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Kaihami, Gilberto Hideo. "Novos reguladores de resposta envolvidos na virulência de Pseudomonas aeruginosa." Universidade de São Paulo, 2018. http://www.teses.usp.br/teses/disponiveis/46/46131/tde-20072018-084306/.
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Os sistemas de sinalização de dois componentes são sistemas prevalentes em bactérias, permitindo a adaptação a diferentes condições ambientais. O sistema de dois componentes classicamente possui uma proteína histidina quinase, o primeiro componente, capaz de reconhecer o estímulo ambiental e fosforilar o regulador de resposta, o segundo componente. Pseudomonas aeruginosa é uma proteobactéria ubíqua, capaz de infectar hospedeiros filogeneticamente distintos. Esse patógeno oportunista apresenta um dos maiores conjuntos de sistemas de dois componentes em bactérias, que permite que ela sobreviva numa grande gama de ambientes, incluindo humanos. P. aeruginosa UCBPP-PA14 apresenta pelo menos 64 histidina quinases e 76 reguladores de resposta codificados em seu genoma. Diversos sistemas de dois componentes já foram correlacionados com a virulência, sendo o sistema GacSA o exemplo melhor caracterizado. Há poucos estudos sistemáticos sobre o envolvimendo dos reguladores de resposta na virulência de P. aeruginosa e os sinais que induzem a ativação dos reguladores de resposta precisam ser encontrados. Para identificar novos reguladores de resposta envolvidos na patogenicidade, infecções in vitro em macrófagos e in vivo em Drosophila melanogaster foram realizadas neste trabalho. Os macrófagos foram infectados com cada mutante dos reguladores de resposta ou com a linhagem selvagem, e a produção da citocina pró-inflamatória TNF-α e o clearance bacteriano foram determinados. Alternativamente, as moscas foram infectadas utilizando-se a estratégia de feeding e a sobrevivência foi verificada. Utilizando-se essas abordagens, a identificação de diversos reguladores de resposta com papel na virulência foi alcançada, além de se corfirmar o papel de reguladores de resposta já estudados. Um dos novos genes envolvidos em virulência, PA14_26570 (nomeado neste trabalho de atvR), codifica um regulador de resposta atípico com substituição no aspartato fosforilável para glutamato, o que usualmente induz um estado sempre ativo. Um mutante não polar em atvR foi construído e macrófagos infectados com a linhagem ΔatvR confirmaram um maior clearance bacteriano e maior produção de TNF-α em comparação aos macrófagos infectados com a linhagem selvagem. Para comprovar a participação de AtvR durante a patogênese, um modelo de pneumonia aguda em camundongos foi utilizado. Camundongos infectados com a linhagem ΔatvR apresentaram uma maior sobrevivência em comparação aos camundongos infectados com a linhagem selvagem. Além disso, os camundongos infectados com ΔatvR apresentaram menor carga bacteriana, aumento no recrutamento de neutrófilos ativados e aumento na produção de citocinas pró-inflamatórias (TNF-α e IFN-γ). Utilizando-se uma abordagem transcritômica (RNA-Seq), foi determindo diversos genes são regulados positivamente na linhagem superexpressando AtvR em relação à linhagem controle. Dentre esses, os clusters de respiração anaeróbia nar, nir, nor e nos estão incluídos. Esse resultado foi confirmado por qRT-PCR e análises fenotípicas, em que a linhagem ΔatvR apresentou menor crescimento e expressão da nitrato redutase durante condições de hipóxia em comparação à linhagem selvagem. Em suma, neste trabalho foi demonstrado que diversos reguladores de resposta são importantes para a virulência de P. aeruginosa em macrófagos in vitro e in vivo em Drosophila, além de caracterizar o regulador de resposta atípico AtvR, que regula a respiração anaeróbica por desnitrificação, permitindo que P. aeruginosa possa infectar e colonizar o hospedeiro com maior eficiência.Two-component systems are widespread in bacteria, allowing the adaptation to environmental changes. A two-component system is classically composed by a sensor kinase that phosphorylates a cognate response regulator. Pseudomonas aeruginosa is a ubiquitous proteobacterium able to cause disease in several hosts. This opportunistic pathogen presents one of the largest sets of two-component systems known in bacteria, which certainly contributes to its ability to thrive in a wide range of environmental settings, including humans. P. aeruginosa UCBPP-PA14 genome codes for at least 64 sensor kinases and 76 response regulators. Some response regulators are already known to be related to virulence, with the GacSA system as the best characterized. There are no systematic studies about the involvement of P. aeruginosa response regulators in virulence. Moreover, the input signal that triggers the response regulator activation is yet to be uncovered for most systems. To find new response regulators involved in virulence, in vitro infections werecarried out using macrophages. Briefly, the macrophages were infected with each response regulator mutant or the wild-type strain, the pro-inflammatory cytokine production (TNF-α) and the bacterial clearance were evaluated. Using this approach, we identified several response regulators involved in virulence, and we also confirmed the involvement of known response regulators in this process. One of the novel virulence-related response regulators, PA14_26570 (named here as AtvR), is an atypical response regulator with a substitution in the phosphorylable aspartate to glutamate, that usually leads to an always-on state. A non-polar mutant was constructed, and macrophage infection with ΔatvR confirmed an increased bacterial clearance as well as a higher TNF-α production as compared to the wild-type strain. To ascertain the role of AtvR during the pathogenic process, an acute pneumonia model was used. Mice infected with ΔatvR showed an increased survival as compared to mice infected with the wildtype strain. In addition, ΔatvR infected mice showed reduced bacterial burden, increased neutrophil recruitment and activation, as well as increased pro-inflammatory cytokine production (TNF-α and IFN-γ). Also, using a transcriptomic approach (RNASeq), we showed that several genes were upregulated in the strain overexpressing AtvR. These genes include the anaerobic respiration clusters nar, nir, nor and nos. This result was confirmed by qRT-PCR and phenotypic analysis, in which ΔatvR showed reduced growth and nitrate reductase expression during hypoxic conditions as compared to the wild-type strain. In conclusion, we have demonstrated that several response regulators are important for P. aeruginosa virulence in vitro. In addition, we further characterized the atypical response regulator AtvR, which regulates anaerobic respiration via denitrification, allowing this bacterium to infect and colonize the host more efficiently.
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Giri-Rachman, Ernawati Arifin. "The CPX two component regulatory system of V. cholerae." Thesis, University of Bristol, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.274636.
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Patel, Jenishkumar. "ENVIRONMENTAL RESPONSES OF TWO-COMPONENT SYSTEMS IN STREPTOCOCCUS SANGUINIS." VCU Scholars Compass, 2010. http://scholarscompass.vcu.edu/etd/2270.
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The gram-positive bacterium Streptococcus sanguinis is a member of human indigenous oral microbialflora and has long been recognized as a key player in the bacterial colonization of the mouth. S. sanguinis is also the most common viridians streptococcal species implicated in infective endocarditis. Although many studies have focused on two-component systems in closely related Streptococcus species such as S. mutans, S. pneumoniae and S. gordonii; the mechanism of the response regulator in S. sanguinis is still unknown. The ability of S. sanguinis to adapt and thrive in hostile environments suggests this bacterium is capable of sensing and responding to various environmental stimuli. The present study clearly demonstrates that a number of RR genes, SSA_0204, SSA_0217, SSA_1810, SSA_1794, and SSA_1842, in S. sanguinis are essential to the recognition and response to various environmental stresses. Results from this study also identified genes SSA_0260, SSA_0261, and SSA-0262, involved in acidic tolerance and suppressed by SSA_0204 response regulator.
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Chang, Yo-Cheng. "Design and implementation of a synthetic biological component based on the bacterial two-component system." Thesis, University of Oxford, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.534147.
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Tomenius, Henrik. "Bacterial virulence and adaptation mediated by two-component system signalling /." Stockholm, 2006. http://diss.kib.ki.se/2006/91-7140-792-8/.
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Quigley, Andrew Michael. "The two-component system controlling inducible glycopeptide resistance in Enterococci." Thesis, University of Warwick, 2010. http://wrap.warwick.ac.uk/3190/.
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VanS and VanR form the two-component regulatory system that controls inducible glycopeptide resistance in Enterococci. Upon induction VanS, a sensor histidine kinase, phosphorylates itself on a conserved histidine residue. This phosphate is transferred to a conserved aspartate residue on VanR. The phosphorylated form of VanR is the transcriptional activator of the vanAHX genes which, when expressed, directly confer vancomycin resistance. VanS also possesses VanR phosphatase activity, providing a mechanism by which to repress vanAHX transcription. This thesis describes approaches used towards the crystallisation of VanS and VanR. These are based upon previous crystallisation studies resulting in full-length VanS crystals which diffracted to 8Å, as well as a cytoplasmic structure of an analogous histidine kinase from Thermatoga maritima (Marina et al., 2005). Full-length and truncated forms of VanSA and full-length VanRA were cloned, expressed in E.coli and purified for crystallisation studies. Autokinase activity was biochemically characterised using radiolabelling and spectrophotomic assays, in tandem with a novel application of mass spectrometry. Site-directed mutagenesis of VanSA, led to the observation that ATP hydrolysis may occur independently of the autokinase function of VanSA. Adenosine 5’ tetraphosphate was also discovered, as a novel product of VanSA. Based upon these data an expanded model for VanS autokinase activity has been proposed. This may be expanded to include the phosphotransfer and phosphatase mechanisms and validated through the measurement of associated product formation. Finally a new mechanism for the control of the VanRS two component system has been proposed. Future studies will validate and expand this model. This work has significantly increased our knowledge of this system providing the tools and foundations that will lead to the elucidation of the way that this two component system functions. This has the potential for the development of novel inhibitors that either complement or supersede existing therapies.
