Статті в журналах: "Type III secretion helper proteins"

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Holeva, Maria C., Kenneth S. Bell, Lizbeth J. Hyman, Anna O. Avrova, Stephen C. Whisson, Paul R. J. Birch, and Ian K. Toth. "Use of a Pooled Transposon Mutation Grid to Demonstrate Roles in Disease Development for Erwinia carotovora subsp. atroseptica Putative Type III Secreted Effector (DspE/A) and Helper (HrpN) Proteins." Molecular Plant-Microbe Interactions® 17, no. 9 (September 2004): 943–50. http://dx.doi.org/10.1094/mpmi.2004.17.9.943.

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Soft rot Erwinia spp., like other closely related plant pathogens, possess a type III secretion system (TTSS) (encoded by the hrp gene cluster) implicated in disease development. We report the sequence of the entire hrp gene cluster and adjacent dsp genes in Erwinia carotovora subsp. atroseptica SCRI1039. The cluster is similar in content and structural organization to that in E. amylovora. However, eight putative genes of unknown function located within the E. carotovora subsp. atroseptica cluster do not have homologues in the E. amylovora cluster. An arrayed set of Tn5 insertional mutants (mutation grid) was constructed and pooled to allow rapid isolation of mutants for any given gene by polymerase chain reaction screening. This novel approach was used to obtain mutations in two structural genes (hrcC and hrcV), the effector gene dspE/A, and the helper gene hrpN. An improved pathogenicity assay revealed that these mutations led to significantly reduced virulence, showing that both the putative E. carotovora subsp. atroseptica TTSS-delivered effector and helper proteins are required for potato infection.

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Yu, H. B., P. S. Srinivasa Rao, H. C. Lee, S. Vilches, S. Merino, J. M. Tomas, and K. Y. Leung. "A Type III Secretion System Is Required for Aeromonas hydrophila AH-1 Pathogenesis." Infection and Immunity 72, no. 3 (March 2004): 1248–56. http://dx.doi.org/10.1128/iai.72.3.1248-1256.2004.

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ABSTRACT Aeromonas hydrophila is a gram-negative opportunistic pathogen in fish and humans. Many bacterial pathogens of animals and plants have been shown to inject anti-host virulence determinants into the hosts via a type III secretion system (TTSS). Degenerate primers based on lcrD family genes that are present in every known TTSS allowed us to locate the TTSS gene cluster in A. hydrophila AH-1. A series of genome walking steps helped in the identification of 25 open reading frames that encode proteins homologous to those in TTSSs in other bacteria. PCR-based analysis showed the presence of lcrD homologs (ascV) in all of the 33 strains of A. hydrophila isolated from various sources. Insertional inactivation of two of the TTSS genes (aopB and aopD) led to decreased cytotoxicity in carp epithelial cells, increased phagocytosis, and reduced virulence in blue gourami. These results show that a TTSS is required for A. hydrophila pathogenesis. This is the first report of sequencing and characterization of TTSS gene clusters from A. hydrophila. The TTSS identified here may help in developing suitable vaccines as well as in further understanding of the pathogenesis of A. hydrophila.

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Morse, Kaitlyn, Junzo Norimine, Guy H. Palmer, Eric L. Sutten, Timothy V. Baszler, and Wendy C. Brown. "Association and Evidence for Linked Recognition of Type IV Secretion System Proteins VirB9-1, VirB9-2, and VirB10 in Anaplasma marginale." Infection and Immunity 80, no. 1 (October 28, 2011): 215–27. http://dx.doi.org/10.1128/iai.05798-11.

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ABSTRACTLike several other bacterial pathogens,Anaplasma marginalehas an outer membrane that induces complete protection from infection and disease. However, the proteins that confer protective immunity and whether protection requires interacting proteins and/or linked T-cell and immunoglobulin G epitopes are not known. Our goal is to target the conserved type IV secretion system (T4SS) to identify conserved, immunogenic membrane proteins that are interacting and linked recognition candidates. Linked recognition is a process by which a B cell is optimally activated by a helper T cell that responds to the same, or physically associated, antigen.A. marginaleT4SS proteins VirB2, VirB4-1, VirB4-2, VirB6-1, VirB7, VirB8-2, VirB9-1, VirB9-2, VirB10, VirB11, and VirD4 were screened for their ability to induce IgG and to stimulate CD4+T cells from outer membrane-vaccinated cattle. VirB9-1, VirB9-2, and VirB10 induced the strongest IgG and T-cell responses in the majority of cattle, although three animals with major histocompatibility complex class II DRB3 restriction fragment length polymorphism types 8/23, 3/16, and 16/27 lacked T-cell responses to VirB9-1, VirB9-1 and VirB9-2, or VirB9-2 and VirB10, respectively. For these animals, VirB9-1-, VirB9-2-, and VirB10-specific IgG production may be associated with T-cell help provided by responses to an interacting protein partner(s). Interacting protein partners indicated by far-Western blotting were confirmed by immunoprecipitation assays and revealed, for the first time, specific interactions of VirB9-1 with VirB9-2 and VirB10. The immunogenicity and interactions of VirB9-1, VirB9-2, and VirB10 justify their testing as a linked protein vaccine againstA. marginale.

