This bacteria got resistant to many antimicrobials within the years, highlighting the S. aureus metchilin-resistant (MRSA). The antimicrobial peptide (AMP) nisin, bacteriocin synthesized by Lactococcus lactis, is one that has been studied in nanoemulsions and nanoparticles. The objective of this study was to synthesize, characterize and evaluate nanoemulsions and gold nanoparticles (AuNPs) with nisin, to verify their antibacterial action, using the Minimum Inhibitory Concentration (MIC), antibiofilm activity, antienterotoxin activity, hemolytic activity, action on bacterial membrane determined by protein leakage, on MRSA ATCC strains, and cell viability in HCT-116 strain by flow citometry. The treatments used were nisin, five nanoemulsions with nisin (Nano-Nis), AuNPs with nisin (AuNPs-Nis), AuNPs with Nano-Nis (AuNPs + Nano-Nis) and AuNPs-Nis with nanoemulsion (AuNPs-Nis + Nano). According to the results obtained for MIC, it was observed that for the MRSA strain ATCC 33591, the number 1 formulation of nanoemulsions was the more efficient betwe... (Complete abstract click electronic access below)
Mestre

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Mollet, Chloé. "Identification et caractérisations physico-chimiques et pharmacologiques de nouvelles molécules bioactives isolées à partir de venins d’animaux : exemple des peptides antimicrobiens." Thesis, Aix-Marseille, 2017. http://www.theses.fr/2017AIXM0352.

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La recherche de nouvelles molécules bioactives utilisables en thérapeutique est un enjeu majeur de santé publique en particulier dans le traitement de certaines maladies telle que les infections bactériennes.La résistance naturelle des bactéries et la surutilisation des antibiotiques ont entraîné la sélection de bactéries pathogènes résistantes à de multiples médicaments. Depuis les dernières décennies, la résistance aux antibiotiques conventionnels a limité les options thérapeutiques, entraînant une augmentation significative de la mortalité et de la morbidité dans les hôpitaux. En outre, depuis 1970, seules deux nouvelles classes d'antibiotiques ont été mises sur le marché. Les venins constituent une source riche de substances naturelles pharmacologiquement actives uniques et novatrices, tels que les peptides antimicrobiens (PAMs) qui représentent une alternative originale pour remédier à ce problème de santé publique.Dans notre étude, parmi les 200 venins d’animaux étudiés pour leurs propriétés antibactériennes, au moins six PAMs ont été isolés à partir d’un venin d’insecte. Le peptide 1 original inhibe la croissance des bactéries Gram positives et négatives mais présente une forte hémotoxicité (IT = 1,6-3,2). La synthèse en phase solide d’analogues structuraux a permis d’identifier R1W8 et I1N11, moins toxiques (IT = 18 et >800 respectivement). Les résultats préliminaires de l’étude du mécanisme d’action suggèrent que ces peptides agissent contre les bactéries par perméabilisation de leur membrane cytoplasmique. Ces peptides peuvent servir de modèles pour l’élaboration de nouveaux agents antimicrobiens
The research for new bioactive molecules which can be used in therapeutic is a major public health issue, particularly in the treatment of certain diseases such as bacterial infections.The natural resistance of bacteria consecutive to overuse of antibiotics have resulted in the selection of pathogenic multi-drug resistant bacteria. Over the last few decades, resistance to conventional antibiotics has limited treatment options, resulting in a significant increase in mortality and morbidity in hospitals. Moreover, since 1970, only two new classes of antibiotics have been placed on the market. Venoms are known to be a rich source of unique and innovative pharmacologically active substances, such as antimicrobial peptides (PAMs), which represent an original alternative to small molecules for the development of new active and non-resistance inducing antibiotics.In our study, among the 200 venoms of animals studied for their antibacterial properties, at least six PAMs were isolated from an insect venom. The original peptide 1 inhibits the growth of Gram positive and negative bacteria but shows a high hemotoxicity (TI = 1,6-3,2). The solid phase synthesis of structural analogs allowed to identify R1W8 and I1N11, less toxic (TI = 18 et >800 respectively). The preliminary results of the action mechanism study suggest that these peptides have a pore-forming action on bacteria cytoplasmic membrane. These peptides can be used as models for the development of new antimicrobial agents

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Nicoud, Quentin. "Study of terminal bacteroid differentiation features during the legume-rhizobium symbiosis Bradyrhizobium diazoefficiens USDA110 nodulation of Aeschynomene afraspera is associated with atypical terminal bacteroid differentiation and suboptimal symbiotic efficiency Sinorhizobium meliloti functions required for resistance to the antimicrobial NCR peptides and bacteroid differentiation." Thesis, université Paris-Saclay, 2021. http://www.theses.fr/2021UPASB007.

