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1
Michel, Gérard P. F., Anthony Aguzzi, Geneviève Ball, Chantal Soscia, Sophie Bleves, and Romé Voulhoux. "Role of fimV in type II secretion system-dependent protein secretion of Pseudomonas aeruginosa on solid medium." Microbiology 157, no. 7 (July 1, 2011): 1945–54. http://dx.doi.org/10.1099/mic.0.045849-0.
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Although classical type II secretion systems (T2SSs) are widely present in Gram-negative bacteria, atypical T2SSs can be found in some species. In Pseudomonas aeruginosa, in addition to the classical T2SS Xcp, it was reported that two genes, xphA and xqhA, located outside the xcp locus were organized in an operon (PaQa) which encodes the orphan PaQa subunit. This subunit is able to associate with other components of the classical Xcp machinery to form a functional hybrid T2SS. In the present study, using a transcriptional lacZ fusion, we found that the PaQa operon was more efficiently expressed (i) on solid LB agar than in liquid LB medium, (ii) at 25 °C than at 37 °C and (iii) at an early stage of growth. These results suggested an adaptation of the hybrid system to particular environmental conditions. Transposon mutagenesis led to the finding that vfr and fimV genes are required for optimal expression of the orphan PaQa operon in the defined growth conditions used. Using an original culturing device designed to monitor secretion on solid medium, the ring-plate system, we found that T2SS-dependent secretion of exoproteins, namely the elastase LasB, was affected in a fimV deletion mutant. Our findings led to the discovery of an interplay between FimV and the global regulator Vfr triggering the modulation of the level of Vfr and consequently the modulation of T2SS-dependent secretion on solid medium.
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Douzi, Badreddine, Alain Filloux, and Romé Voulhoux. "On the path to uncover the bacterial type II secretion system." Philosophical Transactions of the Royal Society B: Biological Sciences 367, no. 1592 (April 19, 2012): 1059–72. http://dx.doi.org/10.1098/rstb.2011.0204.
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Gram-negative bacteria have evolved several secretory pathways to release enzymes or toxins into the surrounding environment or into the target cells. The type II secretion system (T2SS) is conserved in Gram-negative bacteria and involves a set of 12 to 16 different proteins. Components of the T2SS are located in both the inner and outer membranes where they assemble into a supramolecular complex spanning the bacterial envelope, also called the secreton. The T2SS substrates transiently go through the periplasm before they are translocated across the outer membrane and exposed to the extracellular milieu. The T2SS is unique in its ability to promote secretion of large and sometimes multimeric proteins that are folded in the periplasm. The present review describes recently identified protein–protein interactions together with structural and functional advances in the field that have contributed to improve our understanding on how the type II secretion apparatus assembles and on the role played by individual proteins of this highly sophisticated system.
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Strozen, Timothy G., Gang Li, and S. Peter Howard. "YghG (GspSβ) Is a Novel Pilot Protein Required for Localization of the GspSβType II Secretion System Secretin of Enterotoxigenic Escherichia coli." Infection and Immunity 80, no. 8 (May 14, 2012): 2608–22. http://dx.doi.org/10.1128/iai.06394-11.
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ABSTRACTThe enterotoxigenicEscherichia coli(ETEC) pathotype, characterized by the prototypical strain H10407, is a leading cause of morbidity and mortality in the developing world. A major virulence factor of ETEC is the type II secretion system (T2SS) responsible for secretion of the diarrheagenic heat-labile enterotoxin (LT). In this study, we have characterized the two type II secretion systems, designated alpha (T2SSα) and beta (T2SSβ), encoded in the H10407 genome and describe the prevalence of both systems in otherE. colipathotypes. Under laboratory conditions, the T2SSβis assembled and functional in the secretion of LT into culture supernatant, whereas the T2SSαis not. Insertional inactivation of the three genes located upstream ofgspCβ(yghJ,pppA, andyghG) in the atypical T2SSβoperon revealed that YghJ is not required for assembly of the GspDβsecretin or secretion of LT, that PppA is likely the prepilin peptidase required for the function of T2SSβ, and that YghG is required for assembly of the GspDβsecretin and thus function of the T2SSβ. Mutational and physiological analysis further demonstrated that YghG (redesignated GspSβ) is a novel outer membrane pilotin protein that is integral for assembly of the T2SSβby localizing GspDβto the outer membrane, whereupon GspDβforms the macromolecular secretin multimer through which T2SSβsubstrates are translocated.
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Michel, Gérard P. F., Eric Durand, and Alain Filloux. "XphA/XqhA, a Novel GspCD Subunit for Type II Secretion in Pseudomonas aeruginosa." Journal of Bacteriology 189, no. 10 (March 9, 2007): 3776–83. http://dx.doi.org/10.1128/jb.00205-07.
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ABSTRACT The opportunistic human pathogen bacterium Pseudomonas aeruginosa secretes various exoproteins in its surrounding environment. Protein secretion involves different secretory systems, including the type II secretion system, or T2SS, that is one of the most efficient secretory pathways of P. aeruginosa. There are two T2SS in this bacterium, the quorum-sensing-regulated Xcp system and the Hxc system, which is only present under phosphate-limiting conditions. Like T2SS of other bacteria, the Xcp T2SS is species specific, and this specificity mainly involves two proteins, XcpP (GspC family) and the secretin XcpQ (GspD family), which are the gatekeepers of the system. Interestingly, an orphan secretin, XqhA, was previously reported as being able to functionally replace the XcpQ secretin. In this study, we identified another gene, which we named xphA (xcpP homologue A), which is located next to xqhA. We showed that deletion of the xphA gene in an xcpP mutant caused the disappearance of the residual secretion observed in this mutant strain, indicating that the protein XphA plays a role in the secretion process. Our results also revealed that complementation of an xcpP/xcpQ mutant can be obtained with the gene couple xphA/xqhA. The XphA and XqhA proteins (the PAQA subunit) could thus form, together with XcpR-Z, a functional hybrid T2SS. A two-dimensional polyacrylamide gel electrophoresis analysis showed that except for the aminopeptidase PaAP, for which secretion is not restored by the PAQA subunit in the xcpP/xcpQ deletion mutant, each major Xcp-dependent exoprotein is secreted by the new hybrid machinery. Our work supports the idea that components of the GspC/GspD families, such as XphA/XqhA or XcpP/XcpQ, are assembled as a specific tandem within the T2SS. Each of these pairs may thus confer a different level of secretion specificity, as is the case with respect to PaAP. Finally, using a chromosomal xphA-lacZ fusion, we showed that the xphA-xqhA genes are transcribed from an early stage of bacterial growth. We thus suggest that the PAQA subunit might be involved in the secretion process at a different growth stage than XcpP/XcpQ.
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Burtnick, Mary N., Paul J. Brett, and David DeShazer. "Proteomic Analysis of the Burkholderia pseudomallei Type II Secretome Reveals Hydrolytic Enzymes, Novel Proteins, and the Deubiquitinase TssM." Infection and Immunity 82, no. 8 (May 27, 2014): 3214–26. http://dx.doi.org/10.1128/iai.01739-14.
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ABSTRACTBurkholderia pseudomallei, the etiologic agent of melioidosis, is an opportunistic pathogen that harbors a wide array of secretion systems, including a type II secretion system (T2SS), three type III secretion systems (T3SS), and six type VI secretion systems (T6SS). The proteins exported by these systems provideB. pseudomalleiwith a growth advantagein vitroandin vivo, but relatively little is known about the full repertoire of exoproducts associated with each system. In this study, we constructed deletion mutations ingspDandgspE, T2SS genes encoding an outer membrane secretin and a cytoplasmic ATPase, respectively. The secretion profiles ofB. pseudomalleiMSHR668 and its T2SS mutants were noticeably different when analyzed by SDS-PAGE. We utilized liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify proteins present in the supernatants ofB. pseudomalleiMSHR668 andB. pseudomalleiΔgspDgrown in rich and minimal media. The MSHR668 supernatants contained 48 proteins that were either absent or substantially reduced in the supernatants of ΔgspDstrains. Many of these proteins were putative hydrolytic enzymes, including 12 proteases, two phospholipases, and a chitinase. Biochemical assays validated the LC-MS/MS results and demonstrated that the export of protease, phospholipase C, and chitinase activities is T2SS dependent. Previous studies had failed to identify the mechanism of secretion of TssM, a deubiquitinase that plays an integral role in regulating the innate immune response. Here we present evidence that TssM harbors an atypical signal sequence and that its secretion is mediated by the T2SS. This study provides the first in-depth characterization of theB. pseudomalleiT2SS secretome.
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Lu, Connie, Young-un Park, Konstantin Korotkov, Wei Mi, Stewart Turley, Veer Bhatt, Ripal Shah, and Wim Hol. "Multiple approaches towards understanding the type II secretion system." Acta Crystallographica Section A Foundations and Advances 70, a1 (August 5, 2014): C577. http://dx.doi.org/10.1107/s2053273314094224.
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Transport of folded proteins across membranes is a feat accomplished by few biomacromolecular machines. One of the machineries able to do so is the sophisticated type II secretion system (T2SS). It can translocate key virulence factors from the bacterial periplasm into the lumen of the gut of the human host. A prime example is the secretion of cholera toxin by Vibrio cholerae. The T2SS consists of ~12 different proteins, most of these present in multiple copies, organized into three subassemblies: (i) the Inner Membrane Platform; (ii) the Pseudopilus in the periplasm, which acts most likely as a piston pushing exoproteins through the outer membrane pore; (iii) the Outer Membrane Complex, allowing passage of ~100 kDa folded proteins. We have determined crystal structures from more than a dozen T2SS domains, yet, a full understanding of the architecture and mechanism of action of the T2SS remains a formidable challenge. Our approaches include the use of "assistant-multimers" to promote recalcitrant multimer formation and of nanobodies to overcome reluctant crystal formation. The Inner Membrane Platform is interacting with the secretion ATPase GspE which most likely needs to be hexameric for full activity. Full-length GspE co-crystallized with its major partner, the cytoplasmic domain of GspL, revealed a tremendous flexibility of this ATPase, and, most unexpectedly, also the organization of the same linear arrangement of cyto-GspL domains throughout three entirely different crystal forms. Two very different hexamers of GspE were elucidated by linking the GspE subunit to the subunit of Hcp1, which successfully acted as an "assistant hexamer", inducing hexamer formation by GspE. The dodecameric nature of the ~ 850 kDa GspD, the major component of the Outer Membrane Complex, evident in earlier electron microscopy studies, was observed in the dodecameric ring-like helix in crystals of its N-terminal domain. The contacts between GspD and the inner-membrane protein GspC will be discussed as well as the remarkably frequent occurrence of dimers of Inner Membrane Platform domains. How dimers are co-assembled with an ATPase hexamer with C6 symmetry and the Outer Membrane Complex dodecamer with C12 symmetry remains one of the many fascinating outstanding questions of the T2SS.