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Deara, Mohamed Ahmed. "Replacement policies for a two-component system with failure dependence." Thesis, University of Salford, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.366316.
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Mahfouz, Magdy Elsayed. "Molecular characterisation of a two-component regulatory system from Burkholderia pseudomallei." Thesis, University of Plymouth, 2001. http://hdl.handle.net/10026.1/2538.
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Studies were undertaken to clone and characterise a two-component regulatory system from a clinical isolate (204) of the human and animal pathogen Burkholderia pseudomallei. A number of genomic libraries were constructed in E. coli host-vector systems and screened for the presence of a two-component system using oligonucleotide probes based on nucleotide sequence homology. Fragments of genomic DNA were cloned and sequenced and found to possess two open reading frames (ORFs) that overlap with a single nucleotide and are believed to encode a novel two-component regulatory system. A possible promoter region was identified upstream of the two ORFs, mrgR and mrgS, which read in the same direction and may represent an operon. The deduced translation of mrgR reveals a protein, MrgR, which possesses conserved motifs that are consistent with the phosphorylation domains and DNA-binding helix-turn-helix structure of a family of response regulatory proteins. The deduced translation of mrgS reveals that the MrgS protein possesses all the invariant amino acids that characterise other sensor regulatory proteins. Southern hybridisation studies showed that the mrgRS locus was present in 19 isolates of B. pseudomallei from a wide geographical derivation, but not in any closely related bacterial species, including Burkholderia thailandensis. The expression of the two genes was verified using antibodies developed to synthetic peptides based on sequences from the C- and N-terminal regions of MrgR and MrgS, respectively. The specificity of the antibodies was confirmed in Western blotting studies in which almost all of mrgR and the proximal quarter of mrgS were translationally fused with malE (MBP-MrgR and MBP-MrgS) and expressed in E. coli K12. The antibodies were used to probe Western blots of cellular and extracellular extracts of different isolates of B. pseudomallei and identified multiple bands in whole-cell lysates. The sizes of two of these bands were 24 kDa and 115 kDa, which may represent the unprocessed forms of MrgR and MrgS, respectively. It was proposed that the other bands represented either isoforms or degradation products of the full-length proteins. The recognition of all bands was abolished following pre-incubation of the antibodies with the immunising peptide but remained unaffected if an irrelevant peptide, was used for this purpose. Western blot analysis demonstrated that serum antibodies from a patient with acute melioidosis recognised MBP-MrgR but not MBP-MrgS suggesting a possible role for MrgR in the disease process. The expression of mrgR and mrgS was found to be constitutive in B. pseudomallei that had been cultured using different combinations of temperature, pH and NaCl suggesting that the genes perform a number of biological functions. There is some evidence that at 42°C the processing of MrgR and MrgS may be altered and the possible mechanisms for this are discussed. B. pseudomallei grew better at 42°C and pH 5 and less well at 25°C and pH 8 and this was influenced by NaCl concentration partly reflecting the environmental distribution and intracellular nature of the pathogen. Environmental and clinical isolates of B. pseudomallei differed in the pH optimum for growth at 42°C. The DNA flanking the mrgRS locus in isolate 204 was cloned, sequenced, and seven ORFs were identified including a transcriptional regulatory gene similar to bvgR of Bordetella pertussis. Southern blot analysis using three different DNA probes revealed restriction fragment length polymorphisms (RFLPs) in the region downstream of mrgRS. Two distinct RFLP patterns were identified among 16 different isolates of B. pseudomallei. The potential effects of this variation on gene expression and protein function await further investigation.
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Ibrahim, Iskander Mohamed. "Biochemical characterisation of the cyanobacterial Hik2-Rre1 two-component regulatory system." Thesis, Queen Mary, University of London, 2013. http://qmro.qmul.ac.uk/xmlui/handle/123456789/8509.
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Two-component signal transduction systems (TCS) consist of a sensor histidine kinase and a response regulator. TCS are ubiquitous in prokaryotes, but found only in some eukaryotes. TCS mediate adaptation to various environmental changes in bacteria, plants, fungi, and protists. Histidine kinase 2 (Hik2) is a sensor histidine kinase found in all cyanobacteria. The Hik2 homologue known as Chloroplast Sensor Kinase is found in algae and plants, where it is encoded by the nuclear genome and it is targeted to chloroplasts. CSK couples the redox state of the photosynthetic electron transport chain to chloroplast gene transcription. This thesis describes biochemical characterisation of the signal transduction mechanism of Hik2 and its response regulator (Rre) partners in order to clarify the Hik2-Rre two-component signal transduction pathway. Results presented in this thesis illustrate that the autophosphorylation activity of the full-length Hik2 protein is specifically inhibited by sodium ions. An autophosphorylation event of a histidine kinase is the result of homodimerisation and is followed by trans or cis-autophosphorylation of each monomer on its conserved histidine residue. Chemical crosslinking revealed that the Hik2 protein exists predominantly as a phosphorylated (autokinase active) monomer, tetramer, and higher-order oligomeric complexes. The functions of these different oligomeric states of Hik2 are also discussed. From a previous study, which was based on an observation from a yeast two-hybrid assay, Hik2 was proposed to form a two-component pair with Rre1 and RppA. However, no further evidence was presented to support either direct interaction or direct phosphotransfer activity of the Hik2-Rre pair. This thesis confirms interaction of Hik2-Rre1 and Hik2-RppA two-component ! %! pairs using an in vitro pull-down assay and phosphotransfer kinetics. Finally, a model is proposed for the Hik2 based two-component signal transduction pathway.
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Juni, Sarkar. "Characterization of Hpk2-Rrp2, two-component regulatory system in Treponema denticola." VCU Scholars Compass, 2011. http://scholarscompass.vcu.edu/etd/268.
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Treponema denticola levels in the gingival crevice become elevated as periodontal disease develops. Oral treponemes may account for as much as 40% of the total bacterial population in the periodontal pocket. The stimuli that trigger enhanced growth of T. denticola and the mechanisms associated with the transmission of these signals remain to be defined. A hypothesis was set that the T. denticola ORFs tde1970 (histidine kinase) and tde1969 (response regulator) constitute a functional two component regulatory system that regulates, at least in part, responses to the changing environmental conditions associated with the development of periodontal disease. The results presented demonstrate that tde1970 and tde1969 are conserved, universal among T. denticola isolates and transcribed as part of a 7 gene operon in a growth phase dependent manner. Tde1970 undergoes autophosphorylation and transfers phosphate to Tde1969. Henceforth the proteins encoded by these ORFs are designated as Hpk2 and Rrp2 respectively. Hpk2 autophosphorylation kinetics was influenced by environmental conditions and by the presence or absence of a Per Arnt Sim (PAS) domain. It can be concluded that Hpk2 and Rrp2 constitute a functional two-component system that contributes to environmental sensing. This study also sought to determine the molecular basis of Hpk2 function in response to environmental stimuli. Hpk2 was shown to bind hemin via a putative heme-binding domain within the PAS domain. Hemin binding to Hpk2 positively regulated its autokinase ability under anaerobic conditions, suggesting that Hpk2 activation may play a role in the migration of T. denticola away from the aerobic zone deeper into developing periodontal pockets. In this study we have generated point mutations of conserved amino acid residues in the sensor PAS domain of Hpk2 and assessed their role in kinase activation under both aerobic and anaerobic conditions depending on their oligomeric state, hence providing a strong basis to correlate ligand binding, kinase activity and oligomeric states of the protein that may provide stability of these complex interactions. Ultimately this study provides a comparative linkage between the responses of PAS domain to sensory inputs controlling access to its kinase domain within which is contained the dimerization domain, which ultimately leads to fine-tuned control of interactions between Hpk2 dimerization and catalytic domain.
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Barwari, Bala Farhad. "Asymptotic and numerical solutions of a two-component reaction diffusion system." Thesis, University of Nottingham, 2016. http://eprints.nottingham.ac.uk/37231/.
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In this thesis, we study a two-component reaction diffusion system in one and two spatial dimensions, both numerically and asymptotically. The system is related to a nonlocal reaction diffusion equation which has been proposed as a model for a single species that competes with itself for a common resource. In one spatial dimension, we find that this system admits traveling wave solutions that connect the two homogeneous steady states. We also analyse the long-time behaviour of the solutions. Although there exists a lower bound on the speed of travelling wave solutions, we observe that numerical solutions in some regions of parameter space exhibit travelling waves that propagate for an asymptotically long time with speeds below this lower bound. We use asymptotic methods to both verify these numerical results and explain the dynamics of the problem, which include steady, unsteady, spike-periodic travelling and gap-periodic travelling waves. In two spatial dimensions, the numerical solutions of the axisymmetric form of the system are presented. We also establish the existence of a steady axisymmetric solution which takes a form of a circular patch. We then carry out a linear stability analysis of the system. Finally, we perform bifurcation analysis of these patch solutions via a numerical continuation technique and show how these solutions change with respect to variation of one bifurcation parameter.