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La symbiose rhizobium-légumineuse est une intéraction étroite entre plante et bactérie. Au cours de cette symbiose, la bactérie est hébergée par la plante au sein d’organes symbiotiques où elle fixe l’azote atmosphérique pour la plante. Les espèces de légumineuses du groupe des IRLC et des Dalbergioïdes peuvent contrôler les rhizobia symbiotiques et induire un processus de différenciation particulier grâce à la production massive de peptides riches en cystéines (NCR) spécifiques aux nodosités. In vitro, les peptides NCR cationiques ont des activités de perméabilisation de la membrane sur de nombreuses bactéries. La manière dont les rhizobiums s'adaptent pour résister à ce stress intense reste encore aujourd’hui mal compris. Deux axes de recherche principaux ont été menés au cours de cette thèse, tous deux liés à la compréhension de la réponse des bactéries à la différenciation terminale imposée par les peptides NCR. D'un côté, nous avons analysé certaines fonctions bactériennes pour leur rôle dans la résistance à la NCR au cours de l'interaction modèle entre Medicago truncatula et Sinorhizobium meliloti. Dans ce travail, nous avons principalement évalué les fonctions membranaires telles que la synthèse du LPS, le système de réponse aux stress de l’enveloppe et des fonctions d'importation. Nous avons trouvé de nouvelles fonctions qui pourraient être impliquées dans la résistance à la NCR et la différenciation terminale des bactéroïdes.De l'autre côté, nous avons mené une approche multi-omique couplée à des techniques de biologie cellulaire pour caractériser l'interaction mal adaptée entre Bradyrhizobium diazoefficiens USDA110 et Aeschynomene afraspera. Nous avons découvert de nouvelles particularités dans cette interaction avec notamment une différenciation inhabituelle
The legume-rhizobia symbiosis is a close interaction between a plant and bacteria. During this symbiosis, bacteria are hosted by the plants in symbiotic organs called nodules and in which the symbionts fix atmospheric nitrogen for the plants. Legume species from IRLC and Dalbergioid can control symbiotic rhizobia and mediate a particular differentiation process through the massive production of nodule-specific cysteine-rich (NCR) peptides. In vitro, cationic NCR peptides have membrane-permeabilizing activities on many bacteria. How rhizobia adapt to resist this intense stress remains poorly understood. Two main research axes were driven during this thesis, both linked to the understanding of how bacteria react to terminal differentiation imposed by NCR peptides. On one side, we tried to functionally analyze bacterial functions for their role in NCR resistance during the model interaction between Medicago truncatula and Sinorhizobium meliloti. In this work, we mainly assessed membrane functions such as LPS synthesis, Envelope Stress Response, and import functions. We found novel functions that could be involved in NCR resistance and terminal bacteroid differentiation.On the other side, we conducted a multi-omics approach coupled with cell-biology techniques to characterize the ill-adapted interaction between Bradyrhizobium diazoefficiens USDA110 and Aeschynomene afraspera. We discovered new features in this interaction with an unusual differentiation

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Pinto, Flávio Miguel do Carmo. "Alternativas aos antibióticos químicos convencionais: estado-da-arte." Master's thesis, [s.n.], 2013. http://hdl.handle.net/10284/3984.