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Maltz, Michele, and Joerg Graf. "The Type II Secretion System Is Essential for Erythrocyte Lysis and Gut Colonization by the Leech Digestive Tract SymbiontAeromonas veronii." Applied and Environmental Microbiology 77, no. 2 (November 19, 2010): 597–603. http://dx.doi.org/10.1128/aem.01621-10.
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ABSTRACTHemolysin and the type II secretion system (T2SS) have been shown to be important for virulence in many pathogens, but very few studies have shown their importance in beneficial microbes. Here, we investigated the importance of the type II secretion pathway in the beneficial digestive-tract association ofAeromonas veroniiand the medicinal leechHirudo verbanaand revealed a critical role for the hemolysis of erythrocytes. A mutant with a miniTn5insertion inexeM, which is involved in forming the inner membrane platform in the T2SS, was isolated by screening mutants for loss of hemolysis on blood agar plates. A hemolysis assay was used to quantify the mutant's deficiency in lysing sheep erythrocytes and revealed a 99.9% decrease compared to the parent strain. The importance of the T2SS in the colonization of the symbiotic host was assessed. Colonization assays revealed that the T2SS is critical for initial colonization of the leech gut. The defect was tied to the loss of hemolysin production by performing a colonization assay with blood containing lysed erythrocytes. This restored the colonization defect in the mutant. Complementation of the mutant using the promoter region andexeMNrevealed that the T2SS is responsible for secreting hemolysin into the extracellular space and that both the T2SS and hemolysin export by the T2SS are critical for initial establishment ofA. veroniiin the leech gut.
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Zhang, Yichen, Frederick Faucher, and Zongchao Jia. "Insights into minor pseudopilin complexes of the Type II secretion system." Acta Crystallographica Section A Foundations and Advances 70, a1 (August 5, 2014): C585. http://dx.doi.org/10.1107/s2053273314094145.
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The type II secretion system (T2SS) is sophisticated multiprotein machinery that enables Gram-negative pathogens to secrete a wide range of exoproteins, named virulence factors, into the extracellular environments. In Pseudomonas aeruginosa, the Xcp T2SS is responsible for secreting many virulence factors that induce severe infections. In T2SS, the recognition and binding of secreted exoproteins are conducted by a structure called the pseudopilius tip, which is formed by four minor pseudopilins, including XcpU, XcpV, XcpW and XcpX. These minor pseudopilins form a quaternary complex, which is also involved in the initiation and regulation of the pseudopilus assembly. Although individual structures of these four pseudopilins have been revealed in different organisms, the substrate recognition and binding mechanisms have not been clearly elucidated due to the lack of systematic studies on the whole structures of several complexes formed by these pseudopilins. As a result, the understanding of the structures of these protein complexes will provide useful information for unveiling the mystery of the recognition and binding mechanisms. The establishment of the substrate binding model requires the preparation of stable complex(es) of substrates and certain minor pseudopilin(s). In this work, we aim to gradually elucidate the secretion mechanisms by assembling each component to build up the whole architecture. The structure of XcpV in complex with XcpW has been determined, and other complexes, especially the XcpU-containing binary and ternary complexes, have been stably established and purified. The identification of these complex structures will significantly promote our understandings of the type II secretion mechanisms.
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Baldi, Deborah L., Ellen E. Higginson, Dianna M. Hocking, Judyta Praszkier, Rosalia Cavaliere, Catherine E. James, Vicki Bennett-Wood, et al. "The Type II Secretion System and Its Ubiquitous Lipoprotein Substrate, SslE, Are Required for Biofilm Formation and Virulence of Enteropathogenic Escherichia coli." Infection and Immunity 80, no. 6 (March 26, 2012): 2042–52. http://dx.doi.org/10.1128/iai.06160-11.
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ABSTRACTEnteropathogenicEscherichia coli(EPEC) is a major cause of diarrhea in infants in developing countries. We have identified a functional type II secretion system (T2SS) in EPEC that is homologous to the pathway responsible for the secretion of heat-labile enterotoxin by enterotoxigenicE. coli. The wild-type EPEC T2SS was able to secrete a heat-labile enterotoxin reporter, but an isogenic T2SS mutant could not. We showed that the major substrate of the T2SS in EPEC is SslE, an outer membrane lipoprotein (formerly known as YghJ), and that a functional T2SS is essential for biofilm formation by EPEC. T2SS and SslE mutants were arrested at the microcolony stage of biofilm formation, suggesting that the T2SS is involved in the development of mature biofilms and that SslE is a dominant effector of biofilm development. Moreover, the T2SS was required for virulence, as infection of rabbits with a rabbit-specific EPEC strain carrying a mutation in either the T2SS or SslE resulted in significantly reduced intestinal colonization and milder disease.
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Shi, Liang, Shuang Deng, Matthew J. Marshall, Zheming Wang, David W. Kennedy, Alice C. Dohnalkova, Heather M. Mottaz, et al. "Direct Involvement of Type II Secretion System in Extracellular Translocation of Shewanella oneidensis Outer Membrane Cytochromes MtrC and OmcA." Journal of Bacteriology 190, no. 15 (May 23, 2008): 5512–16. http://dx.doi.org/10.1128/jb.00514-08.
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ABSTRACT MtrC and OmcA are cell surface-exposed lipoproteins important for reducing solid metal oxides. Deletions of type II secretion system (T2SS) genes reduced their extracellular release and their accessibility to the proteinase K treatment, demonstrating the direct involvement of T2SS in translocation of MtrC and OmcA to the bacterial cell surface.
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Hwang, Won, Na Yeon Lee, Juri Kim, Mi-Ae Lee, Kun-Soo Kim, Kyu-Ho Lee, and Soon-Jung Park. "Functional Characterization of EpsC, a Component of the Type II Secretion System, in the Pathogenicity of Vibrio vulnificus." Infection and Immunity 79, no. 10 (July 25, 2011): 4068–80. http://dx.doi.org/10.1128/iai.05351-11.
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ABSTRACTEpsC, one of the components comprising the type II secretion system (T2SS), was isolated from a human-pathogenic bacterium,Vibrio vulnificus, to evaluate its role in eliciting virulence. AnespC-deleted mutant ofV. vulnificusdisplayed a reduced cytotoxicity to the human cell line HEp-2 and an attenuated virulence in a mouse model. This mutant exhibited dramatic defects in the secretion of diverse extracellular proteins, such as outer membrane proteins, transporters, and the known secreted factors, notably, a hemolysin (VvhA) and an elastase (VvpE). A defect in its secretion of proteins was restored by intranscomplementation of the intactepsCgene. Analyses of cellular fractions revealed that VvhA and VvpE of theΔepsCmutant were not excreted outside the cell but were present mainly in the periplasmic space. Examination of aV. vulnificusmutant deficient in TolC, a component of the T1SS, showed that it is not involved in the secretion of VvhA and VvpE but that it is necessary for the secretion of another major toxin ofV. vulnificus, RtxA. Therefore, the T2SS is required forV. vulnificuspathogenicity, which is mediated by at least two secreted factors, VvhA and VvpE, via facilitating the secretion and exposure of these factors to host cells.
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Rivas, Amable J., Ana Vences, Matthias Husmann, Manuel L. Lemos, and Carlos R. Osorio. "Photobacterium damselae subsp. damselae Major Virulence Factors Dly, Plasmid-Encoded HlyA, and Chromosome-Encoded HlyA Are Secreted via the Type II Secretion System." Infection and Immunity 83, no. 4 (January 12, 2015): 1246–56. http://dx.doi.org/10.1128/iai.02608-14.
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Photobacterium damselaesubsp.damselaeis a marine bacterium that causes septicemia in marine animals and in humans. Previously, we had determined a major role of pPHDD1 plasmid-encoded Dly (damselysin) and HlyA (HlyApl) and the chromosome-encoded HlyA (HlyAch) hemolysins in virulence. However, the mechanisms by which these toxins are secreted remain unknown. In this study, we found that a mini-Tn10transposon mutant in a plasmidless strain showing an impaired hemolytic phenotype contained an insertion inepsL, a component of a type II secretion system (T2SS). Reconstruction of the mutant by allelic exchange confirmed the specific involvement ofepsLin HlyAchsecretion. In addition, mutation ofepsLin a pPHDD1-harboring strain caused an almost complete abolition of hemolytic activity against sheep erythrocytes, indicating thatepsLplays a major role in secretion of the plasmid-encoded HlyApland Dly. This was further demonstrated by analysis of different combinations of hemolysin gene mutants and by strain-strain complementation assays. We also found that mutation of the putative prepilin peptidase genepilDseverely affected hemolysis, which dropped at levels inferior to those ofepsLmutants. Promoter expression analyses suggested that impairment of hemolysin secretion inepsLandpilDmutants might constitute a signal that affects hemolysin and T2SS gene expression at the transcriptional level. In addition, singleepsLandpilDmutations caused a drastic decrease in virulence for mice, demonstrating a major role of T2SS andpilDinP. damselaesubsp.damselaevirulence.
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Lewenza, Shawn, Laetitia Charron-Mazenod, Shirin Afroj, and Erik van Tilburg Bernardes. "Hyperbiofilm phenotype of Pseudomonas aeruginosa defective for the PlcB and PlcN secreted phospholipases." Canadian Journal of Microbiology 63, no. 9 (September 2017): 780–87. http://dx.doi.org/10.1139/cjm-2017-0244.
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Biofilms are dense communities of bacteria enmeshed in a protective extracellular matrix composed mainly of exopolysaccharides, extracellular DNA, proteins, and outer membrane vesicles (OMVs). Given the role of biofilms in antibiotic-tolerant and chronic infections, novel strategies are needed to block, disperse, or degrade biofilms. Enzymes that degrade the biofilm matrix are a promising new therapy. We screened mutants in many of the enzymes secreted by the type II secretion system (T2SS) and determined that the T2SS, and specifically phospholipases, play a role in biofilm formation. Mutations in the xcp secretion system and in the plcB and plcN phospholipases all resulted in hyperbiofilm phenotypes. PlcB has activity against many phospholipids, including the common bacterial membrane lipid phosphatidylethanolamine, and may degrade cell membrane debris or OMVs in the biofilm matrix. Exogenous phospholipase was shown to reduce aggregation and biofilm formation, suggesting its potential role as a novel enzymatic treatment to dissolve biofilms.