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Misselwitz, Benjamin, Saskia K. Kreibich, Samuel Rout, Bärbel Stecher, Balamurugan Periaswamy, and Wolf-Dietrich Hardt. "Salmonella entericaSerovar Typhimurium Binds to HeLa Cells via Fim-Mediated Reversible Adhesion and Irreversible Type Three Secretion System 1-Mediated Docking." Infection and Immunity 79, no. 1 (October 25, 2010): 330–41. http://dx.doi.org/10.1128/iai.00581-10.

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ABSTRACTThe food-borne pathogenSalmonella entericaserovar Typhimurium invades mammalian epithelial cells. This multistep process comprises bacterial binding to the host cell, activation of theSalmonellatype three secretion system 1 (T1), injection of effector proteins, triggering of host cell actin rearrangements, andS. Typhimurium entry. While the latter steps are well understood, much less is known about the initial binding step. Earlier work had implicated adhesins (but not T1) or T1 (but not other adhesins). We have studied here theSalmonellavirulence factors mediatingS. Typhimurium binding to HeLa cells. Using an automated microscopy assay and isogenicS. Typhimurium mutants, we analyzed the role of T1 and of several known adhesins (Fim, Pef, Lpf, Agf, and Shd) in host cell binding. In wild-typeS. Typhimurium, host cell binding was mostly attributable to T1. However, in the absence of T1, Fim (but not Pef, Lpf, Agf, and Shd) also mediated HeLa cell binding. Furthermore, in the absence of T1 and type I fimbriae (Fim), we still observed residual binding, pointing toward at least one additional, unidentified binding mechanism. Dissociation experiments established that T1-mediated binding was irreversible (“docking”), while Fim-mediated binding was reversible (“reversible adhesion”). Finally, we show that noninvasive bacteria docking via T1 or adhering via Fim can efficiently invade HeLa cells, if actin rearrangements are triggeredin transby a wild-typeS. Typhimurium helper strain. Our data show that binding to HeLa cells is mediated by at least two different mechanisms and that both can lead to invasion if actin rearrangements are triggered.

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D'Souza, S., V. Rosseels, M. Romano, A. Tanghe, O. Denis, F. Jurion, N. Castiglione, A. Vanonckelen, K. Palfliet, and Kris Huygen. "Mapping of Murine Th1 Helper T-Cell Epitopes of Mycolyl Transferases Ag85A, Ag85B, and Ag85C from Mycobacterium tuberculosis." Infection and Immunity 71, no. 1 (January 2003): 483–93. http://dx.doi.org/10.1128/iai.71.1.483-493.2003.

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ABSTRACT BALB/c (H-2d ) and C57BL/6 (H-2b ) mice were infected intravenously with Mycobacterium tuberculosis H37Rv or vaccinated intramuscularly with plasmid DNA encoding each of the three mycolyl transferases Ag85A, Ag85B, and Ag85C from M. tuberculosis. Th1-type spleen cell cytokine secretion of interleukin-2 (IL-2) and gamma interferon (IFN-γ) was analyzed in response to purified Ag85 components and synthetic overlapping peptides covering the three mature sequences. Tuberculosis-infected C57BL/6 mice reacted strongly to some peptides from Ag85A and Ag85B but not from Ag85C, whereas tuberculosis-infected BALB/c mice reacted only to peptides from Ag85A. In contrast, spleen cells from both mouse strains produced elevated levels of IL-2 and IFN-γ following vaccination with Ag85A, Ag85B, and Ag85C DNA in response to peptides of the three Ag85 proteins, and the epitope repertoire was broader than in infected mice. Despite pronounced sequence homology, a number of immunodominant regions contained component specific epitopes. Thus, BALB/c mice vaccinated with all three Ag85 genes reacted against the same amino acid region, 101 to 120, that was also immunodominant for Ag85A in M. bovis BCG-vaccinated and tuberculosis-infected H-2d haplotype mice, but responses were completely component specific. In C57BL/6 mice, a cross-reactive T-cell response was detected against two carboxy-terminal peptides spanning amino acids 241 to 260 and 261 to 280 of Ag85A and Ag85B. These regions were not recognized at all in C57BL/6 mice vaccinated with Ag85C DNA. Our results underline the need for comparative analysis of all three Ag85 components in future vaccination studies.