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Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas
Com a fácil transferência das bactérias quer nos cuidados hospitalares, quer a nível comunitário, com o aumento do número de pessoas a necessitar de cuidados de saúde provocado pelo envelhecimento populacional, com o aumento do número de infeções associadas aos cuidados de saúde, com as viagens internacionais ao dispor de qualquer cidadão, com o uso irracional e exagerado dos antibióticos por parte da população e em explorações animais, a resistência aos antibióticos atingiu um nível tão elevado que coloca em risco a saúde pública mundial. Presentemente torna-se indispensável a adoção de estratégias alternativas que promovam a eliminação destas estirpes resistentes do meio ambiente. Nesta dissertação serão abordadas e analisadas diversas estratégias terapêuticas alternativas aos antibióticos. A terapia fágica é uma delas, e esta recorre à utilização de bacteriófagos estritamente líticos, como alternativa, ou complemento, no tratamento de infeções bacterianas, tendo-se revelado um método seguro pelo facto destas entidades biológicas não possuírem qualquer afinidade para as células eucarióticas. Já a terapia com lisinas estabelece-se como outra opção terapêutica inovadora, sendo que a administração tópica de preparações contendo lisinas recombinantes purificadas, numa concentração na ordem dos nanogramas, em bactérias Gram-positivo, demonstraram elevada capacidade terapêutica, provocando rapidamente a lise da bactéria alvo. Adicionalmente, esta terapia evidencia sinergismo quando combinado com determinados antibióticos já disponíveis no mercado. Outra alternativa terapêutica baseia-se na utilização dos tubos peptídicos formadores de poros. Estes são polipéptidos anfifílicos que provocam disrupção da membrana celular, podendo ser utilizados no combate de infeções bacterianas, fúngicas e víricas, na prevenção de biofilmes e como antitumorais. Curiosamente, as bacteriocinas estabelecem-se como uma estratégia de defesa comum das bactérias contra outros agentes bacterianos, eliminando potenciais oponentes e aumentando o número de nutrientes disponíveis no meio ambiente para o seu próprio crescimento, podendo ser aplicadas na indústria alimentar, como probióticos, e no combate bactérias multirresistentes. A utilização de anticorpos antibacterianos promete ser segura e extremamente eficaz. Já a vacinação estabelece-se como uma das estratégias mais promissoras a nível preventivo. With the easy transfer of bacteria on hospital care and at community level, with the increasing number of people needing health care caused by the aging of population, with the increasing number of infections associated with health care, with international travel available to any citizen, with exaggerated and irrational use of antibiotics by the population and in farm animals, antibiotic resistance has reached such a high level that endangers world public health. Presently it’s essential the adoption of alternative strategies that promote the elimination of these resistant strains from the environment. In this dissertation it will be discussed and analysed several alternative strategies to antibiotics. Phage therapy makes use of lytic bacteriophages as an alternative, or a complement, in the treatment of bacterial infections. It is a safe method because these biological entities don’t possess any affinity to eukaryotic cells. Lysins therapy is recognized as novel therapeutic option, because the topical administration of preparations containing purified recombinant lysins at a concentration in the order of nanograms, in Gram-positive bacteria demonstrated a high potential therapeutic causing immediate lysis of the target bacteria. Additionally, this therapy shows synergism when combined with certain antibiotics already available on the market. Another alternative therapy is based on the use of antimicrobial peptides. These are amphiphilic polypeptides that causes disruption of the bacterial membrane and can be used in treatment of bacterial, fungic and viral infections, in prevention of biofilms formation and as antitumor agents. Interestingly, bacteriocins are a common strategy defence of bacteria against other bacterial agents that eliminates potential opponents and increases the number of available nutrients in the environment for their own growth and can be applied in the food industry, as food preservative, as probiotics and in fighting of multi-resistant bacterial strains. The use of antibacterial antibodies promises to be extremely safe and effective. The vaccination is the most promising preventive strategy.

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Metolina, Patrícia. "Degradação do antibiótico bacitracina zíncica em meio aquoso através de processos oxidativos avançados." Universidade de São Paulo, 2018. http://www.teses.usp.br/teses/disponiveis/3/3137/tde-24092018-082606/.