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Xiao, Ke, Chuanjun Shu, Qin Yan, and Xiao Sun. "Predicting Homogeneous Pilus Structure from Monomeric Data and Sparse Constraints." BioMed Research International 2015 (2015): 1–12. http://dx.doi.org/10.1155/2015/817134.
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Type IV pili (T4P) and T2SS (Type II Secretion System) pseudopili are filaments extending beyond microbial surfaces, comprising homologous subunits called “pilins.” In this paper, we presented a new approach to predict pseudo atomic models of pili combining ambiguous symmetric constraints with sparse distance information obtained from experiments and based neither on electronic microscope (EM) maps nor on accuratea priorisymmetric details. The approach was validated by the reconstruction of the gonococcal (GC) pilus fromNeisseria gonorrhoeae, the type IVb toxin-coregulated pilus (TCP) fromVibrio cholerae, and pseudopilus of the pullulanase T2SS (the PulG pilus) fromKlebsiella oxytoca. In addition, analyses of computational errors showed that subunits should be treated cautiously, as they are slightly flexible and not strictly rigid bodies. A global sampling in a wider range was also implemented and implied that a pilus might have more than one but fewer than many possible intact conformations.
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Fagard, Mathilde, Alia Dellagi, Camille Roux, Claude Périno, Martine Rigault, Virginie Boucher, Vladimir E. Shevchik, and Dominique Expert. "Arabidopsis thaliana Expresses Multiple Lines of Defense to Counterattack Erwinia chrysanthemi." Molecular Plant-Microbe Interactions® 20, no. 7 (July 2007): 794–805. http://dx.doi.org/10.1094/mpmi-20-7-0794.
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Many taxonomically diverse plant species are attacked by Erwinia chrysanthemi, a member of the causal agents of soft-rotting diseases. Symptom development is due to the collective action of pectin-degrading enzymes secreted by the bacterium through a type II secretion system (T2SS). Using Arabidopsis thaliana as a susceptible host, we show that plants respond to E. chrysanthemi 3937 by expressing cell-wall reactions, production of an oxidative burst, and activation of salicylic acid (SA) and jasmonic acid (JA) or ethylene (ET) signaling pathways. We found that the oxidative burst is mainly generated via the expression of the AtrbohD gene, constitutes a barrier of resistance to bacterial attack, and acts independently of the SA-mediated response. To determine the importance of T2SS-secreted proteins in elicitation of these defenses, we used a T2SS deficient mutant and purified enzymatic preparations of representative members of strain 3937 pectate lyase activity. The T2SS-secreted proteins were responsible only partially for the activation of SA and JA or ET signaling pathways observed after infection with the wild-type bacterium and were not involved in the expression of other identified defense reactions. Our study shows the differential role played by pectate lyases isoenzymes in this process and highlights the complexity of the host immune network, which is finely controlled by the bacterium.
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Zhao, Youfu, George W. Sundin, and Dongping Wang. "Construction and analysis of pathogenicity island deletion mutants of Erwinia amylovora." Canadian Journal of Microbiology 55, no. 4 (April 2009): 457–64. http://dx.doi.org/10.1139/w08-147.
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An easy gene-knockout technique, PCR-based one-step inactivation of chromosomal genes, is widely used in Escherichia coli and related enterobacteria to construct mutants. In this study, we adapted this technique to construct genomic island and large operon deletion mutants of Erwinia amylovora , including the 33.4 kb hrp-type III secretion (T3SS) pathogenicity island (PAI1) and the 15.8 kb amylovoran biosynthesis (AMS) operon. Deletion of 2 novel T3SS pathogenicity islands (PAI2 and PAI3) and an operon encoding a type II secretion system (T2SS) demonstrated that these determinants are not involved in virulence in plants. Co-inoculation experiments demonstrated that the hrp-T3SS and AMS deletion mutants could complement each other. These results further confirmed that the one-step inactivation technique can be used to generate large deletions in E. amylovora.
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Liu, Huanli, Shuping Zhang, Mark A. Schell, and Timothy P. Denny. "Pyramiding Unmarked Deletions in Ralstonia solanacearum Shows That Secreted Proteins in Addition to Plant Cell-Wall-Degrading Enzymes Contribute to Virulence." Molecular Plant-Microbe Interactions® 18, no. 12 (December 2005): 1296–305. http://dx.doi.org/10.1094/mpmi-18-1296.
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Ralstonia solanacearum, like many phytopathogenic bacteria, makes multiple extracellular plant cell-wall-degrading enzymes (CWDE), some of which contribute to its ability to cause wilt disease. CWDE and many other proteins are secreted to the milieu via the highly conserved type II protein secretion system (T2SS). R. solanacearum with a defective T2SS is weakly virulent, but it is not known whether this is due to absence of all the CWDE or the loss of other secreted proteins that contribute to disease. These alternatives were investigated by creating mutants of wild-type strain GMI1000 lacking either the T2SS or up to six CWDE and comparing them for virulence on tomato plants. To create unmarked deletions, genomic regions flanking the target gene were polymerase chain reaction (PCR)-amplified, were fused using splice overlap extension PCR, were cloned into a suicide plasmid harboring the sacB counter-selectable marker, and then, were site-specifically introduced into the genome. Various combinations of five deletions (δpehA, δpehB, δpehC, δpme, and δegl) and one inactivated allele (cbhA::aphA-3) resulted in 15 mutants missing one to six CWDE. In soil-drench inoculation assays, virulence of mutants lacking only pectic enzymes (PehA, PehB, PehC, and Pme) was not statistically different from GMI1000, but all the mutants lacking one or both cellulolytic enzymes (Egl or CbhA) wilted plants significantly more slowly than did the wild type. The GMI-6 mutant that lacks all six CWDE was more virulent than the mutant lacking only its two cellulolytic enzymes, and both were significantly more virulent than the T2SS mutant (GMI-D). Very similar results were observed in wounded-petiole inoculation assays, so GMI-6 and GMI-D appear to be less capable of colonizing tomato tissues after invasion. Because the T2SS mutant was much less virulent than the sixfold CWDE mutant, we conclude that other secreted proteins contribute substantially to the ability of R. solanacearum GMI1000 to systemically colonize tomato plants.
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Yang, Ping, Fang-Jing Li, Shi-Wen Huang, Man Luo, Wei Lin, Gao-Qing Yuan, and Qi-Qin Li. "Physiological and Transcriptional Response of Xanthomonas oryzae pv. oryzae to Berberine, an Emerging Chemical Control." Phytopathology® 110, no. 5 (May 2020): 1027–38. http://dx.doi.org/10.1094/phyto-09-19-0327-r.
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Berberine, a botanical drug, has great ability to inhibit the growth of Xanthomonas oryzae pv. oryzae. However, the antibacterial mechanism of berberine against X. oryzae pv. oryzae remains poorly understood. In this study, we investigated the physiological and transcriptional response of X. oryzae pv. oryzae to berberine. When strain X. oryzae pv. oryzae GX13 was treated with berberine (10 µg/ml), the hypersensitive response in tobacco, virulence to rice, pathogen population in the rice xylem, production of extracellular polysaccharide (EPS), and activity of extracellular hydrolases decreased, but the levels of pyruvate and ATP increased. Moreover, biofilm formation was inhibited, and the cell membrane was damaged. Transcriptome sequencing analysis showed downregulated expression of gspD, gspE, and gspF, involved in the type II secretion system (T2SS); hrcC, hrcJ, hrcN, and others, involved in the type III secretion system (T3SS); gumB and gumC, associated with EPS; zapE, ftsQ, and zapA, associated with cell division; lpxH, lpxK, kdtA, and others, associated with the membrane; and pyk, pgk, and mdh, encoding pyruvate kinase, phosphoglycerate kinase, and malate dehydrogenase, respectively. Upregulated expression was observed for nuoA, nuoB, and nuoH, encoding the NADH dehydrogenase complex, and atpF, atpC, and atpB, encoding ATP synthase. An adenylate cyclase (CyaA) fusion assay showed that berberine affects type three effector protein secretion via the T3SS and reduces effector translocation in X. oryzae pv. oryzae. It is speculated that the negative growth and virulence phenotypes of berberine-treated X. oryzae pv. oryzae GX13 may involve differentially expressed genes associated with cytoarchitecture and energy metabolism, and these effects on primary cell function may further dampen virulence and result in differential expression of T3SS- and T2SS-related genes.
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Endoh, Takayuki, and Joanne N. Engel. "CbpA: a Polarly Localized Novel Cyclic AMP-Binding Protein in Pseudomonas aeruginosa." Journal of Bacteriology 191, no. 23 (October 2, 2009): 7193–205. http://dx.doi.org/10.1128/jb.00970-09.
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ABSTRACT In Pseudomonas aeruginosa, cyclic AMP (cAMP) signaling regulates the transcription of hundreds of genes encoding diverse virulence factors, including the type II secretion system (T2SS) and type III secretion system (T3SS) and their associated toxins, type IV pili (TFP), and flagella. Vfr, a cAMP-dependent transcriptional regulator that is homologous to the Escherichia coli catabolite repressor protein, is thought to be the major cAMP-binding protein that regulates these important virulence determinants. Using a bioinformatic approach, we have identified a gene (PA4704) encoding an additional putative cAMP-binding protein in P. aeruginosa PAO1, which we herein refer to as CbpA, for cAMP-binding protein A. Structural modeling predicts that CbpA is composed of a C-terminal cAMP-binding (CAP) domain and an N-terminal degenerate CAP domain and is structurally similar to eukaryotic protein kinase A regulatory subunits. We show that CbpA binds to cAMP-conjugated agarose via its C-terminal CAP domain. Using in vitro trypsin protection assays, we demonstrate that CbpA undergoes a conformational change upon cAMP binding. Reporter gene assays and electrophoresis mobility shift assays defined the cbpA promoter and a Vfr-binding site that are necessary for Vfr-dependent transcription. Although CbpA is highly regulated by Vfr, deletion of cbpA did not affect known Vfr-dependent functions, including the T2SS, the T3SS, flagellum- or TFP-dependent motility, virulence in a mouse model of acute pneumonia, or protein expression profiles. Unexpectedly, CbpA-green fluorescent protein was found to be localized to the flagellated old cell pole in a cAMP-dependent manner. These results suggest that polar localization of CbpA may be important for its function.
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Balloy, V., J. Jyot, G. Jouvion, A. Verma, M. Huerre, R. Ramphal, and M. Chignard. "Both type II (T2SS) and type III (T3SS) secretion systems of Pseudomonas aeruginosa play roles in death due to lung disease." Journal of Cystic Fibrosis 9 (June 2010): S46. http://dx.doi.org/10.1016/s1569-1993(10)60178-6.