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Dogra, Gaurav. "Studies on the role of CheS in Sinorhizobium meliloti chemotaxis." Thesis, Virginia Tech, 2011. http://hdl.handle.net/10919/76842.
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Chemotaxis is the ability of an organism to sense its environment and move towards attractants and away from repellents. The two-component system controlling chemotaxis in bacteria contains a histidine kinase CheA, which is autophosphorylated in response to a signal from a ligand-bound transmembrane methyl-accepting chemotaxis protein. CheA transfers the phosphate group to its cognate response regulator which modulates flagellar rotation. Signal termination by dephosphorylation of the response regulator is necessary for the organism to react rapidly to changes in the environment. The phosphorylated response regulator CheY in Escherichia coli is dephosphorylated by CheZ, a phosphatase; certain organisms, such as Sinorhizobium meliloti, that lack a CheZ homolog have developed alternate methods of signal termination. The signaling chain of S. meliloti contains two response regulators, CheY1 and CheY2, in which CheY2 modulates flagellar rotation and CheY1 causes signal termination by acting as a phosphate sink. In addition to known chemotaxis components, the second gene in the chemotaxis operon of S. meliloti codes a 97 amino acid protein, called CheS. The phenotype of a cheS deletion strain is similar to that of a cheY1 deletion strain. Therefore, the possibility that CheS causes signal termination was explored in this work. The derived amino acid sequence of CheS showed similarities with its orthologs from other °-proteobacteria. Sequence conservation was highest at the centrally located °4 and °5 helices. Earlier observations that CheS localizes at the polar chemotaxis cluster in a CheA-dependent manner were confirmed, and the co-localization of CheS with CheA was demonstrated by fluorescence microscopy. The stable expression of CheS in the presence of CheA was confirmed by immunoblot. The same approach was used to establish the stable expression of CheS only in the presence of the P2 domain of CheA, but not with the P1 or P345 domains. Limited proteolysis followed by mass spectrometry defined CheA163-256 as the CheS binding domain, and this domain overlapped the previously defined CheY2-binding domain, CheA174-316. The role of CheS in the phosphate flux in S. meliloti chemotaxis was analyzed by assays using radio-labeled [?-?°P]ATP. CheS does not play a role in the autophosphorylation of CheA. However, CheS accelerated the rate of CheY1~P dephosphorylation by almost two-fold, but did not affect the rate of CheY2~P dephosphorylation. CheS also does not seem to affect phosphate flow in the retrophosphorylation from CheY2~P to CheA using acetyl [?°P]phosphate as phosphodonor. Since CheS increases the rate of CheY1 dephosphorylation, it can be envisioned that it either increases the association of CheY1 to CheA, increasing the flow of phosphate from CheA to CheY1, or directly accelerates the dephosphorylation of CheY1~P. The presence of a STAS domain and a conserved serine residue in CheS also raises the possibility that CheS may be phosphorylated by a yet unknown kinase, in a mechanism similar to the phosphorylation of Bacillus subtilis SpoIIAA by its cognate kinase SpoIIAB. Phosphorylated CheS may then switch CheA between a kinase or phosphotransferase ON/OFF state or activated CheS may directly interact with CheY1. Further studies are needed to determine the association of CheY1 with CheS to elucidate the mechanism of CheY1 dephosphorylation. This work has confirmed the in vitro association of CheS with CheA, determined the CheS binding domain on CheA, and indicated that CheS accelerates the dephosphorylation of CheY1~P. This has advanced our understanding of the role of CheS in the chemotaxis signaling chain of S. meliloti.Master of Science
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Le, Sage Valerie. "Ligand sensing and signal trasnduction by the two-component system PhoP/PhoQ." Thesis, McGill University, 2009. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=95624.
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The Citrobacter rodentium genome sequence contains a phoPQ operonhomologous (~79% identity) to that of S. typhimurium. We report that C. rodentiumPhoQ senses fluctuations in Mg2+ concentrations and acidic pH. Surprisingly, PhoQwas not activated by the presence of AMPs. However, activation by AMPs is observedwhen C. rodentium PhoP/PhoQ was expressed in as. typhimurium background. Weidentified an outer membrane protease of the omptin family that was responsible forinhibiting PhoQ activation by AMPs. In stark contrast to S. typhimurium, which relieson LPS modifications to resist AMPs, our results suggest that C. rodentium promotesresistance through a PhoP/PhoQ-dependent OM protease to inhibit disruption of theouter membrane by AMPs .La séquence du génome de Citrobacter rodentium présente un opéron phoPQ(~79% identité) homologue à celui de S. typhimurium. Nous avons déterminé quePhoQ de C. rodentium perçoit les variations de pH et en Mg2+ du milieu environnant.De manière surprenante, les PAMs ne causent aucune augmentation d'activité dePhoQ. Néeanmoins, lorsque le système PhoP/PhoQ de C. rodentium est exprimé chezS. typhimurium les PAMs activent PhoQ. Nous avons identifié une protéine de lamembrane externe appartenant à la famille des omptin qui est responsable del'inactivité de PhoQ en présence des P AMs. Ces résultats suggèrent que le mécanismede résistance aux PAMs de C. rodentium serait régulé par le système PhoP/PhoQ et une protéase qui empêcherait la destruction de la membrane externe par les P AMs. Cemécanisme de défense est différent de celui du système PhoP/PhoQ de S. typhimuriumqui repose essentiellement sur des modification du LPS .
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Salazar, Michael E. Jr (Michael Edward). "Induction kinetics of the PhoQ-PhoP two-component system in Escherichia coli." Thesis, Massachusetts Institute of Technology, 2016. http://hdl.handle.net/1721.1/104179.
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Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2016.Cataloged from PDF version of thesis.
Includes bibliographical references.
Cells rely on signal transduction systems to sense and respond to changes in their enviroments. When a stimulus is present, the corresponding signal transduction system will activate and enact the appropriate biological response, often by modulating target gene expression. In many cases, the temporal dynamics of the activation of target gene expression in the presence of constant stimulus is complex, and often exhibits one or several pulses. How these complex temporal dynamics are regulated at the molecular level is unknown for many signal transduction systems. In this thesis, I discuss the molecular regulation of the temporal dynamics of PhoQ-PhoP induction in Escherichia coli. The PhoQ-PhoP pathway is a canonical two-component system that responds to low extracellular Mg'+, certain antimicrobial peptides, and potentially other unknown factors. Upon activation, the bifunctional histidine kinase PhoQ autophosphorylates and subsequently phosphotransfers to the response regulator PhoP, thereby activating it to increase transcription of PhoP target genes. Because PhoQ is bifunctional, PhoQ acts as a phosphatase on phosphorylated PhoP in the absence of stimulus, thereby keeping the system inactivated. When the PhoQ-PhoP system is strongly induced, PhoP target genes exhibit impulse kinetics, meaning gene expression increases to a maximal level and subsequently decreases to an eventual steady state. We discovered that this impulse response is caused by a negative feedback loop in which active PhoP transcribes mgrB, a gene encoding a small membrane protein that interacts directly with PhoQ to repress the output of the system. MgrB selectively inhibits the ability of PhoQ to phosphorylate PhoP, and permits PhoQ to act as a phosphatase on phosphorylated PhoP. This change in PhoQ activity causes a decrease in the level of active PhoP and the level of PhoP target genes. This thesis reveals how negative feedback loops and histidine kinase bifunctionality can drive the kinetics of two-component system induction in bacteria, and more generally explores how cells regulate changes in gene expression over time.
by Michael E. Salazar, Jr.
Ph. D.
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Walker, Jennifer Nicole. "The two-component system, ArlRS, regulates agglutination and pathogenesis in Staphylococcus aureus." Diss., University of Iowa, 2013. https://ir.uiowa.edu/etd/1414.
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Staphylococcus aureus is defined by its ability to agglutinate during exposure to human blood plasma. Although agglutination has long correlated with disease severity, the function of agglutination during infection remains unclear. Increasing evidence suggests the mechanisms of agglutination are highly complex and poorly understood. The goal of this dissertation was to characterize the mechanisms required for S. aureus agglutination in vitro and determine how these factors contribute to pathogenesis.
Chapter II focuses on the development of two in vitro agglutination assays, which allow the process to be measured quantitatively. Through these assays, we confirmed the major factors contributing to agglutination are human fibrinogen and the bacterial surface protein, ClfA. Productive interactions between these two factors are required for agglutination to proceed. Surprisingly, we also identified a novel regulatory system that significantly contributed to agglutination. Inactivation of the ArlRS two-component system (TCS) prevents agglutination in both of the developed assays.