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Lindeberg, Magdalen, Samuel Cartinhour, Christopher R. Myers, Lisa M. Schechter, David J. Schneider, and Alan Collmer. "Closing the Circle on the Discovery of Genes Encoding Hrp Regulon Members and Type III Secretion System Effectors in the Genomes of Three Model Pseudomonas syringae Strains." Molecular Plant-Microbe Interactions® 19, no. 11 (November 2006): 1151–58. http://dx.doi.org/10.1094/mpmi-19-1151.

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Pseudomonas syringae strains translocate large and distinct collections of effector proteins into plant cells via the type III secretion system (T3SS). Mutations in T3SS-encoding hrp genes are unable to elicit the hypersensitive response or pathogenesis in nonhost and host plants, respectively. Mutations in individual effectors lack strong phenotypes, which has impeded their discovery. P. syringae effectors are designated Hop (Hrp outer protein) or Avr (avirulence) proteins. Some Hop proteins are considered to be extracellular T3SS helpers acting at the plant-bacterium interface. Identification of complete sets of effectors and related proteins has been enabled by the application of bioinformatic and high-throughput experimental techniques to the complete genome sequences of three model strains: P. syringae pv. tomato DC3000, P. syringae pv. phaseolicola 1448A, and P. syringae pv. syringae B728a. Several recent papers, including three in this issue of Molecular Plant-Microbe Interactions, address the effector inventories of these strains. These studies establish that active effector genes in P. syringae are expressed by the HrpL alternative sigma factor and can be predicted on the basis of cis Hrp promoter sequences and N-terminal amino-acid patterns. Among the three strains analyzed, P. syringae pv. tomato DC3000 has the largest effector inventory and P. syringae pv. syringae B728a has the smallest. Each strain has several effector genes that appear inactive. Only five of the 46 effector families that are represented in these three strains have an active member in all of the strains. Web-based community resources for managing and sharing growing information on these complex effector arsenals should help future efforts to understand how effectors promote P. syringae virulence.

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Zhao, Yue, Jianjin Shi, Xuyan Shi, Yupeng Wang, Fengchao Wang, and Feng Shao. "Genetic functions of the NAIP family of inflammasome receptors for bacterial ligands in mice." Journal of Experimental Medicine 213, no. 5 (April 25, 2016): 647–56. http://dx.doi.org/10.1084/jem.20160006.

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Biochemical studies suggest that the NAIP family of NLR proteins are cytosolic innate receptors that directly recognize bacterial ligands and trigger NLRC4 inflammasome activation. In this study, we generated Naip5−/−, Naip1−/−, and Naip2−/− mice and showed that bone marrow macrophages derived from these knockout mice are specifically deficient in detecting bacterial flagellin, the type III secretion system needle, and the rod protein, respectively. Naip1−/−, Naip2−/−, and Naip5−/− mice also resist lethal inflammasome activation by the corresponding ligand. Furthermore, infections performed in the Naip-deficient macrophages have helped to define the major signal in Legionella pneumophila, Salmonella Typhimurium and Shigella flexneri that is detected by the NAIP/NLRC4 inflammasome. Using an engineered S. Typhimurium infection model, we demonstrate the critical role of NAIPs in clearing bacterial infection and protecting mice from bacterial virulence–induced lethality. These results provide definitive genetic evidence for the important physiological function of NAIPs in antibacterial defense and inflammatory damage–induced lethality in mice.

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Medina, Eva, Paola Paglia, Thomas Nikolaus, Astrid Müller, Michael Hensel, and Carlos A. Guzmán. "Pathogenicity Island 2 Mutants of Salmonella typhimurium Are Efficient Carriers for Heterologous Antigens and Enable Modulation of Immune Responses." Infection and Immunity 67, no. 3 (March 1, 1999): 1093–99. http://dx.doi.org/10.1128/iai.67.3.1093-1099.1999.