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Solé, Magali, Felix Scheibner, Anne-Katrin Hoffmeister, Nadine Hartmann, Gerd Hause, Annekatrin Rother, Michael Jordan, Martine Lautier, Matthieu Arlat, and Daniela Büttner. "Xanthomonas campestris pv. vesicatoria Secretes Proteases and Xylanases via the Xps Type II Secretion System and Outer Membrane Vesicles." Journal of Bacteriology 197, no. 17 (June 29, 2015): 2879–93. http://dx.doi.org/10.1128/jb.00322-15.
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ABSTRACTMany plant-pathogenic bacteria utilize type II secretion (T2S) systems to secrete degradative enzymes into the extracellular milieu. T2S substrates presumably mediate the degradation of plant cell wall components during the host-pathogen interaction and thus promote bacterial virulence. Previously, the Xps-T2S system fromXanthomonas campestrispv. vesicatoria was shown to contribute to extracellular protease activity and the secretion of a virulence-associated xylanase. The identities and functions of additional T2S substrates fromX. campestrispv. vesicatoria, however, are still unknown. In the present study, the analysis of 25 candidate proteins fromX. campestrispv. vesicatoria led to the identification of two type II secreted predicted xylanases, a putative protease and a lipase which was previously identified as a virulence factor ofX. campestrispv. vesicatoria. Studies with mutant strains revealed that the identified xylanases and the protease contribute to virulence andin plantagrowth ofX. campestrispv. vesicatoria. When analyzed in the related pathogenX. campestrispv. campestris, several T2S substrates fromX. campestrispv. vesicatoria were secreted independently of the T2S systems, presumably because of differences in the T2S substrate specificities of the two pathogens. Furthermore, inX. campestrispv. vesicatoria T2S mutants, secretion of T2S substrates was not completely absent, suggesting the contribution of additional transport systems to protein secretion. In line with this hypothesis, T2S substrates were detected in outer membrane vesicles, which were frequently observed forX. campestrispv. vesicatoria. We, therefore, propose that extracellular virulence-associated enzymes fromX. campestrispv. vesicatoria are targeted to the Xps-T2S system and to outer membrane vesicles.IMPORTANCEThe virulence of plant-pathogenic bacteria often depends on TS2 systems, which secrete degradative enzymes into the extracellular milieu. T2S substrates are being studied in several plant-pathogenic bacteria, includingXanthomonas campestrispv. vesicatoria, which causes bacterial spot disease in tomato and pepper. Here, we show that the T2S system fromX. campestrispv. vesicatoria secretes virulence-associated xylanases, a predicted protease, and a lipase. Secretion assays with the related pathogenX. campestrispv. campestris revealed important differences in the T2S substrate specificities of the two pathogens. Furthermore, electron microscopy showed that T2S substrates fromX. campestrispv. vesicatoria are targeted to outer membrane vesicles (OMVs). Our results, therefore, suggest that OMVs provide an alternative transport route for type II secreted extracellular enzymes.
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Johnson, Tanya L., Ursula Waack, Sara Smith, Harry Mobley, and Maria Sandkvist. "Acinetobacter baumannii Is Dependent on the Type II Secretion System and Its Substrate LipA for Lipid Utilization andIn VivoFitness." Journal of Bacteriology 198, no. 4 (December 14, 2015): 711–19. http://dx.doi.org/10.1128/jb.00622-15.
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ABSTRACTGram-negative bacteria express a number of sophisticated secretion systems to transport virulence factors across the cell envelope, including the type II secretion (T2S) system. Genes for the T2S components GspC through GspN and PilD are conserved among isolates ofAcinetobacter baumannii, an increasingly common nosocomial pathogen that is developing multidrug resistance at an alarming rate. In contrast to most species, however, the T2S genes are dispersed throughout the genome rather than linked into one or two operons. Despite this unique genetic organization, we show here that theA. baumanniiT2S system is functional. Deletion ofgspDorgspEinA. baumanniiATCC 17978 results in loss of secretion of LipA, a lipase that breaks down long-chain fatty acids. Due to a lack of extracellular lipase, thegspDmutant, thegspEmutant, and alipAdeletion strain are incapable of growth on long-chain fatty acids as a sole source of carbon, while their growth characteristics are indistinguishable from those of the wild-type strain in nutrient-rich broth. Genetic inactivation of the T2S system and its substrate, LipA, also has a negative impact onin vivofitness in a neutropenic murine model for bacteremia. Both thegspDandlipAmutants are outcompeted by the wild-type strain as judged by their reduced numbers in spleen and liver following intravenous coinoculation. Collectively, our findings suggest that the T2S system plays a hitherto-unrecognized role inin vivosurvival ofA. baumanniiby transporting a lipase that may contribute to fatty acid metabolism.IMPORTANCEInfections by multidrug-resistantAcinetobacter baumanniiare a growing health concern worldwide, underscoring the need for a better understanding of the molecular mechanisms by which this pathogen causes disease. In this study, we demonstrated thatA. baumanniiexpresses a functional type II secretion (T2S) system that is responsible for secretion of LipA, an extracellular lipase required for utilization of exogenously added lipids. The T2S system and the secreted lipase supportin vivocolonization and thus contribute to the pathogenic potential ofA. baumannii.
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Luo, Qingwei, Pardeep Kumar, Tim J. Vickers, Alaullah Sheikh, Warren G. Lewis, David A. Rasko, Jeticia Sistrunk, and James M. Fleckenstein. "Enterotoxigenic Escherichia coli Secretes a Highly Conserved Mucin-Degrading Metalloprotease To Effectively Engage Intestinal Epithelial Cells." Infection and Immunity 82, no. 2 (November 18, 2013): 509–21. http://dx.doi.org/10.1128/iai.01106-13.
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ABSTRACTEnterotoxigenicEscherichia coli(ETEC) is a leading cause of death due to diarrheal illness among young children in developing countries, and there is currently no effective vaccine. Many elements of ETEC pathogenesis are still poorly defined. Here we demonstrate that YghJ, a secreted ETEC antigen identified in immunoproteomic studies using convalescent patient sera, is required for efficient access to small intestinal enterocytes and for the optimal delivery of heat-labile toxin (LT). Furthermore, YghJ is a highly conserved metalloprotease that influences intestinal colonization of ETEC by degrading the major mucins in the small intestine, MUC2 and MUC3. Genes encoding YghJ and its cognate type II secretion system (T2SS), which also secretes LT, are highly conserved in ETEC and exist in other enteric pathogens, including other diarrheagenicE. coliandVibrio choleraebacteria, suggesting that this mucin-degrading enzyme may represent a shared virulence feature of these important pathogens.
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Sikora, Aleksandra E., Suzanne R. Lybarger, and Maria Sandkvist. "Compromised Outer Membrane Integrity in Vibrio cholerae Type II Secretion Mutants." Journal of Bacteriology 189, no. 23 (September 21, 2007): 8484–95. http://dx.doi.org/10.1128/jb.00583-07.
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ABSTRACT The type II secretion (T2S) system of Vibrio cholerae is a multiprotein complex that spans the cell envelope and secretes proteins important for pathogenesis as well as survival in different environments. Here we report that, in addition to the loss of extracellular secretion, removal or inhibition of expression of the T2S genes, epsC-N, results in growth defects and a broad range of alterations in the outer membrane that interfere with its barrier function. Specifically, the sensitivity to membrane-perturbing agents such as bile salts and the antimicrobial peptide polymyxin B is increased, and periplasmic constituents leak out into the culture medium. As a consequence, the σE stress response is induced. Furthermore, due to the defects caused by inactivation of the T2S system, the Δeps deletion mutant of V. cholerae strain N16961 is incapable of surviving the passage through the infant mouse gastrointestinal tract. The growth defect and leaky outer membrane phenotypes are suppressed when the culture medium is supplemented with 5% glucose or sucrose, although the eps mutants remain sensitive to membrane-damaging agents. This suggests that the sugars do not restore the integrity of the outer membrane in the eps mutant strains per se but may provide osmoprotective functions.
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McCoy-Simandle, Kessler, Catherine R. Stewart, Jenny Dao, Sruti DebRoy, Ombeline Rossier, Paul J. Bryce, and Nicholas P. Cianciotto. "Legionella pneumophila Type II Secretion Dampens the Cytokine Response of Infected Macrophages and Epithelia." Infection and Immunity 79, no. 5 (March 7, 2011): 1984–97. http://dx.doi.org/10.1128/iai.01077-10.
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ABSTRACTThe type II secretion (T2S) system ofLegionella pneumophilais required for the ability of the bacterium to grow within the lungs of A/J mice. By utilizing mutants lacking T2S (lsp), we now document that T2S promotes the intracellular infection of both multiple types of macrophages and lung epithelia. Following infection of macrophages,lspmutants (but not a complemented mutant) elicited significantly higher levels of interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-α), IL-10, IL-8, IL-1β, and MCP-1 within tissue culture supernatants. A similar result was obtained with infected lung epithelial cell lines and the lungs of infected A/J mice. Infection with a mutant specifically lacking the T2S-dependent ProA protease (but not a complementedproAmutant) resulted in partial elevation of cytokine levels. These data demonstrate that the T2S system ofL. pneumophiladampens the cytokine/chemokine output of infected host cells. Upon quantitative reverse transcription (RT)-PCR analysis of infected host cells, anlspFmutant, but not theproAmutant, produced significantly higher levels of cytokine transcripts, implying that some T2S-dependent effectors dampen signal transduction and transcription but that others, such as ProA, act at a posttranscriptional step in cytokine expression. In summary, the impact of T2S on lung infection is a combination of at least three factors: the promotion of growth in macrophages, the facilitation of growth in epithelia, and the dampening of the chemokine and cytokine output from infected host cells. To our knowledge, these data are the first to identify a link between a T2S system and the modulation of immune factors following intracellular infection.
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Moir, Donald T., Ming Di, Erica Wong, Richard A. Moore, Herbert P. Schweizer, Donald E. Woods, and Terry L. Bowlin. "Development and Application of a Cellular, Gain-of-Signal, Bioluminescent Reporter Screen for Inhibitors of Type II Secretion in Pseudomonas aeruginosa and Burkholderia pseudomallei." Journal of Biomolecular Screening 16, no. 7 (May 20, 2011): 694–705. http://dx.doi.org/10.1177/1087057111408605.