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A presença de antibióticos no ecossistema representa um sério risco à saúde humana e animal em virtude do desenvolvimento crescente de resistência bacteriana. Uma vez que a maioria dos antibióticos é persistente à degradação biológica, os processos oxidativos avançados são apontados como uma das tecnologias mais efetivas para decompor esses compostos em águas residuárias. A bacitracina zíncica (Bc-Zn) é um potente antibiótico constituído por uma mistura complexa de peptídeos não-biodegradáveis, conjugados ao zinco. Apesar de ser um antibiótico amplamente consumido na medicina humana e animal, é preocupante a escassez de estudos que investigam sua degradação e destino ambiental. O presente trabalho analisou a degradação da Bc-Zn através dos processos de fotólise direta e UV/H2O2 em diferentes condições de radiação UVC e concentração inicial de H2O2. Os parâmetros cinéticos rendimento quântico da fotólise, constantes cinéticas de pseudo-primeira ordem e constante cinéticas de segunda ordem foram satisfatoriamente estimados pela modelagem do sistema fotoquímico experimental. Os resultados revelaram que a fotólise direta permitiu degradar todos os congêneres da mistura de Bc-Zn nas maiores doses de radiação UVC empregadas. No entanto, não houve remoção de TOC após 120 minutos de irradiação. A adição de H2O2 acelerou substancialmente a fotodegradação do antibiótico, apresentando constantes cinéticas de pseudo-primeira ordem uma ordem de grandeza superiores às obtidas por fotólise direta. Além disso, remoção considerável de até 71% do TOC foi alcançada. A análise estatística demonstrou que a radiação UV foi um fator bem mais significativo para a fotodegradação da Bc-Zn em relação à concentração inicial de H2O2, sendo as melhores condições do processo alcançadas para a maior taxa específica de emissão de fótons (1,11×10-5 Einstein L-1 s-1). Ensaios biológicos com soluções tratadas por fotólise direta e UV/H2O2 indicaram remoção completa da atividade antimicrobiana residual, ainda que os produtos da fotodegradação tenham se mostrado não-biodegradáveis. Análises de toxicidade indicaram que o metal zinco presente no antibiótico é responsável pela a toxicidade no micro-organismo-teste Vibrio fischeri. Estudos adicionais devem ser realizados para identificar os sub-produtos formados, bem como para investigar a degradação da Bc-Zn em efluentes industriais reais.
The presence of antibiotics in ecosystems represents a serious risk to human and animal health, caused by the increase in bacterial resistance. Since most antibiotics resist to biological degradation, advanced oxidation processes are pointed out as the most effective technologies for degrading these compounds in wastewater. Zinc bacitracin (Bc-Zn) is a potent antibiotic with a complex mixture of non-biodegradable peptides conjugated to zinc. Despite being a widely used antibiotic in human and animal medicine, the scarcity of studies dealing with its degradation and environmental fate is a matter of concern. In this work, Bc-Zn degradation by direct photolysis and the UV/H2O2 process was investigated for different UVC radiation conditions and initial H2O2 concentrations. Kinetic parameters, namely the photolysis quantum yield, pseudo-first order kinetic constants and second-order kinetic constants, were satisfactorily estimated from experimental data by modeling the photochemical system. The results showed that all the congeners of the Bc-Zn mixture were photolyzed at the highest UVC doses applied, while no TOC removal was observed after 120 minutes of irradiation. The addition of H2O2 substantially accelerated Bc-Zn photodegradation, with pseudo-first order kinetic constants of one order of magnitude higher than those observed under direct photolysis. In addition, a remarkable removal of up to 71% of TOC was achieved. Statistical analyses showed that UV radiation had a much more important effect on Bc-Zn photodegradation in comparison with initial H2O2 concentration, with the best process conditions achieved for the highest specific photon emission rate (1.11×10-5 Einstein L-1 s-1). Biological assays carried out with the solutions treated by direct photolysis and UV/H2O2 revealed no residual antimicrobial activity, though photodegradation products remained non-biodegradable. In addition, toxicity analyses indicated that the zinc metal present in the antibiotic is responsible for the toxic effect on the test microorganism Vibrio fischeri. Finally, further studies should be performed to identify the by-products formed and to investigate Bc-Zn degradation in real industrial wastewater.

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Montero, Mirabet Maria. "Evaluación de los efectos no intencionados de los transgenes en plantas modificadas genéticamente (MG) resistentes a plagas y diseñadas como biofactorías de péptidos antimicrobianos." Doctoral thesis, Universitat de Girona, 2012. http://hdl.handle.net/10803/97166.