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ABSTRACT The potential use as vaccine delivery system of Salmonella typhimurium strains harboring defined mutations in thesseC (HH104) and sseD (MvP101) genes, which encode putative effector proteins of the type III secretion system ofSalmonella pathogenicity island 2, was evaluated and compared with that of the well-characterized aroA mutant strain SL7207 by using β-galactosidase (β-Gal) as a model antigen. When orally administered to immune-competent or gamma interferon-deficient (IFN-γ−/−) BALB/c mice, both mutants were found to be highly attenuated (50% lethal dose, >109 bacteria). Both strains were also able to efficiently colonize and persist in Peyer’s patches. Immunization with HH104 and MvP101 triggered β-Gal-specific serum and mucosal antibody responses equivalent to or stronger than those observed in SL7207-immunized mice. Although immunoglobulin G2 (IgG2) serum antibodies were dominant in all groups, IgG1 was also significantly increased in mice vaccinated with MvP101 and SL7207. Comparable β-Gal-specific IgA and IgG antibodies were detected in intestinal lavages from mice immunized with the different strains. Antigen-specific CD4+ T-helper cells were generated after vaccination with all vaccine prototypes; however, responses were significantly more efficient when HH104 and MvP101 were used (P < 0.05). Significantly higher levels of IFN-γ were produced by restimulated spleen cells from mice immunized with HH104 than from those vaccinated with the MvP101 or SL7207 derivatives (P ≤ 0.05). Interestingly, the three strains induced major histocompatibility complex class I-restricted CD8+ cytotoxic T cells against β-Gal; however, cytotoxic T-lymphocyte responses were significantly stronger after immunization with HH104 (P < 0.05). These novel S. typhimurium attenuated strains constitute promising delivery systems for vaccine antigens. The qualitative differences observed in the obtained responses with different carriers may be useful for those applications in which a targeted immunomodulation is required.

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Piazza, Roxane M. F., Sabine Delannoy, Patrick Fach, Halha O. Saridakis, Margareth Z. Pedroso, Letícia B. Rocha, Tânia A. T. Gomes, Mônica A. M. Vieira, Lothar Beutin, and Beatriz E. C. Guth. "Molecular and Phenotypic Characterization of Escherichia coli O26:H8 among Diarrheagenic E. coli O26 Strains Isolated in Brazil." Applied and Environmental Microbiology 79, no. 22 (August 23, 2013): 6847–54. http://dx.doi.org/10.1128/aem.01693-13.

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ABSTRACTEscherichia colistrains of serogroup O26 comprise two distinct groups of pathogens, characterized as enteropathogenicE. coli(EPEC) and enterohemorrhagicE. coli(EHEC). Among the several genes related to type III secretion system-secreted effector proteins,espKwas found to be highly specific for EHEC O26:H11 and itsstx-negative derivative strains isolated in European countries.E. coliO26 strains isolated in Brazil from infant diarrhea, foods, and the environment have consistently been shown to lackstxgenes and are thus considered atypical EPEC. However, no further information related to their genetic background is known. Therefore, in this study, we aimed to discriminate and characterize these Brazilian O26stx-negative strains by phenotypic, genetic, and biochemical approaches. Among 44 isolates confirmed to be O26 isolates, most displayed flagellar antigen H11 or H32. Out of the 13 nonmotile isolates, 2 tested positive forfliCH11, and 11 werefliCH8positive. The identification of genetic markers showed that several O26:H11 and all O26:H8 strains tested positive forespKand could therefore be discriminated as EHEC derivatives. The presence of H8 among EHEC O26 and itsstx-negative derivative isolates is described for the first time. The interaction of three isolates with polarized Caco-2 cells and with intestinal biopsy specimen fragmentsex vivoconfirmed the ability of the O26 strains analyzed to cause attaching-and-effacing (A/E) lesions. The O26:H32 strains, isolated mostly from meat, were considered nonvirulent. Knowledge of the virulence content ofstx-negative O26 isolates within the same serotype helped to avoid misclassification of isolates, which certainly has important implications for public health surveillance.

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Coburn, Bryan, Inna Sekirov, and B. Brett Finlay. "Type III Secretion Systems and Disease." Clinical Microbiology Reviews 20, no. 4 (October 2007): 535–49. http://dx.doi.org/10.1128/cmr.00013-07.

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SUMMARY Type III secretion systems (T3SSs) are complex bacterial structures that provide gram-negative pathogens with a unique virulence mechanism enabling them to inject bacterial effector proteins directly into the host cell cytoplasm, bypassing the extracellular milieu. Although the effector proteins vary among different T3SS pathogens, common pathogenic mechanisms emerge, including interference with the host cell cytoskeleton to promote attachment and invasion, interference with cellular trafficking processes, cytotoxicity and barrier dysfunction, and immune system subversion. The activity of the T3SSs correlates closely with infection progression and outcome, both in animal models and in human infection. Therefore, to facilitate patient care and improve outcomes, it is important to understand the T3SS-mediated virulence processes and to target T3SSs in therapeutic and prophylactic development efforts.