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The type II secretion (T2S) system in gram-negative bacteria comprises the Sec and Tat pathways for translocating proteins into the periplasm and an outer membrane secretin for transporting proteins into the extracellular space. To discover Sec/Tat/T2S pathway inhibitors as potential new therapeutics, the authors used a Pseudomonas aeruginosa bioluminescent reporter strain responsive to SecA depletion and inhibition to screen compound libraries and characterize the hits. The reporter strain placed a luxCDABE operon under regulation of a SecA depletion-responsive upregulated promoter in a secA deletion background complemented with an ectopic lac-regulated secA copy. Bioluminescence was indirectly proportional to the isopropyl-β-D-thiogalactopyranoside concentration and stimulated by azide, a known SecA ATPase inhibitor. A total of 96 compounds (0.1% of 73 000) were detected as primary hits due to stimulation of luminescence with a z score ≥5. Direct secretion assays of the nine most potent hits, representing five chemical scaffolds, revealed that they do not inhibit SecA-mediated secretion of β-lactamase into the periplasm but do inhibit T2S-mediated extracellular secretion of elastase with IC50 values from 5 to 25 µM. In addition, seven of the nine compounds also inhibited the T2S-mediated extracellular secretion of phospholipase C by P. aeruginosa and protease activity by Burkholderia pseudomallei.
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Karaba, Sara M., Richard C. White, and Nicholas P. Cianciotto. "Stenotrophomonas maltophilia Encodes a Type II Protein Secretion System That Promotes Detrimental Effects on Lung Epithelial Cells." Infection and Immunity 81, no. 9 (June 17, 2013): 3210–19. http://dx.doi.org/10.1128/iai.00546-13.
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ABSTRACTThe Gram-negative bacteriumStenotrophomonas maltophiliais increasingly identified as a multidrug-resistant pathogen, being associated with pneumonia, among other infections. Despite this increasing clinical problem, the genetic and molecular basis ofS. maltophiliavirulence is quite minimally defined. We now report that strain K279a, the first clinical isolate ofS. maltophiliato be sequenced, encodes a functional type II protein secretion (T2S) system. Indeed, mutants of K279a that contain a mutation in thexpslocus exhibit a loss of at least seven secreted proteins and three proteolytic activities. Unlike culture supernatants from the parental K279a, supernatants from multiplexpsmutants also failed to induce the rounding, detachment, and death of A549 cells, a human lung epithelial cell line. Supernatants of thexpsmutants were also unable to trigger a massive rearrangement in the host cell's actin cytoskeleton that was associated with K279a secretion. In all assays, a complementedxpsFmutant behaved as the wild type did, demonstrating that Xps T2S is required for optimal protein secretion and the detrimental effects on host cells. The activities that were defined as being Xps dependent in K279a were evident among other respiratory isolates ofS. maltophilia. Utilizing a similar type of genetic analysis, we found that a second T2S system (Gsp) encoded by the K279a genome is cryptic under all of the conditions tested. Overall, this study represents the first examination of T2S inS. maltophilia, and the data obtained indicate that Xps T2S likely plays an important role inS. maltophiliapathogenesis.
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Rasti, Elnaz, and Angela Brown. "Cholera Toxin Encapsulated within Several Vibrio cholerae O1 Serotype Inaba Outer Membrane Vesicles Lacks a Functional B-Subunit." Toxins 11, no. 4 (April 6, 2019): 207. http://dx.doi.org/10.3390/toxins11040207.
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Cholera toxin (CT), the major virulence factor of Vibrio cholerae, is an AB5 toxin secreted through the type II secretion system (T2SS). Upon secretion, the toxin initiates endocytosis through the interaction of the B pentamer with the GM1 ganglioside receptor on small intestinal cells. In addition to the release of CT in the free form, the bacteria secrete CT in association with outer membrane vesicles (OMVs). Previously, we demonstrated that strain 569B releases OMVs that encapsulate CT and which interact with host cells in a GM1-independent mechanism. Here, we have demonstrated that OMV-encapsulated CT, while biologically active, does not exist in an AB5 form; rather, the OMVs encapsulate two enzymatic A-subunit (CTA) polypeptides. We further investigated the assembly and secretion of the periplasmic CT and found that a major fraction of periplasmic CTA does not participate in the CT assembly process and instead is continuously encapsulated within the OMVs. Additionally, we found that the encapsulation of CTA fragments in OMVs is conserved among several Inaba O1 strains. We further found that under conditions in which the amount of extracellularly secreted CT increases, the concentration of OMV-encapsulated likewise CTA increases. These results point to a secondary mechanism for the secretion of biologically active CT that does not depend on the CTB-GM1 interaction for endocytosis.
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Mudrak, Benjamin, and Meta J. Kuehn. "Specificity of the Type II Secretion Systems of Enterotoxigenic Escherichia coli and Vibrio cholerae for Heat-Labile Enterotoxin and Cholera Toxin." Journal of Bacteriology 192, no. 7 (January 22, 2010): 1902–11. http://dx.doi.org/10.1128/jb.01542-09.
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ABSTRACT The Gram-negative type II secretion (T2S) system is a multiprotein complex mediating the release of virulence factors from a number of pathogens. While an understanding of the function of T2S components is emerging, little is known about what identifies substrates for export. To investigate T2S substrate recognition, we compared mutations affecting the secretion of two highly homologous substrates: heat-labile enterotoxin (LT) from enterotoxigenic Escherichia coli (ETEC) and cholera toxin (CT) from Vibrio cholerae. Each toxin consists of one enzymatic A subunit and a ring of five B subunits mediating the toxin's secretion. Here, we report two mutations in LT's B subunit (LTB) that reduce its secretion from ETEC without global effects on the toxin. The Q3K mutation reduced levels of secreted LT by half, and as with CT (T. D. Connell, D. J. Metzger, M. Wang, M. G. Jobling, and R. K. Holmes, Infect. Immun. 63:4091-4098, 1995), the E11K mutation impaired LT secretion. Results in vitro and in vivo show that these mutants are not degraded more readily than wild-type LT. The Q3K mutation did not significantly affect CT B subunit (CTB) secretion from V. cholerae, and the E11A mutation altered LT and CTB secretion to various extents, indicating that these toxins are identified as secretion substrates in different ways. The levels of mutant LTB expressed in V. cholerae were low or undetectable, but each CTB mutant expressed and secreted at wild-type levels in ETEC. Therefore, ETEC's T2S system seems to accommodate mutations in CTB that impair the secretion of LTB. Our results highlight the exquisitely fine-tuned relationship between T2S substrates and their coordinate secretion machineries in different bacterial species.
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Llanos Salinas, Samantha Paulina, Luz Olivia Castillo Sánchez, Giselle Castañeda Miranda, Ernesto Armando Rodríguez Reyes, Liliana Ordoñez López, Rodrigo Mena Bañuelos, Luz Elena Alcaraz Sosa, et al. "GspD, The Type II Secretion System Secretin of Leptospira, Protects Hamsters against Lethal Infection with a Virulent L. interrogans Isolate." Vaccines 8, no. 4 (December 14, 2020): 759. http://dx.doi.org/10.3390/vaccines8040759.
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The wide variety of pathogenic Leptospira serovars and the weak protection offered by the available vaccines encourage the search for protective immunogens against leptospirosis. We found that the secretin GspD of the type II secretion system (T2S) of Leptospira interrogans serovar Canicola was highly conserved amongst pathogenic serovars and was expressed in vivo during infection, as shown by immunohistochemistry. Convalescent sera of hamsters, dogs, and cows showed the presence of IgG antibodies, recognizing a recombinant version of this protein expressed in Escherichia coli (rGspDLC) in Western blot assays. In a pilot vaccination study, a group of eight hamsters was immunized on days zero and 14 with 50 µg of rGspDLC mixed with Freund’s incomplete adjuvant (FIA). On day 28 of the study, 1,000 LD50 (Lethal Dose 50%) of a virulent strain of Leptospira interrogans serovar Canicola (LOCaS46) were inoculated by an intraoral submucosal route (IOSM). Seventy-five percent protection against disease (p = 0.017573, Fisher’s exact test) and 50% protection against infection were observed in this group of vaccinated hamsters. In contrast, 85% of non-vaccinated hamsters died six to nine days after the challenge. These results suggest the potential usefulness of the T2S secretin GspD of Leptospira as a protective recombinant vaccine against leptospirosis.
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Douet, Vanessa, Dominique Expert, Frédéric Barras, and Béatrice Py. "Erwinia chrysanthemi Iron Metabolism: the Unexpected Implication of the Inner Membrane Platform within the Type II Secretion System." Journal of Bacteriology 191, no. 3 (October 31, 2008): 795–804. http://dx.doi.org/10.1128/jb.00845-08.
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ABSTRACT The type II secretion (T2S) system is an essential device for Erwinia chrysanthemi virulence. Previously, we reported the key role of the OutF protein in forming, along with OutELM, an inner membrane platform in the Out T2S system. Here, we report that OutF copurified with five proteins identified by matrix-assisted laser desorption ionization-time of flight analysis as AcsD, TogA, SecA, Tsp, and DegP. The AcsD protein was known to be involved in the biosynthesis of achromobactin, which is a siderophore important for E. chrysanthemi virulence. The yeast two-hybrid system allowed us to gain further evidence for the OutF-AcsD interaction. Moreover, we showed that lack of OutF produced a pleiotropic phenotype: (i) altered production of the two siderophores of E. chrysanthemi, achromobactin and chrysobactin; (ii) hypersensitivity to streptonigrin, an iron-activated antibiotic; (iii) increased sensitivity to oxidative stress; and (iv) absence of the FbpA-like iron-binding protein in the periplasmic fraction. Interestingly, outE and outL mutants also exhibited similar phenotypes, but, outD and outJ mutants did not. Moreover, using the yeast two-hybrid system, several interactions were shown to occur between components of the T2S system inner membrane platform (OutEFL) and proteins involved in achromobactin production (AcsABCDE). The OutL-AcsD interaction was also demonstrated by Ni2+ affinity chromatography. These results fully confirm our previous view that the T2S machinery is made up of three discrete blocks. The OutEFLM-forming platform is proposed to be instrumental in two different processes essential for virulence, protein secretion and iron homeostasis.
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Yamazaki, Akihiro, Jin Li, William C. Hutchins, Lixia Wang, Jincai Ma, A. Mark Ibekwe, and Ching-Hong Yang. "Commensal Effect of Pectate Lyases Secreted fromDickeya dadantiion Proliferation ofEscherichia coliO157:H7 EDL933 on Lettuce Leaves." Applied and Environmental Microbiology 77, no. 1 (November 12, 2010): 156–62. http://dx.doi.org/10.1128/aem.01079-10.