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Genetically modified crops are submitted to strict regulation to ensure the safety of consumers and the environment. To complement the comparison between GM plants and their counterparts, in the present Thesis, we evaluated the possible unexpected effects of the transgene on the host plant, by means of transcriptomic technologies. More exactly, we studied three pathogen-resistant GM rice lines: S-afp, expressing constitutively the antifungal protein AFP; and S-bp217 and S-bp213, expressing undecapeptide BP100 derivatives, which were developed in the UdG in the context of this Thesis. Although the high phytotoxicity of the BP100 derivatives on the host plant the transcriptional changes observed in S-afp, S-bp217 and S-bp213 compared to the conventional line Senia were similar that those observed in other GM crops, of other species and with different transgenes, and only the half of them was attributed to the insertion and/or expression of the transgene.
Les plantes modificades genèticament (MG) destinades a comercialització estan sotmeses a estricta legislació per garantir la seguretat del consumidor i del medi ambient. Per complementar la comparativa entre plantes MG i convencionals, en aquesta tesi s’ha abordat l’avaluació dels possibles efectes no esperats del transgèn sobre la planta hoste, mitjançant tècniques de transcriptòmica. Concretament s’han estudiat línies d'arròs MG que presenten fenotips de resistència a patògens: S-afp, que expressa constitutivament la proteïna antifúngica AFP, i S-bp213 i S-bp217, que expressen derivats de l’undecapèptid BP100, desenvolupat a la UdG, que s’han obtingut també en el marc d’aquesta tesi. Malgrat l’elevada fitotoxicitat dels derivats de BP100 enfront la planta hoste, els canvis transcripcionals de S-afp, S-bp213 i S-bp217 respecte la línia convencional Senia són similars als observats en altres events MG, de diferents espècies i amb diferents transgens; i només la meitat d’ells s’ha atribuit a la presència o expressió del transgèn.

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44

Hoang, Ky Van. "Mechanisms of Antimicrobial Peptide Resistance in Campylobacter." 2010. http://trace.tennessee.edu/utk_graddiss/887.

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Campylobacter is the major bacterial cause of human gastroenteritis in the United States and other developed countries. Poultry are considered a main source of human Campylobacter infections. Thus, reduction of Campylobacter load in poultry is significant in food safety and public health. However, no effective measure is commercially available to prevent Campylobacter colonization in poultry to date. Antimicrobial peptides (AMPs) are short and bactericidal peptides widely present in intestine to limit bacterial infections. Recently, AMPs have been increasingly recognized as a novel class of antibiotics (peptide antibiotics) to control foodborne pathogens. Notably, several potent anti-Campylobacter bacteriocins, a group of AMPs produced by commensal bacteria, dramatically reduced C. jejuni colonization in chickens and are being directed toward on-farm control of this pathogen to protect public health. As an important strategy to evade killing by potential peptide antibiotics and by host innate defense, AMP resistance mechanisms in C. jejuni are critical to understand, but are still unknown. In this dissertation, molecular basis of Campylobacter resistance to polymyxin B, the anti-Campylobacter bacteriocins (BCNs), and a chicken host defense AMP (fowlicidin-1) was comprehensively examined using both in vitro and in vivo systems. Although polymyxin B has been successfully used as a model peptide to study AMP resistance in other Gram-negative bacteria, functional genomics examination in this study suggested that polymyxin B is not a good surrogate to study Campylobacter resistance to physiologically relevant AMPs. Campylobacter only developed low-level BCN resistance with low frequency in vitro and in vivo; the acquired BCN resistance was not stable in Campylobacter. Genomic examination of two BCN resistant mutants using DNA microarray and random transposon mutagenesis revealed that the multidrug efflux pump CmeABC contributes to both intrinsic and acquired resistance of Campylobacter to the BCNs. Random transposon mutagenesis and targeted site-directed mutagenesis identified four genes (cbrR, tig, cjaB, and cj1583c) involved in Campylobacter resistance to fowlicidin-1. These genes were also required for optimal colonization of Campylobacter in chickens. Together, the findings from this dissertation revealed uniqueness and complexity of AMP resistance in Campylobacter and will enable us to develop more sustainable peptide antibiotics and novel intervention strategies to prevent and control Campylobacter infections in humans and animal reservoirs. Key words: Campylobacter, antimicrobial peptide resistance, polymyxin B, bacteriocins, fowlicidins

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45

Mount, Kristy Lee Beavers. "The Haemophilus ducreyi SAP Transporter Contributes to Antimicrobial Peptide Resistance." Thesis, 2009. http://hdl.handle.net/1805/1951.