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Hanson, Brett R., Anatoly Slepenkin, Ellena M. Peterson, and Ming Tan. "Chlamydia trachomatis Type III Secretion Proteins Regulate Transcription." Journal of Bacteriology 197, no. 20 (July 27, 2015): 3238–44. http://dx.doi.org/10.1128/jb.00379-15.

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ABSTRACTThe Scc4 protein (CT663) of the pathogenic bacteriumChlamydiahas been described as a type III secretion (T3S) chaperone as well as an inhibitor of RNA polymerase. To examine if these roles are connected, we first investigated physical interactions betweenChlamydia trachomatisScc4 and the T3S chaperone Scc1 and a T3S substrate, CopN. In a yeast 3-hybrid assay, Scc4, Scc1, and CopN were all required to detect an interaction, which suggests that these proteins form a trimolecular complex. We also detected interactions between any two of these three T3S proteins in a pulldown assay using only recombinant proteins. We next determined whether these interactions affected the function of Scc4 as an inhibitor of RNA transcription. UsingEscherichia colias a heterologousin vivosystem, we demonstrated that expression ofC. trachomatisScc4 led to a drastic decrease in transcript levels for multiple genes. However, coexpression of Scc4 with Scc1, CopN, or both alleviated Scc4-mediated inhibition of transcription. Scc4 expression also severely impairedE. coligrowth, but this growth defect was reversed by coexpression of Scc4 with Scc1, CopN, or both, suggesting that the inhibitory effect of Scc4 on transcription and growth can be antagonized by interactions between Scc4, Scc1, and CopN. These findings suggest that the dual functions of Scc4 may serve as a bridge to link T3S and the regulation of gene expression inChlamydia.IMPORTANCEThis study investigates a novel mechanism for regulating gene expression in the pathogenic bacteriumChlamydia. TheChlamydiatype III secretion (T3S) chaperone Scc4 has been shown to inhibit transcription by RNA polymerase. This study describes physical interactions between Scc4 and the T3S proteins Scc1 and CopN. Furthermore,ChlamydiaScc1 and CopN antagonized the inhibitory effects of Scc4 on transcription and growth in a heterologousEscherichia colisystem. These results provide evidence that transcription inChlamydiacan be regulated by the T3S system through interactions between T3S proteins.

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Galán, Jorge E. "SnapShot: Effector Proteins of Type III Secretion Systems." Cell 130, no. 1 (July 2007): 192.e1–192.e2. http://dx.doi.org/10.1016/j.cell.2007.06.042.

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Hueck, Christoph J. "Type III Protein Secretion Systems in Bacterial Pathogens of Animals and Plants." Microbiology and Molecular Biology Reviews 62, no. 2 (June 1, 1998): 379–433. http://dx.doi.org/10.1128/mmbr.62.2.379-433.1998.

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SUMMARY Various gram-negative animal and plant pathogens use a novel, sec-independent protein secretion system as a basic virulence mechanism. It is becoming increasingly clear that these so-called type III secretion systems inject (translocate) proteins into the cytosol of eukaryotic cells, where the translocated proteins facilitate bacterial pathogenesis by specifically interfering with host cell signal transduction and other cellular processes. Accordingly, some type III secretion systems are activated by bacterial contact with host cell surfaces. Individual type III secretion systems direct the secretion and translocation of a variety of unrelated proteins, which account for species-specific pathogenesis phenotypes. In contrast to the secreted virulence factors, most of the 15 to 20 membrane-associated proteins which constitute the type III secretion apparatus are conserved among different pathogens. Most of the inner membrane components of the type III secretion apparatus show additional homologies to flagellar biosynthetic proteins, while a conserved outer membrane factor is similar to secretins from type II and other secretion pathways. Structurally conserved chaperones which specifically bind to individual secreted proteins play an important role in type III protein secretion, apparently by preventing premature interactions of the secreted factors with other proteins. The genes encoding type III secretion systems are clustered, and various pieces of evidence suggest that these systems have been acquired by horizontal genetic transfer during evolution. Expression of type III secretion systems is coordinately regulated in response to host environmental stimuli by networks of transcription factors. This review comprises a comparison of the structure, function, regulation, and impact on host cells of the type III secretion systems in the animal pathogens Yersinia spp., Pseudomonas aeruginosa, Shigella flexneri, Salmonella typhimurium, enteropathogenic Escherichia coli, and Chlamydia spp. and the plant pathogens Pseudomonas syringae, Erwinia spp., Ralstonia solanacearum, Xanthomonas campestris, and Rhizobium spp.