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ABSTRACTThe outbreaks caused by enterohemorrhagicEscherichia coliO157:H7 on leafy greens have raised serious and immediate food safety concerns. It has been suggested that several phytopathogens aid in the persistence and proliferation of the human enteropathogens in the phyllosphere. In this work, we examined the influence of virulence mechanisms ofDickeya dadantii3937, a broad-host-range phytopathogen, on the proliferation of the human pathogenE. coliO157:H7 EDL933 (EDL933) on postharvest lettuce by coinoculation of EDL933 withD. dadantii3937 derivatives that have mutations in virulence-related genes. A type II secretion system (T2SS)-deficient mutant ofD. dadantii3937, A1919 (ΔoutC), lost the capability to promote the multiplication of EDL933, whereas Ech159 (ΔrpoS), a stress-responsive σ factor RpoS-deficient mutant, increased EDL933 proliferation on lettuce leaves. A spectrophotometric enzyme activity assay revealed that A1919 (ΔoutC) was completely deficient in the secretion of pectate lyases (Pels), which play a major role in plant tissue maceration. In contrast to A1919 (ΔoutC), Ech159 (ΔrpoS) showed more than 2-fold-greater Pel activity than the wild-typeD. dadantii3937. Increased expression ofpelD(encodes an endo-pectate lyase) was observed in Ech159 (ΔrpoS)in planta. These results suggest that the pectinolytic activity ofD. dadantii3937 is the dominant determinant of enhanced EDL933 proliferation on the lettuce leaves. In addition, RpoS, the general stress response σ factor involved in cell survival in suboptimal conditions, plays a role in EDL933 proliferation by controlling the production of pectate lyases inD. dadantii3937.
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Tyson, Jessica Y., Paloma Vargas, and Nicholas P. Cianciotto. "The novel Legionella pneumophila type II secretion substrate NttC contributes to infection of amoebae Hartmannella vermiformis and Willaertia magna." Microbiology 160, no. 12 (December 1, 2014): 2732–44. http://dx.doi.org/10.1099/mic.0.082750-0.
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The type II protein secretion (T2S) system of Legionella pneumophila secretes over 25 proteins, including novel proteins that have no similarity to proteins of known function. T2S is also critical for the ability of L. pneumophila to grow within its natural amoebal hosts, including Acanthamoeba castellanii, Hartmannella vermiformis and Naegleria lovaniensis. Thus, T2S has an important role in the natural history of legionnaires’ disease. Our previous work demonstrated that the novel T2S substrate NttA promotes intracellular infection of A. castellanii, whereas the secreted RNase SrnA, acyltransferase PlaC, and metalloprotease ProA all promote infection of H. vermiformis and N. lovaniensis. In this study, we determined that another novel T2S substrate that is specific to Legionella, designated NttC, is unique in being required for intracellular infection of H. vermiformis but not for infection of N. lovaniensis or A. castellanii. Expanding our repertoire of amoebal hosts, we determined that Willaertia magna is susceptible to infection by L. pneumophila strains 130b, Philadelphia-1 and Paris. Furthermore, T2S and, more specifically, NttA, NttC and PlaC were required for infection of W. magna. Taken together, these data demonstrate that the T2S system of L. pneumophila is critical for infection of at least four types of aquatic amoebae and that the importance of the individual T2S substrates varies in a host cell-specific fashion. Finally, it is now clear that novel T2S-dependent proteins that are specific to the genus Legionella are particularly important for L. pneumophila infection of key, environmental hosts.
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Giltner, Carmen L., Ylan Nguyen, and Lori L. Burrows. "Type IV Pilin Proteins: Versatile Molecular Modules." Microbiology and Molecular Biology Reviews 76, no. 4 (November 29, 2012): 740–72. http://dx.doi.org/10.1128/mmbr.00035-12.
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SUMMARYType IV pili (T4P) are multifunctional protein fibers produced on the surfaces of a wide variety of bacteria and archaea. The major subunit of T4P is the type IV pilin, and structurally related proteins are found as components of the type II secretion (T2S) system, where they are called pseudopilins; of DNA uptake/competence systems in both Gram-negative and Gram-positive species; and of flagella, pili, and sugar-binding systems in the archaea. This broad distribution of a single protein family implies both a common evolutionary origin and a highly adaptable functional plan. The type IV pilin is a remarkably versatile architectural module that has been adopted widely for a variety of functions, including motility, attachment to chemically diverse surfaces, electrical conductance, acquisition of DNA, and secretion of a broad range of structurally distinct protein substrates. In this review, we consider recent advances in this research area, from structural revelations to insights into diversity, posttranslational modifications, regulation, and function.
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Kumar B, Abishek, Bency Thankappan, Angayarkanni Jayaraman, and Akshita Gupta. "Evaluation of Antibiotic Tolerance in Pseudomonas aeruginosa for Aminoglycosides and Its Predicted Gene Regulations through In-Silico Transcriptomic Analysis." Microbiology Research 12, no. 3 (July 29, 2021): 630–45. http://dx.doi.org/10.3390/microbiolres12030045.
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Pseudomonas aeruginosa causes chronic infections, such as cystic fibrosis, endocarditis, bacteremia, and sepsis, which are life-threatening and difficult to treat. The lack of antibiotic response in P. aeruginosa is due to adaptive resistance mechanism, which prevents the entry of antibiotics into the cytosol of the cell to achieve tolerance. Among the different groups of antibiotics, aminoglycosides are used as a parenteral antibiotic for the treatment of P. aeruginosa. This study aimed to determine the kinetics of antibiotic tolerance and gene expression changes in P. aeruginosa exposed to amikacin, gentamicin, and tobramycin. These antibiotics were exposed to P. aeruginosa at their MICs and the experimental setup was monitored for 72 h, followed by the measurement of optical density every 12 h. The growth of P. aeruginosa in the MICs of antibiotics represented the kinetics of antibiotic tolerance in amikacin, gentamicin, and tobramycin. The transcriptomic profile of antibiotic exposed P. aeruginosa PA14 was taken from the Gene Expression Omnibus (GEO), NCBI as microarray datasets. The gene expressions of two datasets were compared by test versus control. Tobramycin-exposed P. aeruginosa failed to develop tolerance in MICs of 0.5 µg/mL, 1 µg/mL, and 1.5 µg/mL, whereas amikacin- and gentamicin-treated P. aeruginosa developed tolerance. This illustrated the superior in vitro response of tobramycin over gentamicin and amikacin. Further, in silico transcriptomic analysis of tobramycin-treated P. aeruginosa resulted in differentially expressed genes (DEGs), enriched in 16s rRNA methyltransferase E, B, and L, alginate biosynthesis genes, and several proteins of the type II secretion system (T2SS) and type III secretion system (T3SS). The regulation of mucA in alginate biosynthesis, and gidB in RNA methyltransferases, suggested an increased antibiotic response and a low probability of developing resistance during tobramycin treatment. The use of tobramycin as a parenteral antibiotic with its synergistic combination might combat P. aeruginosa with increased response.
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Tran, Nini, Ryszard A. Zielke, Oliver B. Vining, Mark D. Azevedo, Donald J. Armstrong, Gary M. Banowetz, Kerry L. McPhail, and Aleksandra E. Sikora. "Development of a Quantitative Assay Amenable for High-Throughput Screening to Target the Type II Secretion System for New Treatments against Plant-Pathogenic Bacteria." Journal of Biomolecular Screening 18, no. 8 (April 11, 2013): 921–29. http://dx.doi.org/10.1177/1087057113485426.
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Plant-pathogenic bacteria are the causative agents of diseases in important agricultural crops and ornamental plants. The severe economic burden of these diseases requires seeking new approaches for their control, particularly because phytopathogenic bacteria are often resistant to available treatments. The type II secretion (T2S) system is a key virulence factor used by major groups of phytopathogenic bacteria. The T2S machinery transports many hydrolytic enzymes responsible for degradation of the plant cell wall, thus enabling successful colonization and dissemination of the bacteria in the plant host. The genetic inactivation of the T2S system leads to loss of virulence, which strongly suggests that targeting the T2S could enable new treatments against plant-pathogenic bacteria. Accordingly, we have designed and optimized an assay to identify small-molecule inhibitors of the T2S system. This assay uses a double parametric output: measurement of bacterial growth and the enzymatic activity of cellulase, which is secreted via the T2S pathway in our model organism Dickeya dadantii. The assay was evaluated by screening natural extracts, culture filtrates isolated from rhizosphere bacteria, and a collection of pharmaceutically active compounds in LOPAC1280. The calculated Z′ values of 0.63, 0.63, and 0.58, respectively, strongly suggest that the assay is applicable for a high-throughput screening platform.
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Johnson, Tanya L., Maria E. Scott, and Maria Sandkvist. "Mapping Critical Interactive Sites within the Periplasmic Domain of the Vibrio cholerae Type II Secretion Protein EpsM." Journal of Bacteriology 189, no. 24 (October 5, 2007): 9082–89. http://dx.doi.org/10.1128/jb.01256-07.
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ABSTRACT The type II secretion (T2S) system is present in many gram-negative species, both pathogenic and nonpathogenic, where it supports the delivery of a variety of toxins, proteases, and lipases into the extracellular environment. In Vibrio cholerae, the T2S apparatus is composed of 12 Eps proteins that assemble into a multiprotein complex that spans the entire cell envelope. Two of these proteins, EpsM and EpsL, are key components of the secretion machinery present in the inner membrane. In addition to likely forming homodimers, EpsL and EpsM have been shown to form a stable complex in the inner membrane and to protect each other from proteolytic degradation. To identify and map the specific regions of EpsM involved in protein-protein interactions with both another molecule of EpsM and EpsL, we tested the interactions of deletion constructs of EpsM with full-length EpsM and EpsL by functional characterization and copurification as well as coimmunoprecipitation. Analysis of the truncated EpsM mutants revealed that the region of EpsM from amino acids 100 to 135 is necessary for EpsM to form homo-oligomers, while residues 84 to 99 appear to be critical for a stable interaction with EpsL.
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Zielke, Ryszard A., Ryan S. Simmons, Bo R. Park, Mariko Nonogaki, Sarah Emerson, and Aleksandra E. Sikora. "The Type II Secretion Pathway in Vibrio cholerae Is Characterized by Growth Phase-Dependent Expression of Exoprotein Genes and Is Positively Regulated by σE." Infection and Immunity 82, no. 7 (April 14, 2014): 2788–801. http://dx.doi.org/10.1128/iai.01292-13.