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Indiana University-Purdue University Indianapolis (IUPUI)
Haemophilus ducreyi is the causative agent of the genital ulcer disease chancroid, which has been shown to facilitate the transmission of HIV. H. ducreyi is likely exposed to multiple sources of antimicrobial peptides in vivo. APs are small, cationic molecules with both bactericidal and immunomodulatory functions. Because H. ducreyi is able to establish and maintain an infection in an environment rich with antimicrobial peptides, we hypothesized that the bacterium was resistant to the bactericidal effects of these peptides. Using a 96-well AP bactericidal assay, we examined H. ducreyi susceptibility to eight human APs likely to be encountered at the site of infection, including the α-defensins human neutrophil peptide-1, human neutrophil peptide-2, human neutrophil peptide-3, and human defensin 5, the β-defensins human β defensin-2, human beta defensin-3, and human beta defensin-4, and the human cathelicidin, LL-37. H. ducreyi survival was compared to the survival of Escherichia coli ML35, a strain known to be susceptible to several antimicrobial peptides. H. ducreyi was significantly more resistant than E. coli ML35 to the bactericidal effects of all peptides tested. Furthermore, we found that representative class I and class II strains of H. ducreyi were each resistant to APs of each functional category, indicating that resistance to antimicrobial peptides could represent a conserved method of pathogenesis for H. ducreyi as a species. The H. ducreyi genome contains a homolog for the Sap influx transporter. To study the role of the H. ducreyi Sap transporter in AP resistance, we generated an isogenic sapA mutant and used the 96-well AP bactericidal assay to compare the AP susceptibility profiles of wild-type H. ducreyi, the sapA mutant and the sapA trans-complement to α-defensins, β-defensins, and LL-37. We observed a 25% decrease in the survival of the sapA mutant when it was exposed to LL-37. These findings suggest that the H. ducreyi Sap transporter plays a role in H. ducreyi resistance to LL-37, but it is likely that other AP resistance mechanisms co-exist within the bacterium.

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46

Shen, Chun-Jung, and 沈春榮. "Proteomic analysis on the antimicrobial peptide-resistance mechanism of Vibrio parahaemolyticus." Thesis, 2008. http://ndltd.ncl.edu.tw/handle/06928103018115929340.

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碩士
國立宜蘭大學
生物技術研究所碩士班
96
Vibrio parahaemolyticus is a halophilic gram-negative bacterium that occurs naturally in estuarine environments world-wide. V. parahaemolyticus is also a universal marine pathogen causing vibriosis in fish, shrimp and shellfish. Consumption of uncooked seafood contaminated with a virulent strain of V. parahaemolyticus can cause bacterial gastroenteritis of human. The extensive use of classical antibiotics has led to the growing emergence of many resistant strains of pathogenic microorganisms. Therefore, the development of novel therapeutic agents that could overcome the resistance problem has become critical. Antimicrobial peptides (AMPs) is widespread present in all kinds of organisms. AMPs are of greatest potential to represent such a new class of antibiotics. In the present study, we measured the minimal inhibitory concentrations (MICs) of several AMPs against V. parahaemolyticus. Three synthetic AMPs (Q4, Q6 and H1) and a natural one from fish (Pleurocidin) have best antibacterial activity and selectivity. Incubation with serially increased concentrations of AMPs starting from MIC/2 during bacterial culture passages were performed for selection of resistant clones. We compared the differential expression patterns of the outer- and inner- membrane proteins (OMPs and IMPs) among wild-type and the resistant strains by two-dimensional gel electrophoresis (2DE). Protein spots significantly altered were then subjected to mass spectrometry analysis and protein identification using Mascot program. Two OMPs (TolC and flagellin) and Five IMPs (transcription elongation factor NusA, long-chain fatty acid transport protein, translation elongation factor Tu, α-subunit of F1-ATP synthase and dihydrolipoic dehydrogenase) were identified. Real-time quantitative polymerase chain reaction (RT-qPCR) were also performed to determine the mRNA level of the above proteins. In conclusion, V. parahaemolyticus may obtain their resistance against AMPs through multidrug efflux transporter and repair of damaged cell mambrane by membrane biosynthesis.

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47

McPhee, Joseph B. "Regulation of polymyxin B and cationic antimicrobial peptide resistance in pseudomonas aeruginosa." Thesis, 2006. http://hdl.handle.net/2429/18192.