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LeBlanc, Marc-André, Morgan R. Fink, Thomas T. Perkins, and Marcelo C. Sousa. "Type III secretion system effector proteins are mechanically labile." Proceedings of the National Academy of Sciences 118, no. 12 (March 15, 2021): e2019566118. http://dx.doi.org/10.1073/pnas.2019566118.

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Multiple gram-negative bacteria encode type III secretion systems (T3SS) that allow them to inject effector proteins directly into host cells to facilitate colonization. To be secreted, effector proteins must be at least partially unfolded to pass through the narrow needle-like channel (diameter <2 nm) of the T3SS. Fusion of effector proteins to tightly packed proteins—such as GFP, ubiquitin, or dihydrofolate reductase (DHFR)—impairs secretion and results in obstruction of the T3SS. Prior observation that unfolding can become rate-limiting for secretion has led to the model that T3SS effector proteins have low thermodynamic stability, facilitating their secretion. Here, we first show that the unfolding free energy (ΔGunfold0) of two Salmonella effector proteins, SptP and SopE2, are 6.9 and 6.0 kcal/mol, respectively, typical for globular proteins and similar to published ΔGunfold0 for GFP, ubiquitin, and DHFR. Next, we mechanically unfolded individual SptP and SopE2 molecules by atomic force microscopy (AFM)-based force spectroscopy. SptP and SopE2 unfolded at low force (Funfold ≤ 17 pN at 100 nm/s), making them among the most mechanically labile proteins studied to date by AFM. Moreover, their mechanical compliance is large, as measured by the distance to the transition state (Δx‡ = 1.6 and 1.5 nm for SptP and SopE2, respectively). In contrast, prior measurements of GFP, ubiquitin, and DHFR show them to be mechanically robust (Funfold > 80 pN) and brittle (Δx‡ < 0.4 nm). These results suggest that effector protein unfolding by T3SS is a mechanical process and that mechanical lability facilitates efficient effector protein secretion.

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Markham, Aaron P., Susan E. Birket, William D. Picking, Wendy L. Picking, and C. Russell Middaugh. "pH sensitivity of type III secretion system tip proteins." Proteins: Structure, Function, and Bioinformatics 71, no. 4 (January 3, 2008): 1830–42. http://dx.doi.org/10.1002/prot.21864.

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Kim, Jaewha, Kyungseop Ahn, Sungran Min, Jinghua Jia, Unhwan Ha, Donghai Wu, and Shouguang Jin. "Factors triggering type III secretion in Pseudomonas aeruginosa." Microbiology 151, no. 11 (November 1, 2005): 3575–87. http://dx.doi.org/10.1099/mic.0.28277-0.

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The type III secretion system of Pseudomonas aeruginosa is tightly regulated by various environmental signals, such as low calcium and contact with the host cell. However, the exact signals triggering type III secretion are unknown. The present study describes the finding that secretion of P. aeruginosa type III effector molecules requires protein factors from serum and L broth, designated type III secretion factors (TSFs), in addition to the low-calcium environment. In the absence of TSF or calcium chelator EGTA, basal levels of type III effector molecules are accumulated intracellularly. Addition of TSF and EGTA together effectively triggers the secretion of pre-existing effector molecules in a short time, even before the active expression of type III genes; thus, active type III gene expression does not seem to be a prerequisite for type III secretion. A search for TSF molecules in serum and L broth resulted in the identification of albumin and casein as the functional TSF molecules. Although there is no clear sequence similarity between albumin and casein, both proteins are known to have a low-affinity, high-capacity calcium-binding property. Tests of well-studied calcium-binding proteins seemed to indicate that low-affinity calcium-binding proteins have TSF activity, although the requirement of low-affinity calcium-binding ability for the TSF activity is not clear. P. aeruginosa seems to have evolved a sensing mechanism to detect target cells for type III injection through host-derived proteins in combination with a low-calcium signal. Disruption of the bacterial ability to sense low calcium or TSF might be a valid avenue to the effective control of this bacterial pathogen.

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Pan, Ning J., Michael J. Brady, John M. Leong, and Jon D. Goguen. "Targeting Type III Secretion in Yersinia pestis." Antimicrobial Agents and Chemotherapy 53, no. 2 (November 17, 2008): 385–92. http://dx.doi.org/10.1128/aac.00670-08.