Studies in Chapter III focused on characterizing the mechanism by which ArlRS inhibits agglutination. To examine regulation, quantitative PCR identified the major output of the ArlRS system as the gene ebh. Surprisingly, transcript levels of known extracellular matrix (ECM) binding proteins did not change. Characterization of ebh indicated that overexpression in an arlRS mutant is the major factor responsible for preventing agglutination. Deletion of ebh restores the ability of the arlRS mutant to agglutinate in both gravity and flow-based agglutination assays. Fluorescence microscopy of clumps indicates wildtype cells bind and incorporate fluorescently labeled human fibrinogen (Fg) displaying co-localization with the clumps. Surprisingly, arlRS mutants also bound human Fg, but these interactions were not productive for clumping, suggesting successful agglutination is more complex than binding ECM proteins. These studies indicate that ArlRS regulates agglutination through a unique mechanism that depends on the surface protein Ebh.
Studies in Chapter IV were performed to determine the role ArlRS played in pathogenesis. A rabbit model of infective endocarditis and sepsis was employed to assess ArlRS virulence because this model has been shown to require agglutination for disease progression. Mutants in arlRS displayed reduced virulence in the rabbit model of infective endocarditis, which correlated with the mutant's inability to form a vegetation of the heart valve. These studies provide further insight into the importance of S. aureus agglutination during infection and define a mechanism of regulation through a novel surface protein.
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Daza, Oscar Eduardo. "Simulation and Validation of Two-Component Flow in a Void Recirculation System." DigitalCommons@CalPoly, 2011. https://digitalcommons.calpoly.edu/theses/504.
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Nuclear power plants rely on the Emergency Core Cooling System (ECCS) to cool down the reactor core in case of an accident. Occasionally, air is entrained into the suction piping of ECCS causing voids that decrease pumping efficiency, and consequently damage the pumps. In an attempt to minimize the amount of voids entering the suction side of the pump in ECCS, a Void Recirculation System (VRS) experiment was conducted for a proof of concept purpose. While many studies have been oriented in studying two-component flow behavior in ECCS, none of them propose a solution to minimize air entrainment. As a consequence, there are no simulation models that use computational fluid dynamics to address gas entrainment solutions in ECCS. The objectives of this thesis are to (1) simulate and investigate two-component air-water flow in a VRS that minimizes the amount of air in piping systems, using RELAP5/MOD3 as the computational tool, and (2) to validate the numerical results with respect to experimental results and observations.
A one-dimensional model of the VRS was built in RELAP5, in which eight different scenarios (replicating those from the VRS experiment) were simulated for a period of 150 seconds. Four Froude numbers of 0.8, 1.0, 1.3 and 1.6 were evaluated in two different pipe configurations, and the experimental data obtained from the VRS experiment was used to validate the numerical results obtained from these simulations. It was concluded that air recirculation occurs indefinitely throughout the entire 150 seconds of the simulation for Froude numbers up to 1.3; while for a Froude number of 1.6, air recirculation occurs for approximately 100 seconds and ceases after 125 seconds of the simulation. An average air reduction effectiveness of 90% was found for all simulation scenarios. The VRS model was successfully validated and can be used to investigate the effects of air entrainment in suction piping.
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Pinto, Cecilia de Agrela. "The two-component system of a novel copper resistant operon of Marinobacter hydrocarbonoclasticus." Master's thesis, Faculdade de Ciências e Tecnologia, 2012. http://hdl.handle.net/10362/10062.
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Dissertation for the Master’s Degree in Structural and Functional BiochemistryThe majority of bacterial heavy metal resistance systems are regulated by twocomponent signal transduction systems. Stimuli from the environment interact with the histidine kinase, which in turn activates the response regulator by phosphorylation. The effector domain of the response regulator then binds to DNA, eliciting the specific response. Analysis of the Marinobacter hydrocarbonoclasticus genome revealed the presence of genes, copXAB, that code for proteins associated with copper response. The biochemical characterization of the two-component signal transduction system, copSR, is of interest due to the vital role it plays in the regulation of expression of the copXAB operon. The genes that encode for the CopR and CopS_C (cytosolic sensor domain of CopS) proteins were heterologously expressed in E.coli and expression was optimized for the production of soluble protein using LB medium. Due to solubility problems, the genes that code for these proteins were cloned as hexahistidine or glutathione S-transferase fusion proteins. CopR and its domains were optimally expressed at 16°C for 16 and 3 h after induction, respectively, whilst CopS_C was expressed at 37°C during 3 h after induction. Proteins were purified using different chromatographic strategies, most of them using affinity chromatography. The yields of pure protein per liter of growth culture obtained after complete purification from the soluble cellular extract were: 0.14 to 0.23 mg/L for CopR; 0.42 mg/L, CopR_NHis6; for the CopR_CHis6 it was 0.16 mg/L and 4.2 mg/L of CopS_C. The molecular mass of each protein was determined by gel filtration, 31 kDa for CopR, 17.5 kDa for CopR_NHis6, 15.1 kDa for CopR_CHis6 and 38.2 kDa for CopS_C. In the case of CopS_C there is the possibility that a dimer is formed, which should be evaluated. From the evaluation of disulfide bonds, using SDS PAGE and PAGE gels, all proteins or protein domains appeared to be monomers when in the presence of β-MEtOH. Circular dichroism evaluated the state of folding of the CopS_C and CopR proteins, which were shown to be folded in which the α-helix structures predominate. A model structure for CopR was also determined which agrees with this analysis. However, in the case of the CopR domains, the data obtained merely indicate folding, due to the low concentrations of the proteins. Phosphorylation and electrophoresis mobility shift assays of the CopR protein were, for the most part, inconclusive. However, in the absence of BSA, formation of the CopR:DNA complex in a gel filtration column is observed, though requires additional evaluation.
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Al-Meer, Jehan Abdulla. "Molecular analysis of the ompR/envZ two-component regulatory system in Bortadella species." Thesis, Imperial College London, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.285150.
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Patel, Isha Rameshbhai. "Evaluation of the role of BarA-UvrY two-component system in escherichia coli." College Park, Md. : University of Maryland, 2004. http://hdl.handle.net/1903/1564.
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Thesis (M.S.) -- University of Maryland, College Park, 2004.Thesis research directed by: Virginia-Maryland Regional College of Veterinary Medicine. Maryland Campus. Title from t.p. of PDF. Includes bibliographical references. Published by UMI Dissertation Services, Ann Arbor, Mich. Also available in paper.
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Wang, Nancy S. "DhkA, A two-component system histidine kinase gene that functions in Dictyostelium development /." Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 1997. http://wwwlib.umi.com/cr/ucsd/fullcit?p9735271.
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Mioro, Miriam Kanyua. "Designing a Two Component System for Enzyme Immobilization Using a Modified Chitosan Support." Youngstown State University / OhioLINK, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=ysu15946615388307.
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Lin, Yi-Shiuan, Arthur Shaw, Shi-Gang Wang, Chia-Chen Hsu, I.-W. Teng, Min-Jen Tseng, Tim Huang, Ching-Shih Chen, Yu-Wei Leu, and Shu-Huei Hsiao. "Identification of novel DNA methylation inhibitors via a two-component reporter gene system." BioMed Central, 2011. http://hdl.handle.net/10150/610175.
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BACKGROUND:Targeting abnormal DNA methylation represents a therapeutically relevant strategy for cancer treatment as demonstrated by the US Food and Drug Administration approval of the DNA methyltransferase inhibitors azacytidine and 5-aza-2'-deoxycytidine for the treatment of myelodysplastic syndromes. But their use is associated with increased incidences of bone marrow suppression. Alternatively, procainamide has emerged as a potential DNA demethylating agent for clinical translation. While procainamide is much safer than 5-aza-2'-deoxycytidine, it requires high concentrations to be effective in DNA demethylation in suppressing cancer cell growth. Thus, our laboratories have embarked on the pharmacological exploitation of procainamide to develop potent DNA methylation inhibitors through lead optimization.METHODS:We report the use of a DNA methylation two-component enhanced green fluorescent protein reporter system as a screening platform to identify novel DNA methylation inhibitors from a compound library containing procainamide derivatives.RESULTS:A lead agent IM25, which exhibits substantially higher potency in GSTp1 DNA demethylation with lower cytotoxicity in MCF7 cells relative to procainamide and 5-aza-2'-deoxycytidine, was identified by the screening platform.CONCLUSIONS:Our data provide a proof-of-concept that procainamide could be pharmacologically exploited to develop novel DNA methylation inhibitors, of which the translational potential in cancer therapy/prevention is currently under investigation.
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Mack, Lydia Eileen. "Understanding the Regulatory Mechanism of BfmR in Acinetobacter baumannii ATCC 19606T." Miami University / OhioLINK, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=miami1561671121598468.
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Affandi, Trisiani, and Trisiani Affandi. "Structural and Functional Characterization of the Histidine Kinase CusS in Escherichia coli." Diss., The University of Arizona, 2016. http://hdl.handle.net/10150/622937.