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Pseudomonas aeruginosa is a Gram-negative, opportunistic pathogen that is noted for its environmental ubiquity, its metabolic potential and its intrinsic resistance to a wide variety of antimicrobials, detergents, dyes, and biocides. These properties are consequences of a large (6.3 Mb) genome containing ~5500 genes of which 9.4% encode regulatory proteins. One of the largest classes of regulators in the P. aeruginosa genome is the two-component regulators. This work describes the contribution of two two-component regulatory systems, PhoP-PhoQ and PmrA-PmrB to Mg²⁺-limitation induced polymyxin B and cationic antimicrobial peptide resistance. Both of these systems respond to limiting Mg²⁺ and cause increased transcription of an eight-gene operon, pmrHFIJKLM-ugd, that is responsible for the addition of aminoarabinose to 1 and 4' phosphates on Lipid A. In addition, the PmrA-PmrB system regulates a three gene operon, PA4773-PA4775 that also contributes to polymyxin B and cationic antimicrobial peptide resistance. In addition to regulating polymyxin B and cationic antimicrobial peptide resistance, the PhoP-PhoQ system also directly regulates several small ORFs, one of which PA0921 contributes to swimming motility via an unknown mechanism. Similarly, PmrA-PmrB regulate other phenotypes, including the growth of P. aeruginosa in the presence of Fe³⁺. This growth phenotype occurs through gene products encoded by the feoAB operon. Interestingly, all genes identified in this study that are PmrA-PmrB regulated are also regulated by the presence of sub-inhibitory concentrations of cationic antimicrobial peptides. The regulation of PA4773-PA4775 and pmrHFIJKLM-ugd via cationic peptides is mostly independent of the PmrA-PmrB and PhoP-PhoQ systems. This observation explains why adaptive resistance to cationic antimicrobial peptides occurs and suggests that another, as yet unidentified, regulator is responsible for the detection of cationic antimicrobial peptides. A third regulatory system, PxrRS, is also identified. Mutants in this system show increased susceptibility to cationic antimicrobial peptides and polymyxin B. This susceptibility was not due to loss of regulation of the PA4773-PA4775 or pmrHFIJKLM-ugd. Microarray analysis demonstrated downregulation of a number of heat-shock proteins, as well as two operons potentially involved in efflux. The combined downregulation of heat-shock proteins involved in response to cellular stress and efflux systems suggests that intrinsic cationic peptide resistance is altered in these mutants.
Science, Faculty of
Microbiology and Immunology, Department of
Graduate

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48

Trombley, Michael Patrick. "Phosphoethanolamine transferases in Haemophilus ducreyi modify lipid A and contribute to human defensin resistance." Thesis, 2015. http://hdl.handle.net/1805/7915.

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Indiana University-Purdue University Indianapolis (IUPUI)
Haemophilus ducreyi resists the cytotoxic effects of human antimicrobial peptides (APs), including α-defensins, β-defensins, and the cathelicidin LL-37. Resistance to LL-37, mediated by the sensitive to antimicrobial peptide (Sap) transporter, is required for H. ducreyi virulence in humans. Cationic APs are attracted to the negatively charged bacterial cell surface. In other gram-negative bacteria, modification of lipopolysaccharide or lipooligosaccharide (LOS) by the addition of positively charged moieties, such as phosphoethanolamine (PEA), confers AP resistance by means of electrostatic repulsion. H. ducreyi LOS has PEA modifications at two sites, and we identified three genes (lptA, ptdA, and ptdB) in H. ducreyi with homology to a family of bacterial PEA transferases. We generated non-polar, unmarked mutants with deletions in one, two, or all three putative PEA transferase genes. Mutants with deletions in two PEA transferase genes were significantly more susceptible to β-defensins, and the triple mutant was significantly more susceptible to both α- and β-defensins, but not LL-37; complementation of all three genes restored parental levels of AP resistance. Deletion of all three PEA transferase genes also resulted in a significant increase in the negativity of the mutant cell surface, suggesting these three genes contribute to the addition of positively charged moieties on the cell surface. Mass spectrometric analysis revealed that LptA was required for PEA modification of lipid A; PtdtA and PtdB did not affect PEA modification of LOS. In human inoculation experiments, the triple mutant was as virulent as its parent strain. While this is the first identified mechanism of resistance to α-defensins in H. ducreyi, our in vivo data suggest that resistance to cathelicidin may be more important than defensin resistance to H. ducreyi pathogenesis.

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LO, YUAN-TING, and 羅元廷. "Study of Elevated Disease Resistance of Prawn by Feeding Antimicrobial Peptide-expressing Recombinant Yeast Strains." Thesis, 2018. http://ndltd.ncl.edu.tw/handle/rupxg3.