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ABSTRACTVibrio cholerae, an etiological agent of cholera, circulates between aquatic reservoirs and the human gastrointestinal tract. The type II secretion (T2S) system plays a pivotal role in both stages of the lifestyle by exporting multiple proteins, including cholera toxin. Here, we studied the kinetics of expression of genes encoding the T2S system and its cargo proteins. We have found that under laboratory growth conditions, the T2S complex was continuously expressed throughoutV. choleraegrowth, whereas there was growth phase-dependent transcriptional activity of genes encoding different cargo proteins. Moreover, exposure ofV. choleraeto different environmental cues encountered by the bacterium in its life cycle induced transcriptional expression of T2S. Subsequent screening of aV. choleraegenomic library suggested that σEstress response, phosphate metabolism, and the second messenger 3′,5′-cyclic diguanylic acid (c-di-GMP) are involved in regulating transcriptional expression of T2S. Focusing on σE, we discovered that the upstream region of the T2S operon possesses both the consensus σEand σ70signatures, and deletion of the σEbinding sequence prevented transcriptional activation of T2S by RpoE. Ectopic overexpression of σEstimulated transcription of T2S in wild-type and isogenic ΔrpoEstrains ofV. cholerae, providing additional support for the idea that the T2S complex belongs to the σEregulon. Together, our results suggest that the T2S pathway is characterized by the growth phase-dependent expression of genes encoding cargo proteins and requires a multifactorial regulatory network to ensure appropriate kinetics of the secretory traffic and the fitness ofV. choleraein different ecological niches.
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Sikora, Aleksandra E., Sinem Beyhan, Michael Bagdasarian, Fitnat H. Yildiz, and Maria Sandkvist. "Cell Envelope Perturbation Induces Oxidative Stress and Changes in Iron Homeostasis in Vibrio cholerae." Journal of Bacteriology 191, no. 17 (June 19, 2009): 5398–408. http://dx.doi.org/10.1128/jb.00092-09.
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ABSTRACT The Vibrio cholerae type II secretion (T2S) machinery is a multiprotein complex that spans the cell envelope. When the T2S system is inactivated, cholera toxin and other exoproteins accumulate in the periplasmic compartment. Additionally, loss of secretion via the T2S system leads to a reduced growth rate, compromised outer membrane integrity, and induction of the extracytoplasmic stress factor RpoE (A. E. Sikora, S. R. Lybarger, and M. Sandkvist, J. Bacteriol. 189:8484-8495, 2007). In this study, gene expression profiling reveals that inactivation of the T2S system alters the expression of genes encoding cell envelope components and proteins involved in central metabolism, chemotaxis, motility, oxidative stress, and iron storage and acquisition. Consistent with the gene expression data, molecular and biochemical analyses indicate that the T2S mutants suffer from internal oxidative stress and increased levels of intracellular ferrous iron. By using a tolA mutant of V. cholerae that shares a similar compromised membrane phenotype but maintains a functional T2S machinery, we show that the formation of radical oxygen species, induction of oxidative stress, and changes in iron physiology are likely general responses to cell envelope damage and are not unique to T2S mutants. Finally, we demonstrate that disruption of the V. cholerae cell envelope by chemical treatment with polymyxin B similarly results in induction of the RpoE-mediated stress response, increased sensitivity to oxidants, and a change in iron metabolism. We propose that many types of extracytoplasmic stresses, caused either by genetic alterations of outer membrane constituents or by chemical or physical damage to the cell envelope, induce common signaling pathways that ultimately lead to internal oxidative stress and misregulation of iron homeostasis.
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Tyson, Jessica Y., Meghan M. Pearce, Paloma Vargas, Sreya Bagchi, Brendan J. Mulhern, and Nicholas P. Cianciotto. "Multiple Legionella pneumophila Type II Secretion Substrates, Including a Novel Protein, Contribute to Differential Infection of the Amoebae Acanthamoeba castellanii, Hartmannella vermiformis, and Naegleria lovaniensis." Infection and Immunity 81, no. 5 (February 19, 2013): 1399–410. http://dx.doi.org/10.1128/iai.00045-13.
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ABSTRACTType II protein secretion (T2S) byLegionella pneumophilais required for intracellular infection of host cells, including macrophages and the amoebaeAcanthamoeba castellaniiandHartmannella vermiformis. Previous proteomic analysis revealed that T2S byL. pneumophila130b mediates the export of >25 proteins, including several that appeared to be novel. Following confirmation that they are unlike known proteins, T2S substrates NttA, NttB, and LegP were targeted for mutation.nttAmutants were impaired for intracellular multiplication inA. castellaniibut notH. vermiformisor macrophages, suggesting that novel exoproteins which are specific toLegionellaare especially important for infection. Because the importance of NttA was host cell dependent, we examined a panel of T2S substrate mutants that had not been tested before in more than one amoeba. As a result, RNase SrnA, acyltransferase PlaC, and metalloprotease ProA all proved to be required for optimal intracellular multiplication inH. vermiformisbut notA. castellanii. Further examination of anlspFmutant lacking the T2S apparatus documented that T2S is also critical for infection of the amoebaNaegleria lovaniensis. Mutants lacking SrnA, PlaC, or ProA, but not those deficient for NttA, were defective inN. lovaniensis. Based upon analysis of a double mutant lacking PlaC and ProA, the role of ProA inH. vermiformiswas connected to its ability to activate PlaC, whereas inN. lovaniensis, ProA appeared to have multiple functions. Together, these data document that the T2S system exports multiple effectors, including a novel one, which contribute in different ways to the broad host range ofL. pneumophila.
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Erova, Tatiana E., Lakshmi Pillai, Amin A. Fadl, Jian Sha, Shaofei Wang, Cristi L. Galindo, and Ashok K. Chopra. "DNA Adenine Methyltransferase Influences the Virulence of Aeromonas hydrophila." Infection and Immunity 74, no. 1 (January 2006): 410–24. http://dx.doi.org/10.1128/iai.74.1.410-424.2006.
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ABSTRACT Among the various virulence factors produced by Aeromonas hydrophila, a type II secretion system (T2SS)-secreted cytotoxic enterotoxin (Act) and the T3SS are crucial in the pathogenesis of Aeromonas-associated infections. Our laboratory molecularly characterized both Act and the T3SS from a diarrheal isolate, SSU of A. hydrophila, and defined the role of some regulatory genes in modulating the biological effects of Act. In this study, we cloned, sequenced, and expressed the DNA adenine methyltransferase gene of A. hydrophila SSU (dam AhSSU) in a T7 promoter-based vector system using Escherichia coli ER2566 as a host strain, which could alter the virulence potential of A. hydrophila. Recombinant Dam, designated as M.AhySSUDam, was produced as a histidine-tagged fusion protein and purified from an E. coli cell lysate using nickel affinity chromatography. The purified Dam had methyltransferase activity, based on its ability to transfer a methyl group from S-adenosyl-l-methionine to N6-methyladenine-free lambda DNA and to protect methylated lambda DNA from digestion with DpnII but not against the DpnI restriction enzyme. The dam gene was essential for the viability of the bacterium, and overproduction of Dam in A. hydrophila SSU, using an arabinose-inducible, PBAD promoter-based system, reduced the virulence of this pathogen. Specifically, overproduction of M.AhySSUDam decreased the motility of the bacterium by 58%. Likewise, the T3SS-associated cytotoxicity, as measured by the release of lactate dehydrogenase enzyme in murine macrophages infected with the Dam-overproducing strain, was diminished by 55% compared to that of a control A. hydrophila SSU strain harboring the pBAD vector alone. On the contrary, cytotoxic and hemolytic activities associated with Act as well as the protease activity in the culture supernatant of a Dam-overproducing strain were increased by 10-, 3-, and 2.4-fold, respectively, compared to those of the control A. hydrophila SSU strain. The Dam-overproducing strain was not lethal to mice (100% survival) when given by the intraperitoneal route at a dose twice that of the 50% lethal dose, which within 2 to 3 days killed 100% of the animals inoculated with the A. hydrophila control strain. Taken together, our data indicated alteration of A. hydrophila virulence by overproduction of Dam.
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Rajeshwari, R., Gopaljee Jha, and Ramesh V. Sonti. "Role of an In Planta-Expressed Xylanase of Xanthomonas oryzae pv. oryzae in Promoting Virulence on Rice." Molecular Plant-Microbe Interactions® 18, no. 8 (August 2005): 830–37. http://dx.doi.org/10.1094/mpmi-18-0830.
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Xanthomonas oryzae pv. oryzae is the causal agent of bacterial leaf blight, a serious disease of rice. We demonstrated earlier that the type II secretion system (T2S) is important for virulence of X. oryzae pv. oryzae and that several proteins, including a xylanase, are secreted through this system. In this study, the xynB gene encoding for the secreted xylanase was cloned as a 6.9-kb Eco RI fragment (pRR7) that also included a paralog called xynA. As in X. oryzae pv. oryzae, xynA and xynB are adjacent to each other in X. axonopodis pv. citri, whereas only the xynA homolog is present in X. campestris pv. campestris. Mutations in xynB but not xynA affect secreted xylanase activity. Western blot analysis using anti-XynB antibodies on exudates from infected rice leaves indicated that this xylanase is expressed during in planta growth. Another T2S-secreted protein was identified to be a lipase/esterase (LipA) based on the sequence tags obtained by tandem mass spectrometry analysis and biochemical assays. Mutations in either xynB or lipA partially affected virulence. However, a lipA-xynB double mutant was significantly reduced for virulence, and the pRR7 clone containing an intact xynB gene could complement the virulence-deficient phenotype of the lipA-xynB mutant. Our results suggest that there is functional redundancy among the T2S secreted proteins of X. oryzae pv. oryzae in promoting virulence on rice.
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Ho, Theresa D., Brigid M. Davis, Jennifer M. Ritchie, and Matthew K. Waldor. "Type 2 Secretion Promotes Enterohemorrhagic Escherichia coli Adherence and Intestinal Colonization." Infection and Immunity 76, no. 5 (March 3, 2008): 1858–65. http://dx.doi.org/10.1128/iai.01688-07.
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ABSTRACT Enterohemorrhagic Escherichia coli (EHEC) is a noninvasive food-borne pathogen that colonizes the distal ileum and colon. Proteins encoded in the EHEC locus of enterocyte effacement (LEE) pathogenicity island are known to contribute to this pathogen's adherence to epithelial cells and intestinal colonization. The role of non-LEE-encoded proteins in these processes is not as clear. We found that the Z2053 gene (designated adfO here), a gene located in a cryptic EHEC prophage, exhibits similarity to adherence and/or colonization factor genes found in several other enteric pathogens. An EHEC adfO mutant exhibited marked reductions in adherence to HeLa cells and in the secretion of several proteins into the supernatant. YodA, one of these secreted proteins, was found to be a substrate of the EHEC pO157-encoded type 2 secretion system (T2SS). Both the T2SS and YodA proved to be essential for EHEC adherence to cultured HeLa cell monolayers. Using an infant rabbit model of infection, we found that the adfO mutation did not affect colonization but that the colonization of an etpC (T2SS) mutant was reduced ∼5-fold. A strain deficient in YodA had a more severe colonization defect; however, this strain also exhibited a growth defect in vitro. Overall, our findings indicate that the pO157-encoded T2SS contributes to EHEC adherence and intestinal colonization and thus show that EHEC pathogenicity depends on type 2 secretion as well as type 3 secretion.