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Bacteria may live in harsh environments where they face changing and new conditions. Therefore, the ability to maintain homeostasis in cells may be vital for survival. Transition metals such as iron, zinc, and copper are essential nutrients for cell survival, but become toxic if in excess amount. In order to survive, bacteria have developed defensive mechanisms to protect themselves. Copper and silver levels need to be carefully maintained within cells to balance cellular needs with potential toxicity. This dissertation focuses on the Cus copper and silver efflux system in E. coli. The E. coli cus system is composed of two divergently transcribed operons, cusCFBA and cusRS. The cusCFBA genes encode for a tripartite metal efflux pump CusCBA and a metallochaperone CusF. The cusRS genes encode a two-component system CusS-CusR that regulates the expression of the cusCFBA genes in response to elevated levels of copper or silver in the periplasm. The histidine kinase CusS senses and binds to metals on its periplasmic sensor domain and transduces signal into the cytoplasm to further communicate with its cognate response regulator CusR through histidyl-aspartyl phosphotransfer event. CusR then outputs cellular response by activating the upregulation of the cusCFBA genes, which then turn on the CusCBA efflux pump to eliminate excess copper or silver in the periplasm. While bacterial two-component systems have been widely studied, the mechanisms of ligand-induced signal transduction by histidine kinases remain unclear. It is now known that cusS is essential for copper and silver resistance, and CusS directly binds metal ions in the periplasmic sensor domain and dimerizes upon metal binding. Thus, the goal of this research is to characterize the metal binding properties in the sensor domain, and to elucidate the signal transduction and autophosphorylation mechanisms of CusS upon metal binding. The data from this work reveal that there are two distinct metal binding sites, interface and internal binding sites, in the sensor domain of CusS, and the interface binding site is functionally more important in metal resistance in E. coli. Furthermore, metal-induced dimerization through the interface metal binding site plays an important role in CusS kinase activity. Together, these findings aid in our understanding of the molecular details in metal binding within the sensor domain of CusS. Based on these data, we propose a model for the signal transduction mechanism and histidine phosphorylation mechanism of the histidine kinase CusS.
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Belzil-Lacasse, Christian. "Study of Dissipative Spots In Three-Component Reaction-Difussion Systems on Two-Dimensional Domains." Thesis, Université d'Ottawa / University of Ottawa, 2016. http://hdl.handle.net/10393/34257.
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Dissipative spots are found in physical experiments of many branches of natural science. In this thesis we use three-component reaction-diffusion systems on two-dimensional domains in order to generate these patterns. Using a dynamical system approach we proceed with a Fourier analysis on a linearized reaction-diffusion system in order to provide the bifurcation conditions for a given homogeneous state. We validate our results and establish it's limitations through numerical experiments. We report very interesting behavior during these simulations, notably hysteresis and multi-stability. We will then turn our attention to the relatively unexplored phenomenon of rotating spots. Based on previous work done for spiral waves, we investigate the effect of translational symmetry-breaking on a rotating spot mainly through careful numerical analysis.
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Tatke, Gorakh Digambar. "Elucidating The Role of MifS-MifR Two-Component System in Regulating Pseudomonas aeruginosa Pathogenicity." FIU Digital Commons, 2016. http://digitalcommons.fiu.edu/etd/3002.
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Pseudomonas aeruginosa is a Gram-negative, metabolically versatile, opportunistic pathogen that exhibits a multitude of virulence factors, and is extraordinarily resistant to a gamut of clinically significant antibiotics. This ability is in part mediated by two-component systems (TCS) that play a crucial role in regulating virulence mechanisms, metabolism and antibiotic resistance. Our sequence analysis of the P. aeruginosa PAO1 genome revealed the presence of two open reading frames, mifS and mifR, which encodes putative TCS proteins, a histidine sensor kinase MifS and a response regulator MifR, respectively. This two-gene operon was found immediately upstream of the poxAB operon, where poxB encodes a chromosomal ß-lactamase, hinting at the role of MifSR TCS in regulating antibiotic resistance. However, loss of mifSR had no effect on the antibiotic resistance profile when compared to P. aeruginosa parent PAO1 strain. Subsequently, our phenotypic microarray data (BioLOG) and growth profile studies indicated the inability of mifSR mutants to grow in α-ketoglutarate (α-KG), a key tricarboxylic acid (TCA) cycle intermediate, as a sole carbon source. To date, very little is known about the physiology of P. aeruginosa when provided with α-KG as its sole carbon source and the role of MifS and MifR TCS in virulence. Importantly, in the recent years, α-KG has gained notoriety for its newly identified role as a signaling molecule in addition to its conventional role in metabolism. This led us to hypothesize that MifSR TCS is involved in α-KG utilization and virulence in P. aeruginosa. Using mifS, mifR and mifSR clean in-frame deletion strains, our study demonstrates that the MifSR TCS modulates the expression P. aeruginosa kgtP (PA5530) and pcaT (PA0229) genes encoding putative α-KG permeases. In addition, our study shows that the MifSR-regulation of these transporters requires functional sigma factor RpoN (σ54). Loss of mifSR in the presence of α-KG, resulted in differential regulation of P. aeruginosa key virulence determinants including biofilm formation, motility, cell cytoxicity and the production of pyocyanin and pyoverdine. Involvement of multiple regulators and transporters suggests the presence of an intricate circuitry in the transport of α-KG and its importance in P. aeruginosa survival. This is further supported by the α-KG-dependent MifSR regulation of multiple virulence mechanisms. Simultaneous regulation of multiple mechanisms involved in P. aeruginosa pathogenesis suggests a complex mechanism of MifSR action. Understanding the physiological cues and regulation would provide a better stratagem to fight often indomitable P. aeruginosa infections.
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Quaranta, Davide. "Analysis and Molecular Characterization of an Unusual Copper Inducible Homeostasis Mechanism in Pseudomonas putida KT2440." Diss., The University of Arizona, 2009. http://hdl.handle.net/10150/194391.
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The purpose of this research was to identify and characterize novel molecular mechanisms in copper homeostasis. Pseudomonas putida KT2440 is a soil bacterium studied for its potential use in bioremediation of soils contaminated with aromatic organic contaminants. The cinAQ operon was analyzed. cinAQ is transcribed in presence of copper. The product of cinA is a periplasmic azurin-like protein with a methionine and histidine rich region, characterized by a high redox potential (456 ±4 mV). CinQ was shown to be a pyridine nucleotide-dependent nitrile oxidoreductase that catalyzes the reduction of preQ₀ to preQ₁, the first committed step in the biosynthetic pathway leading to the production of the unusual nucleotide queuosine. Gene disruption of cinQ in Pseudomonas putida KT2440 and in Pseudomonas aeruginosa PAO1 did not result in a significant increase in copper sensitivity on disk assays. Furthermore, a P. putida KT2440 cinA mutant also did not present a greater sensitivity to copper on disk assays while cinA mutants in Pseudomonas aeruginosa PAO1 presented increased toxicity to copper compared to the wild-type. CinA is by sequence similarity proposed to be an electron shuttle, and was shown to be upregulated in the presence of copper. Increasing CinA levels in the periplasm after copper stress may represent a mechanism used to regenerate the multicopper oxidase CopA (involved in Cu(I) to Cu(II) oxidation). Alternatively, CinA could act as an electron shuttle that takes part in an alternative electron transport chain once redox active copper is available, or it could represent a periplasmic copper chaperon. CinQ is involved in the biosynthesis of the rare hyper-modified nucleotide queuosine, found in the wobble position of several tRNAs, and required to avoid the readthrough of the stop codon UAG. Transcription of cinAQ was shown to be under the control of the two component system CinR-CinS. CinS is a histidine kinase, with a sensor domain located in the periplasm. CinR is the cognate response regulator that activates transcription of genes upon phosphorylation from CinS. The CinR-CinS two component system was shown to be responsive to 0.5 LM copper. CinS displayed very high metal specificity and elicited a response only in the presence of copper and silver, but not other metals. Modeling of the CinS protein structure, performed using Swiss Model and using the periplasmic sensor DcuS from Escherichia coli as a template, identified a potential copper binding site, containing H37 and H147. Sequence alignment of copper sensing histidine kinases further identified other conserved residues in the periplasmic domain. Site-Directed Mutagenesis was used to generate CinS mutants that were tested for their ability to activate the cinAQ promoter in presence of Cu. When challenged with copper CinS mutant H37R and H147R had an almost 10 fold reduction in copper sensitivity compared to the wild-type, indicating a possible role in Cu coordination. Other CinS mutants responded similarly to the wild-type in the presence of 10 μM of Cu.
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Kočan, Martina. "Regulation of the phosphate starvation response in Corynebacterium glutamicum by the PhoRS two-component system." Jülich : Forschungszentrum, Zentralbibliothek, 2005. http://deposit.d-nb.de/cgi-bin/dokserv?idn=973908785.
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Eversmeyer, Lauren Michelle. "TWO-COMPONENT REGULATORY SYSTEM IN PSEUDOMONAS AERUGINOSA: COMPLEMENTING RPEB IN WILD-TYPE AND NEGATIVE MUTANTS." Thesis, The University of Arizona, 2009. http://hdl.handle.net/10150/192321.
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Bourret, Travis John. "The PhoPQ two-component regulatory system : at the crossroads of nitrosative stress and Salmonella pathogenesis /." Connect to full text via ProQuest. Limited to UCD Anschutz Medical Campus, 2008. http://proquest.umi.com/pqdweb?did=1545957681&sid=1&Fmt=6&clientId=18952&RQT=309&VName=PQD.
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Thesis (Ph.D. in Microbiology) -- University of Colorado Denver, 2008.Typescript. Includes bibliographical references (leaves 112-132). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;
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Williams, Danielle A. "The AlgZ/R Two-Component System Is Responsible for Attenuation of Virulence in Pseudomonas aeruginosa." Digital Commons @ East Tennessee State University, 2017. https://dc.etsu.edu/etd/3340.