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碩士
東吳大學
微生物學系
106
In order to reduce the cost of the production for antibacterial peptides in aquaculture, to establish recombinant strains that exhibit the effect of both antibacterial peptides and probiotics is our aim. In this study, the cDNA of an antibacterial peptide ALF B9, which is a mutant form of MrALF from freshwater Macrobrachium rosenbergii, was transformed into the yeast Kluyveromyces maxianus (KM), a human probiotic, to express ALF B9. By feeding method, prawn M. rosenbergii was the model animal to evaluate the possibility of using KM and the recombinant strain expressing ALF B9 as prawn probiotics. The non-secreting strain rKM-B9, secreting strain rKM+B9 and KM were continuously fed to shrimp for five days. The mortality of shrimp in KM, rKM-B9, rKM+B9, KM+YPG and rKM+B9+YPG were 50%, 38%, 47% 42% and 36%, respectively. There result were significantly lower than the control group (73%) of the non feeding strains. Bacterial DNA was extracted from the shrimp feces before and after uptaking KM and rKM-B9 and the Next Generation Sequencing was used to analyze the bacterial composition. The results showed that the intestinal bacreial community in shrimp feces after uptaking KM and rKM-B9 were highly different from those in feces from before uptaking KM and rKM-B9. In addition, the relative abundance of pathogen L. garvieae derived from feces was reduced after the shrimp uptake KM and rKM-B9. The possible physiological factors of shrimp resistance to pathogens include proPO and Pon , gene associated with antioxidant SOD-Mn and GPx, the gene associated with stress HSP70, the activity of specific phenol oxidizing enzymes (phenoloxidase, PO), and superoxide dismutase (Superoxide dismutase, SOD). The result that the mRNA expression of five genes of shrimp uptaking KM, rKM-B9, and rKM+B9 decreased significantly (P < 0.05). The activity of activated PO (POS) and POT in the plasma and the activity of POT were higher than that of shrimp before feeding, and the SOD activity of the shrimp in each feeding group was significantly higher than that of the shrimp before feeding or after stopping. According to the above results, two recombinant strains of recombinant ALF B9 resistant to microbial peptide, rKM-B9 and rKM+B9, were superior to the original strain KM. These strains could enhance the PO activity in the shrimp's immune related enzymes and the activity of antioxidant enzyme SPD, and reduce the number of pathogenic bacteria in the shrimp's intestines. Therefore, it is believed that these strains have potential to be a probiotics for shrimp production.

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50

Di, Pierro Erica Jacqueline. "Unexpected biochemistry determines endotoxin structure in two enteric gram-negatives." Thesis, 2015. http://hdl.handle.net/2152/30448.

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Most gram-negative organisms require lipopolysaccharide and its membrane anchor, lipid A, for growth and survival. Also known as endotoxin, lipid A is synthesized via a nine-step enzymatic process, culminating in a conserved hexa-acylated, bis-phosphorylated disaccharide of glucosamine. This framework is often altered by condition- or species-specific lipid A modifications, which change the biochemical properties of the molecule in response to and to defend against environmental stress signals. Here, we expound on two stories in different gram-negative organisms, both involving novel or unanticipated biochemistry that impacts lipid A structure. First, the missing acyltransferase in the Epsilonproteobacterium Helicobacter pylori lipid A biosynthesis pathway is identified. This enzyme transfers a secondary acyl chain to the 3'-linked primary acyl chain of lipid A like E. coli LpxM, but shares almost no sequence similarity with the E. coli acyltransferase. It is reannotated as LpxJ and demonstrated to possess an unprecedented ability to act before the 2'-secondary acyltransferase, LpxL, as well as the 3-deoxy-D-manno-octulosonic acid transferase, KdtA. LpxJ is one member of a large class of acyltransferases found in a diverse range of organisms that lack an E. coli LpxM homolog, suggesting that LpxJ participates in lipid A biosynthesis in place of an LpxM homolog. The second story focuses on regulation of modifications to endotoxin structure that occur after the conserved biosynthesis pathway. E. coli pmrD is shown to be required for PmrAB-dependent lipid A modifications in conditions that exclusively activate PhoPQ; this result proves that PmrD connects PhoPQ and PmrAB despite previous reports that it is an inactive connector in this organism. Further, RNA sequencing and polymyxin B survival assays solidify the role of E. coli pmrD in influencing expression of pmrA and its target genes and promoting survival during exposure to cationic antimicrobial peptides. Notably, the presence of an unknown factor or system capable of activating pmrD to promote lipid A modification in the absence of the PhoPQ system is also revealed. In all, the findings presented here expand our understanding of alternative approaches to lipid A biosynthesis and the complex systems that regulate modifications of this dynamic molecule.

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Dissertations / Theses on the topic 'Antimicrobial peptide resistance'

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