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Jha, Gopaljee, Ramanan Rajeshwari, and Ramesh V. Sonti. "Functional Interplay Between Two Xanthomonas oryzae pv. oryzae Secretion Systems in Modulating Virulence on Rice." Molecular Plant-Microbe Interactions® 20, no. 1 (January 2007): 31–40. http://dx.doi.org/10.1094/mpmi-20-0031.
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The type II (T2S) and type III (T3S) secretion systems are important for virulence of Xanthomonas oryzae pv. oryzae, causal agent of bacterial leaf blight of rice. The T3S of gram-negative bacterial plant pathogens has been shown to suppress host defense responses, including programmed cell death reactions, whereas the T2S is involved in secreting cell-wall-degrading enzymes. Here, we show that a T3S-deficient (T3S¯) mutant of X. oryzae pv. oryzae can induce a basal plant defense response seen as callose deposition, immunize rice against subsequent X. oryzae pv. oryzae infection, and cause cell-death-associated nuclear fragmentation. A T2S¯ T3S¯ double mutant exhibited a substantial reduction in the ability to evoke these responses. We purified two major effectors of the X. oryzae pv. oryzae T2S and characterized them to be a cellulase (ClsA) and a putative cellobiosidase (CbsA). The purified ClsA, CbsA, and lipase/esterase (LipA; a previously identified T2S effector) proteins induced rice defense responses that were suppressible by X. oryzae pv. oryzae in a T3S-dependent manner. These defense responses also were inducible by the products of the action of these purified proteins on rice cell walls. We further show that a CbsA¯ mutant or a ClsA¯ LipA¯ double mutant are severely virulence deficient. These results indicate that the X. oryzae pv. oryzae T2S secretes important virulence factors, which induce innate rice defense responses that are suppressed by T3S effectors to enable successful infection.
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Peabody, Christopher R., Yong Joon Chung, Ming-Ren Yen, Dominique Vidal-Ingigliardi, Anthony P. Pugsley, and Milton H. Saier. "Type II protein secretion and its relationship to bacterial type IV pili and archaeal flagella." Microbiology 149, no. 11 (November 1, 2003): 3051–72. http://dx.doi.org/10.1099/mic.0.26364-0.
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Homologues of the protein constituents of the Klebsiella pneumoniae (Klebsiella oxytoca) type II secreton (T2S), the Pseudomonas aeruginosa type IV pilus/fimbrium biogenesis machinery (T4P) and the Methanococcus voltae flagellum biogenesis machinery (Fla) have been identified. Known constituents of these systems include (1) a major prepilin (preflagellin), (2) several minor prepilins (preflagellins), (3) a prepilin (preflagellin) peptidase/methylase, (4) an ATPase, (5) a multispanning transmembrane (TM) protein, (6) an outer-membrane secretin (lacking in Fla) and (7) several functionally uncharacterized envelope proteins. Sequence and phylogenetic analyses led to the conclusion that, although many of the protein constituents are probably homologous, extensive sequence divergence during evolution clouds this homology so that a common ancestry can be established for all three types of systems for only two constituents, the ATPase and the TM protein. Sequence divergence of the individual T2S constituents has occurred at characteristic rates, apparently without shuffling of constituents between systems. The same is probably also true for the T4P and Fla systems. The family of ATPases is much larger than the family of TM proteins, and many ATPase homologues function in capacities unrelated to those considered here. Many phylogenetic clusters of the ATPases probably exhibit uniform function. Some of these have a corresponding TM protein homologue although others probably function without one. It is further shown that proteins that compose the different phylogenetic clusters in both the ATPase and the TM protein families exhibit unique structural characteristics that are of probable functional significance. The TM proteins are shown to have arisen by at least two dissimilar intragenic duplication events, one in the bacterial kingdom and one in the archaeal kingdom. The archaeal TM proteins are twice as large as the bacterial TM proteins, suggesting an oligomeric structure for the latter.
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Somvanshi, Vishal S., Poorna Viswanathan, Janette L. Jacobs, Martha H. Mulks, George W. Sundin, and Todd A. Ciche. "The Type 2 Secretion Pseudopilin, gspJ, Is Required for Multihost Pathogenicity of Burkholderia cenocepacia AU1054." Infection and Immunity 78, no. 10 (July 26, 2010): 4110–21. http://dx.doi.org/10.1128/iai.00558-10.
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ABSTRACT Burkholderia cenocepacia AU1054 is an opportunistic pathogen isolated from the blood of a person with cystic fibrosis. AU1054 is a multihost pathogen causing rapid pathogenicity to Caenorhabditis elegans nematodes. Within 24 h, AU1054 causes greater than 50% mortality, reduced growth, emaciated body, distended intestinal lumen, rectal swelling, and prolific infection of the nematode intestine. To determine virulence mechanisms, 3,000 transposon mutants were screened for attenuated virulence in nematodes. Fourteen virulence-attenuated mutants were isolated, and the mutant genes were identified. These genes included paaA, previously identified as being required for full virulence of B. cenocepacia K56-2. Six mutants were restored in virulence by complementation with their respective wild-type gene. One of these contained an insertion in gspJ, predicted to encode a pseudopilin component of the type 2 secretion system (T2SS). Nematodes infected with AU1054 gspJ had fewer bacteria present in the intestine than those infected with the wild type but still showed rectal swelling. The gspJ mutant was also defective in pathogenicity to onion and in degradation of polygalacturonic acid and casein. This result differs from previous studies where no or little role was found for T2SS in Burkholderia virulence, although virulence factors such as zinc metalloproteases and polygalacturonase are known to be secreted by the T2SS. This study highlights strain specific differences in B. cenocepacia virulence mechanisms important for understanding what enables environmental microbes to function as opportunistic pathogens.
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Konjufca, Vjollca, Mark Jenkins, Shifeng Wang, Maria Dolores Juarez-Rodriguez, and Roy Curtiss. "Immunogenicity of Recombinant Attenuated Salmonella enterica Serovar Typhimurium Vaccine Strains Carrying a Gene That Encodes Eimeria tenella Antigen SO7." Infection and Immunity 76, no. 12 (September 22, 2008): 5745–53. http://dx.doi.org/10.1128/iai.00897-08.
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ABSTRACT Recombinant attenuated Salmonella vaccines against avian coccidiosis were developed to deliver Eimeria species antigens to the lymphoid tissues of chickens via the type 3 secretion system (T3SS) and the type 2 secretion system (T2SS) of Salmonella. For antigen delivery via the T3SS, the Eimeria tenella gene encoding sporozoite antigen SO7 was cloned downstream of the translocation domain of the Salmonella enterica serovar Typhimurium sopE gene in the parental pYA3868 and pYA3870 vectors to generate pYA4156 and pYA4157. Newly constructed T3SS vectors were introduced into host strain χ8879 (ΔphoP233 ΔsptP1033::xylE ΔasdA16), an attenuated derivative of the highly virulent UK-1 strain. The SopE-SO7 fusion protein was secreted by the T3SS of Salmonella. The vector pYA4184 was constructed for delivery of the SO7 antigen via the T2SS. The SO7 protein was toxic to Salmonella when larger amounts were synthesized; thus, the synthesis of this protein was placed under the control of the lacI repressor gene, whose expression in turn was dependent on the amount of available arabinose in the medium. The pYA4184 vector was introduced into host strain χ9242 (ΔphoP233 ΔasdA16 ΔaraBAD23 ΔrelA198::araC PBAD lacI TT [TT is the T4ipIII transcription terminator]). In addition to SO7, for immunization and challenge studies we used the EAMZ250 antigen of Eimeria acervulina, which was previously shown to confer partial protection against E. acervulina challenge when it was delivered via the T3SS. Immunization of chickens with a combination of the SO7 and EAMZ250 antigens delivered via the T3SS induced superior protection against challenge by E. acervulina. In contrast, chickens immunized with SO7 that was delivered via the T2SS of Salmonella were better protected from challenge by E. tenella.
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Rybin, V. O., B. D. Uhal, L. A. Russo, and D. E. Rannels. "ADP ribosylation of type II pulmonary epithelial cell G proteins." American Journal of Physiology-Lung Cellular and Molecular Physiology 260, no. 6 (June 1, 1991): L539—L547. http://dx.doi.org/10.1152/ajplung.1991.260.6.l539.
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Secretion of pulmonary surfactant by type II pulmonary epithelial cells (T2P) is regulated by receptor-mediated mechanisms. In other systems, coupling of receptor-linked signals to intracellular events involves guanine nucleotide-binding proteins (G proteins), but the specific role of G proteins in T2P signaling pathways is poorly defined. The present studies begin to address the role of G proteins in transmembrane signaling in these pneumocytes. Membrane preparations from purified T2Ps demonstrated ADP ribosylation of specific substrates by pertussis, cholera, and botulinum toxins (PT, CT, and BT, respectively). Toxin-dependent T2P substrate labeling from 32P-labeled NAD was dependent on time and membrane protein concentration. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography showed ADP ribosylation of membrane substrates of the following molecular masses: PT, 40/41 kDa; CT, 47/51 kDa; BT, 22 kDa. BT-dependent ADP ribosylation of a 22-kDa cytosolic substrate was also observed. Pretreatment of cultured T2P with the individual toxins led to ADP ribosylation of their respective specific substrates in a time-dependent fashion. In cells pretreated with PT or CT, substrates for the complimentary toxins remained available for subsequent ADP ribosylation in vitro. This result supports the specificity of the toxin effects. Basal secretion of the major phospholipid of pulmonary surfactant, disaturated phosphatidylcholine (DSPC) was unaffected in T2P treated with PT, but was stimulated in cells exposed to CT or BT. Neither CT nor BT altered release of lactate dehydrogenase. In cells treated with AMP or with isoproterenol DSPC secretion was stimulated six- to eightfold; preexposure of the cells to CT reduced the response to either agonist by 70%.(ABSTRACT TRUNCATED AT 250 WORDS)
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Peter Howard, S. "Assembly of the type II secretion system." Research in Microbiology 164, no. 6 (July 2013): 535–44. http://dx.doi.org/10.1016/j.resmic.2013.03.018.
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Cianciotto, Nicholas P. "Type II secretion: a protein secretion system for all seasons." Trends in Microbiology 13, no. 12 (December 2005): 581–88. http://dx.doi.org/10.1016/j.tim.2005.09.005.
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