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Pseudomonas aeruginosa is an important opportunistic pathogen. Many P. aeruginosa virulence factors are regulated by the AlgZ/R two component system. AlgZ is the sensor histidine kinase which phosphorylates AlgR, the response regulator. AlgR activates transcription of different gene targets based upon its phosphorylation state. The genes that encode AlgZ and AlgR are transcribed in an operon. While regulation of algR expression has been well studied, regulation of algZ expression has not. Using a pilW mutant in concert with algZTF-lacZ transcriptional fusion, we conducted a transposon mutagenesis to identify algZ regulators. We identified an unknown autoregulatory loop. The type IV pilus minor pilins prevent the phosphorylation of AlgR by AlgZ . This inhibition of the AlgZ/R system subsequently down-regulates both the expression of the fimU operon and the algZ/R operon. Because AlgR regulates virulence, it is possible that virulence can also be reduced by targeting activation of the AlgZ/R system.
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Joshi, Gauri Suresh. "Regulation of CO2 fixation in Rhodopseudomonas palustris mediated by a unique two-component regulatory system." The Ohio State University, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=osu1273605616.
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Marszalek, Marta Anna. "The DosRST two-component system of Mycobacterium tuberculosis: Characterizing the activation mechanism of DosR response regulator as a potential target for novel antimycobacterial drugs." Doctoral thesis, Universitat Ramon Llull, 2014. http://hdl.handle.net/10803/275939.
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La tuberculosi, la malaltia infecciosa causada per Mycobacterium tuberculosis, es un problema de salut global que provoca aproximadament 2 milions de morts anuals. Un terç de la població mundial es troba crònicament infectada amb Mycobacterium tuberculosis però no mostra símptomes clínics encara que té un risc d’un 10% de desenvolupar la malaltia, la qual cosa representa un reservori incontrolable de tuberculosi. En aquesta condició asimptomàtica, coneguda com a tuberculosi latent, Mycobacterium tuberculosis es localitza en lesions granulomatoses a l’hoste i és resistent als medicaments antimicobacterians existents en l’actualitat.
En bacteris, els sistemes reguladors de dos components son un conjunt de proteïnes implicades en l’adaptació a canvis en l’entorn del microorganisme. Un sistema de dos components típic consta d’una histidina quinasa unida a membrana que té un paper essencial com a sensor dels canvis ambientals i un regulador transcripcional citosòlic que exerceix la seva funció controlant l’expressió de gens diana. Aquest parell de proteïnes funciona com un interruptor molecular que controla diferents respostes adaptatives a canvis a l’ambient cel•lular. Mycobacterium tuberculosis té 11 sistemes de dos components complets. El sistema DosRST, composat per un regulador transcripcional, DosR, i dues histidines quinases, DosS i DosT, té un paper estel•lar en l’adaptació de Mycobacterium tuberculosis a la tuberculosi latent. DosS i DosT s’autofosforilen en residus conservats d’histidina i ambdues proteïnes transfereixen aquesta un unitat de fosfat al residu d’àcid aspàrtic en posició 54 del regulador DosR. La fosforilació d’Asp54 és un interruptor que activa DosR i incrementa la seva afinitat per als promotors dels gens que regula. Les treonines 198 i 205 de DosR tenen un paper crucial en la dimerització de DosR i en la seva unió al DNA. Les dinàmiques moleculars realitzades amb la proteïna salvatge DosR i amb versions mutants mostren diferències notables en la formació del dímer actiu. També mostren una reducció o abolició completa de les interaccions proteïna-DNA a causa de les repulsions generades pels residus mutants carregats negativament. Encara més, les substitucions en Thr198 i Thr205 tenen un important efecte en la fosforilació química i enzimàtica de DosR així com també en la seva defosforilació catalitzada per DosS.
El sistema de dos components DosRST és una bona diana per al desenvolupament de nous compostos amb activitat antimicobacteriana contra formes dorments de Mycobacterium tuberculosis. S’ha iniciat un programa de recerca dirigit al desenvolupament d’inhibidors específics de la proteïna reguladors DosR, usant com a punt de partida compostos comercials estructuralment relacionats amb un derivat fenilcumarínic que ha estat descrit com a molècula que interfereix en la interacció DosR-DNA.La tuberculosis, la enfermedad infecciosa causada por Mycobacterium tuberculosis, es un problema de salud global que provoca aproximadamente 2 millones de muertes anuales. Un tercio de la población mundial se encuentra crónicamente infectada con Mycobacterium tuberculosis pero no muestra síntomas clínicos aunque tiene un riesgo de un 10% de desarrollar la enfermedad, lo que representa un reservorio incontrolable de tuberculosis. En esta condición asintomática, conocida como tuberculosis latente, Mycobacterium tuberculosis se localiza en lesiones granulomatosas en el huésped y es resistente a los medicamentos antimicobacterianos existentes en la actualidad. En bacterias, los sistemas regulatorios de dos componentes son un conjunto de proteínas implicadas en la adaptación a cambios en el entorno del microorganismo. Un sistema de dos componentes típico consta de una histidina quinasa unida a membrana que tiene un papel esencial como sensor de los cambios ambientales y un regulador transcripcional citosólico que ejerce su función controlando la expresión de genes diana. Este par de proteínas funciona como un interruptor molecular que controla distintas respuestas adaptativas a cambios en el ambiente celular. Mycobacterium tuberculosis tiene 11 sistemas de dos componentes completos. El sistema DosRST, compuesto por un regulador transcripcional, DosR, y dos histidinas quinasas, DosS y DosT, juega un papel estelar en la adaptación de Mycobacterium tuberculosis a la tuberculosis latente. DosS y DosT se autofosforilan en residuos conservados de histidina y ambas proteínas transfieren esta unidad de fosfato al residuo de ácido aspártico en posición 54 del regulador DosR. La fosforilación de Asp54 es un interruptor que activa a DosR e incrementa su afinidad por los promotores de los genes que regula. Las treoninas 198 y 205 de DosR juegan un papel crucial en la dimerización de DosR y en su unión al DNA. Las dinámicas moleculares realizadas con la proteína salvaje DosR y con versiones mutantes muestran diferencias notables en la formación del dímero activo. También muestran una reducción o abolición completa de las interacciones proteína-DNA a causa de las repulsiones generadas por los residuos mutantes cargados negativamente. Más aún, las sustituciones en Thr198 y Thr205 tienen un importante efecto en la fosforilación química y enzimática de DosR así como también en su defosforilaición catalizada por DosS. El sistema de dos componentes DosRST es una buena diana para el desarrollo de nuevos compuestos con actividad antimicobacteriana contra formas durmientes de Mycobacterium tuberculosis. Se ha iniciado un programa de investigación dirigido al desarrollo de inhibidores específicos de la proteína reguladora DosR, usando como punto de partida compuestos comerciales estructuralmente relacionados con un derivado fenilcumarínico que ha sido descrito como molécula que interfiere en la interacción DosR-DNA.
Tuberculosis, the infectious disease caused by Mycobacterium tuberculosis, is a global health problem with approximately two million deaths annually. One-third of the world population is chronically infected with Mycobacterium tuberculosis but do not show clinical symptoms although there is a 10% risk to development active disease, representing an uncontrollable reservoir of tuberculosis. In this asymptomatic condition, referred to as latent tuberculosis, Mycobacterium tuberculosis is located within granulomatous lesions in the host and is resistant to currently available antimycobacterial drugs. Two-component regulatory systems in bacteria are a major class of signal transduction proteins involved in adaptation to environmental changes. Typical system contains a membrane-bound histidine kinase that plays a crucial role in sensing environmental stimuli, and a cytosolic response regulator. This pair of proteins functions as a molecular switch that controls diverse adaptive environmental responses. Mycobacterium tuberculosis has eleven complete two-component systems. The DosRST system, composed of a response regulator, DosR, and two histidine kinases, DosS and DosT, plays a key role in Mycobacterium tuberculosis adaptation to latent tuberculosis. DosS and DosT autophosphorylate at conserved histidine residues and both proteins transfer this phosphor moiety to aspartic acid residue 54 of DosR. The phosphorylation of Asp54 serves as a switch to activate DosR and to increase the affinity for its cognate DNA promoters. Threonines 198 and 205 of DosR play a crucial role in DosR dimerization and DNA binding. The molecular dynamics with wild type and mutant version of DosR show different stability in the formation of the active DosR dimer. They also show the reduction or the abolishment of protein-DNA interactions because of the repulsions generated by negatively charged mutated residues. Moreover, substitutions in threonines 198 and 205 of DosR have a relevant effect on the chemical and enzymatic phosphorylation of DosR and on its DosS-catalysed dephosphorylation. The DosRST two-component system is a good target for the development of novel antimycobacterial drugs against dormant forms of M. tuberculosis. A structure-based discovery programme of inhibitors of DosR response regulator has been initiated with commercially available compounds showing a certain degree of similarity with a phenylcoumarin derivative previously described as DosR-DNA interfering molecule.
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Trinh, My. "Role of Two-Component System Response Regulators in Virulence of Streptococcus pneumoniae TIGR4 in Infective Endocarditis." VCU Scholars Compass, 2011. http://scholarscompass.vcu.edu/etd/2374.