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ABSTRACT Yersinia pestis, the causative agent of plague, utilizes a plasmid-encoded type III secretion system (T3SS) to aid it with its resistance to host defenses. This system injects a set of effector proteins known as Yops (Yersinia outer proteins) into the cytosol of host cells that come into contact with the bacteria. T3SS is absolutely required for the virulence of Y. pestis, making it a potential target for new therapeutics. Using a novel and simple high-throughput screening method, we examined a diverse collection of chemical libraries for small molecules that inhibit type III secretion in Y. pestis. The primary screening of 70,966 compounds and mixtures yielded 421 presumptive inhibitors. We selected eight of these for further analysis in secondary assays. Four of the eight compounds effectively inhibited Yop secretion at micromolar concentrations. Interestingly, we observed differential inhibition among Yop species with some compounds. The compounds did not inhibit bacterial growth at the concentrations used in the inhibition assays. Three compounds protected HeLa cells from type III secretion-dependent cytotoxicity. Of the eight compounds examined in secondary assays, four show good promise as leads for structure-activity relationship studies. They are a diverse group, with each having a chemical scaffold not only distinct from each other but also distinct from previously described candidate type III secretion inhibitors.

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Ebanks, Roger O., Leah C. Knickle, Michel Goguen, Jessica M. Boyd, Devanand M. Pinto, Michael Reith, and Neil W. Ross. "Expression of and secretion through the Aeromonas salmonicida type III secretion system." Microbiology 152, no. 5 (May 1, 2006): 1275–86. http://dx.doi.org/10.1099/mic.0.28485-0.

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Aeromonas salmonicida subsp. salmonicida is the aetiological agent of furunculosis, a disease of farmed and wild salmonids. The type III secretion system (TTSS) is one of the primary virulence factors in A. salmonicida. Using a combination of differential proteomic analysis and reverse transcriptase (RT)-PCR, it is shown that A. salmonicida A449 induces the expression of TTSS proteins at 28 °C, but not at its more natural growth temperature of 17 °C. More modest increases in expression occur at 24 °C. This temperature-induced up-regulation of the TTSS in A. salmonicida A449 occurs within 30 min of a growth temperature increase from 16 to 28 °C. Growth conditions such as low-iron, low pH, low calcium, growth within the peritoneal cavity of salmon and growth to high cell densities do not induce the expression of the TTSS in A. salmonicida A449. The only other known growth condition that induces expression of the TTSS is growth of the bacterium at 16 °C in salt concentrations ranging from 0·19 to 0·38 M NaCl. It is also shown that growth at 28 °C followed by exposure to low calcium results in the secretion of one of the TTSS effector proteins. This study presents a simple in vitro model for the expression of TTSS proteins in A. salmonicida.

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Warawa, Jonathan, B. Brett Finlay, and Brendan Kenny. "Type III Secretion-Dependent Hemolytic Activity of Enteropathogenic Escherichia coli." Infection and Immunity 67, no. 10 (October 1, 1999): 5538–40. http://dx.doi.org/10.1128/iai.67.10.5538-5540.1999.

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ABSTRACT Enteropathogenic Escherichia coli (EPEC) was found to exhibit a type III secretion-dependent, contact-mediated, hemolytic activity requiring the EspA, EspB, and EspD secreted proteins. EspB and EspD display homology to pore-forming molecules. Our data suggest a mechanism to explain the requirement for all three Esp proteins in the transfer of EPEC proteins, such as Tir, into target cells.

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20

Araujo-Garrido, Juan Luis, Joaquín Bernal-Bayard, and Francisco Ramos-Morales. "Type III Secretion Effectors with Arginine N-Glycosyltransferase Activity." Microorganisms 8, no. 3 (March 2, 2020): 357. http://dx.doi.org/10.3390/microorganisms8030357.

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Type III secretion systems are used by many Gram-negative bacterial pathogens to inject proteins, known as effectors, into the cytosol of host cells. These virulence factors interfere with a diverse array of host signal transduction pathways and cellular processes. Many effectors have catalytic activities to promote post-translational modifications of host proteins. This review focuses on a family of effectors with glycosyltransferase activity that catalyze addition of N-acetyl-d-glucosamine to specific arginine residues in target proteins, leading to reduced NF-κB pathway activation and impaired host cell death. This family includes NleB from Citrobacter rodentium, NleB1 and NleB2 from enteropathogenic and enterohemorrhagic Escherichia coli, and SseK1, SseK2, and SseK3 from Salmonella enterica. First, we place these effectors in the general framework of the glycosyltransferase superfamily and in the particular context of the role of glycosylation in bacterial pathogenesis. Then, we provide detailed information about currently known members of this family, their role in virulence, and their targets.