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Streptococci resident in the oral cavity have been linked to infective endocarditis (IE). While viridans streptococci are commonly studied and associated with IE, less research has been focused on Streptococcus pneumoniae. Two-component systems (TCSs), consisting of a histidine kinase (HK) protein and response regulator (RR) protein, are bacterial signaling systems that may mediate S. pneumoniae TIGR4 strain virulence in IE. To test this hypothesis, TCS RR mutants of TIGR4 were examined in vivo through use of rabbit models. There were 14 RR proteins identified and 13 RR mutants synthesized because SP_1227 was found to be essential. The requirement of the 13 RRs for S. pneumoniae growth in IE models was assessed by quantifying mutants after overnight inoculation in IE infected rabbits through use of real time PCR (qPCR), colony enumeration on antibiotic selection plates, and competitive index assays. Real time PCR pinpointed several candidate virulence factors. Candidate RR SP_0798 was selected to be further examined. In the in vivo model, mutant SP_0798 grew significantly less than our control mutant SP_1678, which encodes a hypothetical protein and grew at a comparable rate to wild-type TIGR4 strains. Literature and databases identified SP_0798 as the ciaR gene, which has roles in regulating many diverse cellular functions. Our data suggests that RR SP_0798 is a virulence factor of S. pneumoniae TIGR4 strain in IE. This research may place more emphasis on virulence factors and lead to novel methods to combat pneumococcal endocarditis.
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Lucas, Darren Edward. "Coordinated Regulation of Salmonella Virulence Genes by the BarA/SirA Two-Component System and the Csr Global Regulatory System." The Ohio State University, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=osu1374087620.
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Flack, Caralyn E. "Mutagenesis and structural analysis of the Staphylococcus Aureus Sae two-component system reveals the intricate nature of virulence regulation." Diss., University of Iowa, 2014. https://ir.uiowa.edu/etd/2206.
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Two-component systems (TCSs) are highly conserved across bacteria and are used to rapidly sense and respond to changing environmental conditions. The human pathogen Staphylococcus aureus uses the S. aureus exoprotein expression (sae) TCS to sense host signals and activate transcription of virulence factors essential to pathogenesis. Despite its importance, the mechanism by which the sae sensor kinase SaeS recognizes specific host stimuli is unknown. This thesis describes topology and mutagenesis studies of the sensing domain of SaeS, including basal expression and inducer-dependent phenotypes. Meanwhile, investigation of the sae auxiliary protein SaeP has identified a novel DNA binding function for this surface expressed lipoprotein that may be involved in fine-tuning the activity of the sae system. Overall, these structure-function studies provide insight into the sae signal transduction mechanism and raise some new questions regarding the role the sae system plays in the larger regulatory network S. aureus uses to control expression of its secreted virulence factors.
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Gellatly, Shaan Lae. "Regulation of the PhoP-PhoQ two-component system in Pseudomonas aeruginosa and its role in virulence." Thesis, University of British Columbia, 2012. http://hdl.handle.net/2429/42991.
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Pseudomonas aeruginosa is an opportunistic bacterial pathogen that can cause severe
infections in individuals with underlying medical conditions. P. aeruginosa primarily infects at
epithelial surfaces where it interacts initially via type IV pili, flagella and LPS. Two component
regulatory systems control many aspects of pseudomonal physiology and mediate adaptation to
environmental changes including those that occur in the host. This thesis outlines the
contributions of these systems to the cytotoxicity to epithelial cells and sheds light on the
regulation mediated by the two-component sensor PhoQ. Systems that contributed to cytotoxicity
fell into several themes including motility, cyclic-di-GMP regulation, and carbon and nitrogen
utilization. Several genes controlled by PhoQ were shown to be dysregulated during infection of
lung epithelial cells, including upregulation of oprH-phoP-phoQ, the lipid A modification gene
arnB, and downregulation of a lipid A deacylase, pagL. Consistent with this, lipid A from a
phoQ mutant grown in varying magnesium concentrations displayed alterations. LPS of the
phoQ mutant revealed increased inflammatory properties as demonstrated by increased secretion
of the cytokines IL6, TNFalpha , and IL10 from PBMCs. The decrease in cytotoxicity of a phoQ
mutant correlated with a decrease in secretion of lipases and proteases when co-incubated with
cultured epithelial cells. These results suggest that the PhoP-PhoQ system might adapt the
bacterium to lung epithelia and that this might contribute to and be exacerbated by the selective
pressure of inhaled polymyxin therapeutics.
Unlike most sensor kinases that phosphorylate their cognate response regulators, PhoQ of P.
aeruginosa appears to act only as a phosphatase of its cognate regulator PhoP. Here it was
demonstrated that PhoP was not activated by PhoQ in a luminescence reporter screen. The sensor
kinase, RoxS, involved in regulation of the cyanide insensitive oxidase, was revealed as a
candidate phosphodonor to PhoP. Since mutation of roxS was able to reduce but not eliminate
expression from the oprH-phoP-phoQ operon, it is conceivable that other sensors contribute to
PhoP phosphorylation. It was also demonstrated that PhoP contributed to the known polymyxin
resistance of a phoQ mutant but only partially to cytotoxicity. These results emphasize the
complexity of the PhoP-PhoQ regulatory system.
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Palaniyandi, Senthilkumar. "Regulation of virulence by BarA-UvrY two-component system and LuxS in extraintestinal pathogenic (escherichia coli)." College Park, Md.: University of Maryland, 2007. http://hdl.handle.net/1903/7782.
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Thesis (M.S.) -- University of Maryland, College Park, 2007.Thesis research directed by: Virginia-Maryland Regional College of Veterinary Medicine. Maryland Campus. Title from t.p. of PDF. Includes bibliographical references. Published by UMI Dissertation Services, Ann Arbor, Mich. Also available in paper.
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Forson, Benedicta. "Biochemical Characterization of the Two-component Monooxygenase System; Isobutylamine N-hydroxylase (IBAH) and Flavin Reductase (FRED)." Thesis, Virginia Tech, 2016. http://hdl.handle.net/10919/81454.
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Isobutylamine N-hydroxylase (IBAH) and flavin reductase (FRED) from Streptomyces viridifaciens are part of a two-component flavin-dependent monooxygenase enzyme system that catalyze the conversion of isobutylamine (IBA) to isobutylhydroxylamine (IBHA), a key step in the formation of valanimycin, an azoxy antibiotic. In this work, we present the over-expression, purification and biochemical characterization of this two-component enzyme system. IBAH and FRED were expressed and purified to homogeneity as separate proteins. FRED exhibited the oxidoreductase activity by catalyzing the oxidation of NADPH. The hydroxylation activity of IBAH was confirmed using liquid chromatography – mass spectrometry (LC-MS). Steady state kinetic data showed an oxidation activity of the monooxygenase component which proceeded at 1.97 ± 0.06 s⁻¹ as measured from oxygen consumption and in product formation, the rate was 0.012 ± 0.001 s⁻¹ , suggesting a high degree of uncoupling between product formation and oxygen consumption. In pre-steady state kinetic characterization studies, the FRED-catalyzed reduction of FAD by NADPH occurred at a rate of 10.0 ± 0.2 s⁻¹ and the KM was 490 ± 40 µM. The rate of reduction was ~1.5-fold decreased in the presence of substrate IBA whiles the KM was 500 ± 50 µM. NADH showed a markedly reduced rate of reduction with a kred of 0.34 ± 0.03 s⁻¹ with an apparent KM of 3000 ± 500 µM. The rate of flavin re-oxidation in the absence of monooxygenase IBAH was 4.79 × 10⁻⁹ M-1 s⁻¹. Our results suggest a reaction mechanism for the IBAH monooxygenase system controlled by the oxidation half reaction that may be modulated by a complex formation between the reductase and monooxygenase components.Master of Science in Life Sciences
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Izumitsu, Kosuke. "Studies on two-component signaling system in osmotic adaptation and fungicide sensitivity of plant pathogenic fungi." Kyoto University, 2010. http://hdl.handle.net/2433/120463.
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Kyoto University (京都大学)0048
新制・課程博士
博士(農学)
甲第15420号
農博第1805号
新制||農||978(附属図書館)
学位論文||H22||N4519(農学部図書室)
27898
京都大学大学院農学研究科地域環境科学専攻
(主査)教授 二井 一禎, 教授 舟川 晋也, 教授 渡邊 隆司
学位規則第4条第1項該当
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Yeboah, Kwasi. "Identification of Transcription Regulators of the AlgZ/R Two-Components Regulatory System in Pseudomonas aeruginosa." Digital Commons @ East Tennessee State University, 2021. https://dc.etsu.edu/etd/3854.
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Pseudomonas aeruginosa is an opportunistic pathogen that express a plethora of virulence components controlled through two-component regulatory systems that allow for sensing and responding to environmental stimuli. This study was aimed at identifying transcription regulators of algZ that encodes the histidine sensor kinase (AlgZ) of the AlgZR two-component regulatory system. To understand how the algZ gene is transcriptionally controlled, transposon mutagenesis was used to create a mutant library with varying algZ expression based on their b-Galactosidase activity. The gene PA3327 was identified as a potential regulator of algZ expression using arbitrary PCR. This gene encodes a probable non-ribosomal peptide synthetase responsible for the biosynthesis of secondary metabolites such as antibiotics. Further experiments are required to understand how PA3327 transcriptionally regulates algZ expression and its physiological role in the organism. Because the AlgZ/R system regulates virulence, it is possible to attenuate virulence by targeting the expression of algZ gene.
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Tomkovič, Jiří [Verfasser], and Markus K. [Akademischer Betreuer] Oberthaler. "A driven two-component BEC: Chaos in a Macroscopic Quantum System / Jiří Tomkovič. Betreuer: Markus K. Oberthaler." Heidelberg : Universitätsbibliothek Heidelberg, 2013. http://d-nb.info/106105425X/34.
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Svensson, Sarah Lauren. "Molecular mechanisms of Campylobacter jejuni survival : characterization of the CprRS two-component regulatory system and biofilm formation." Thesis, University of British Columbia, 2012. http://hdl.handle.net/2429/42969.
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Schramke, Hannah [Verfasser], and Kirsten [Akademischer Betreuer] Jung. "Stimulus perception and signal transduction in the KdpD/KdpE two-component system / Hannah Schramke ; Betreuer: Kirsten Jung." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2016. http://d-nb.info/1137835346/34.
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Urbanic, Kevin William. "Identification and initial characterization of the MtrAB two-component signal transduction system of Mycobacterium avium subspecies paratuberculosis." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2001. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp04/MQ58384.pdf.
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Kolar, Stacey Lynn. "The Role and Regulation of NsaRS: a Cell-Envelope Stress Sensing Two-Component System in Staphylococcus aureus." Scholar Commons, 2012. http://scholarcommons.usf.edu/etd/4104.
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Abstract
S. aureus has 16 predicted two-component systems (TCS) that respond to a range of environmental stimuli, and allow for adaptation to stresses. Of these 16, three have no known function, and are not homologous to any other TCS found in closely related organisms. NsaRS is one such element, and belongs to the intramembrane-sensing histidine kinase (IM-HK) family, which is conserved within the Firmicutes. The regulators are defined by a small sensing domain within their histidine kinase, suggesting that they do not sense external signals, but stress in or at the membrane. Our characterization of NsaRS in this work reveals that, as with other IM-HK TCS, it responds to cell-envelope damaging antibiotics, including phosphomycin, ampicillin, nisin, gramicidin, CCCP and penicillin G. Additionally; we reveal that NsaRS regulates a downstream transporter, NsaAB, during nisin-induced stress. Phenotypically, nsaS mutants display a 200-fold decreased ability to develop resistance to another cell-wall targeting antibiotic, bacitracin. Microarray analysis reveals the transcription of 245 genes is altered in a nsaS mutant, with the vast majority down-regulated. Included within this list are genes involved in transport, drug-resistance, cell-envelope synthesis, transcriptional regulation, amino acid metabolism and virulence. Using ICP-MS, a decrease in intracellular divalent metal ions was observed in an nsaS mutant, when grown under low abundance conditions. Characterization of cells using electron microscopy reveals that nsaS mutants also have alterations in cell-envelope structure. Finally, a variety of virulence related phenotypes are impaired in nsaS mutants, including biofilm formation, resistance to killing by human macrophages and survival in whole human blood. Thus NsaRS is important in sensing cell wall damage in S. aureus, and functions to reprogram gene expression to modify cell-envelope architecture, facilitating adaptation and survival. Interestingly, in our microarray analysis, we observed a more than 30-fold decrease in transcription of an ABC transporter, SACOL2525/2526, in the nsaS mutant. This transporter bears strong homology to nsaAB, and is currently uncharacterized. Exploration of the role of SACOL2525/2526 revealed that, along with NsaRS, it too responds to cell-envelope damaging antibiotics. Specifically, its expression was induced by phosphomycin, daptomycin, penicillin G, ampicillin, oxacillin, D-cycloserine and CCCP. Mutation of this transporter results in increased sensitivity to the antibacterial agent daptomycin, and decreased sensitivity to free fatty acids. These findings are perhaps explained by altered membrane fluidity in the mutant strain, as the transporter null-strain is more readily killed in the presence of organic solvents, such as toluene. In addition, SACOL2525/2526 mutants have a decreased ability to form spontaneous mutants in response to several other peptidoglycan synthesis targeting antibiotics, suggesting a role for SACOL2525/2526 in antibiotic resistance. Inactivation of this transporter alters the cell envelope, and produces similar effects to those observed with the nsaS mutant, with increased capsule production, that may provide resistance to lysostaphin. Interestingly, the nsaS microarray revealed that this TCS negatively regulates only 34 genes, including 6 out of the 10 major secreted proteases. Despite a number of reports in the literature describing these enzymes as virulence factors, the data is often conflicting. Therefore, the contribution of proteases to CA-MRSA pathogenesis was investigated, by constructing a strain lacking all 10 extracellular protease genes. Analysis of this strain using murine models of infection reveals secreted proteases significantly impact virulence in both localized and systemic infections. Additionally, inactivation of these enzymes strongly influences survival in whole human blood, and increases sensitivity to antimicrobial peptides. Using a proteomics approach, we demonstrate that the contribution of secreted proteases to pathogenicity is related to differential processing of a large number of surface-associated virulence factors and secreted toxins. Collectively these findings provide a unique insight into the role of secreted proteases in CA-MRSA infections.
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Carlsson, Carin. "Modeling and Experimental Validation of a Rankine Cycle Based Exhaust WHR System for Heavy Duty Applications." Thesis, Linköpings universitet, Fordonssystem, 2012. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-81737.
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To increase the efficiency of the engine is one of the biggest challenges for heavy vehicles. One possible method is the Rankine based Waste Heat Recovery. Crucial for Rankine based Waste Heat Recovery is to model the temperature and the state of the working fluid. If the state of the working fluid is not determined, not only the efficiency of the system could be decreased, the components of thesystem might be damaged.A Simulink model based on the physical components in a system developed by Scania is proposed. The model for the complete system is validated against a reference model developed by Scania, and the component models are further validated against measurement data. The purpose of the model is to enable model based control, which is not possible with the reference model. The main focus on the thesis is to model the evaporation and condensation to determine state and temperature of the working fluid. The developed model is compared to a reference model with little differences for while stationary operating for both the components and the complete system. The developed model also follows the behavior from measurement data. The thesis shows that two phase modeling in Simulink is possible with models based on the physical components.
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Sakaida, Masaru. "Disorder-induced quantum phenomena in inhomogeneous optical lattices." 京都大学 (Kyoto University), 2016. http://hdl.handle.net/2433/215289.
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Allam, Sabry. "Acoustic modelling and testing of advanced exhaust system components for automotive engines." Doctoral thesis, KTH, Aeronautical and Vehicle Engineering, 2004. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-49.
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The increased use of the diesel engine in the passenger car, truck and bus market is due to high efficiency and lower fuel costs. This growing market share has brought with it several environmental issues for instance soot particle emission. Different technologies to remove the soot have been developed and are normally based on some kind of soot trap. In particular for automobiles the use of diesel particulate traps or filters (DPF:s) based on ceramic monolithic honeycombs are becoming a standard. This new exhaust system component will affect the acoustics and also work as a muffler. To properly design exhaust systems acoustic models for diesel particulate traps are needed. The first part of this thesis considers the modelling of sound transmission and attenuation for traps that consist of narrow channels separated by porous walls. This work has resulted in two new models an approximate 1-D model and a more complete model based on the governing equations for a visco-thermal fluid. Both models are expressed as acoustic 2-ports which makes them suitable for implementation in acoustic software for exhaust systems analysis. The models have been validated by experiments on clean filters at room temperature with flow and the agreement is good. In addition the developed filter models have been used to set up a model for a complete After Treatment Device (ATD) for a passenger car. The unit consisted of a chamber which contained both a diesel trap and a Catalytic Converter (CC). This complete model was also validated by experiments at room temperature. The second part of the thesis focuses on experimental techniques for plane wave decomposition in ducts with flow. Measurements in ducts with flow are difficult since flow noise (turbulence) can strongly influence the data. The difficulties are also evident from the lack of good published in-duct measurement data, e.g., muffler transmission loss data, for Mach-numbers above 0.1-0.2. The first paper in this part of the thesis investigates the effect of different microphone mountings and signal processing techniques for suppressing flow noise. The second paper investigates in particular flow noise suppression techniques in connection with the measurement of acoustic 2-ports. Finally, the third paper suggests a general wave decomposition procedure using microphone arrays and over-determination. This procedure can be used to determine the full plane wave data, e.g., the wave amplitudes and complex wave numbers k+ and k-. The new procedure has been applied to accurately measure the sound radiation from an unflanged pipe with flow. This problem is of interest for correctly determining the radiated power from an engine exhaust outlet. The measured data for the reflection coefficient and end correction have been compared with the theory of Munt [33] and the agreement is excellent. The measurements also produced data for the damping value (imaginary part of the wavenumber) which were compared to a model suggested by Howe [13]. The agreement is good for a normalized boundary layer thickness less than 30-40
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Occhino, Deborah Ann. "Vibrio cholerae iron transport : characterization of two tonB systems and components of a heme transport system /." Digital version accessible at:, 1998. http://wwwlib.umi.com/cr/utexas/main.
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