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21

Sorg, Joseph A., Nathan C. Miller, Melanie M. Marketon, and Olaf Schneewind. "Rejection of Impassable Substrates by Yersinia Type III Secretion Machines." Journal of Bacteriology 187, no. 20 (October 15, 2005): 7090–102. http://dx.doi.org/10.1128/jb.187.20.7090-7102.2005.

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ABSTRACT Type III machines of pathogenic Yersinia spp. transport Yop proteins across the bacterial envelope into host cells. Translational fusions of yopE to the dihydrofolate reductase gene (dhfr) or the β-galactosidase gene (lacZ) generate hybrid proteins that block type III injection of Yop proteins into host cells, consistent with the canonical view that impassable DHFR and LacZ hybrids jam secretion machines. Mutations in repressors of posttranscriptional gene regulation, Yersinia enterocolitica yscM1 and yscM2 as well as Yersinia pestis lcrQ, relieve the YopE-DHFR-imposed blockade and restore type III injection into host cells. Genetic suppression of the type III blockade does not, however, promote YopE-DHFR secretion. A model is proposed whereby rejection of YopE-DHFR from the secretion pathway inhibits type III gene expression.

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22

Ince, Dilek, Fayyaz S. Sutterwala, and Timothy L. Yahr. "Secretion of Flagellar Proteins by the Pseudomonas aeruginosa Type III Secretion-Injectisome System." Journal of Bacteriology 197, no. 12 (April 6, 2015): 2003–11. http://dx.doi.org/10.1128/jb.00030-15.

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ABSTRACTThe opportunistic pathogenPseudomonas aeruginosautilizes an injectisome-type III secretion system (injectisome-T3SS) to elicit cytotoxicity toward epithelial cells and macrophages. Macrophage killing results from the cytotoxic properties of the translocated effector proteins (ExoS, ExoT, ExoU, and ExoY) and inflammasome-mediated induction of pyroptosis. Inflammasome activation can occur following Nlrc4-mediated recognition of cytosolic translocated flagellin (FliC). In the present study, we demonstrate that FliC is a secretion substrate of both the injectisome- and flagellum-associated T3SSs. Molecular analyses indicate that the first 20 amino-terminal residues of FliC are sufficient for secretion by the injectisome-T3SS and that the first 100 residues are sufficient for translocation of FliC into host cells. Although maximal inflammasome activation requires FliC, activation can also occur in the absence of FliC. This prompted us to examine whether other flagellar components might also be translocated into cells to elicit inflammasome activation. Indeed, we find that the flagellar cap (FliD), hook-associated (FlgK and FlgL), hook (FlgE), and rod (FlgE) proteins are secretion substrates of the injectisome-T3SS. None of these proteins, however, result in increased inflammasome activation when they are overexpressed in afliCmutant and appear to be translocated into host cells. While a role in inflammasome activation has been excluded, these data raise the possibility that flagellar components, which are highly conserved between different bacterial species, trigger other specific host responses from the extracellular milieu or contribute to the pathogenesis ofP. aeruginosa.IMPORTANCEThe inflammasome is a host defense mechanism that recognizes invading bacteria and triggers an inflammatory immune response. The opportunistic pathogenP. aeruginosaproduces both inflammasome agonists and antagonists. In this study, we demonstrate that overexpression of an agonist suppresses the activity of an antagonist, thereby resulting in inflammasome activation. Since the relative expression levels of agonists and antagonists likely vary between strains, these differences could be important predictors of whether a particularP. aeruginosastrain elicits inflammasome activation.

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23

Troisfontaines, Paul, and Guy R. Cornelis. "Type III Secretion: More Systems Than You Think." Physiology 20, no. 5 (October 2005): 326–39. http://dx.doi.org/10.1152/physiol.00011.2005.

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The type III secretion (T3S) pathway allows bacteria to inject effector proteins into the cytosol of target animal or plant cells. T3S systems evolved into seven families that were distributed among Gram-negative bacteria by horizontal gene transfer. There are probably a few hundred effectors interfering with control and signaling in eukaryotic cells and offering a wealth of new tools to cell biologists.

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24

Collmer, A., J. L. Badel, A. O. Charkowski, W. L. Deng, D. E. Fouts, A. R. Ramos, A. H. Rehm, et al. "Pseudomonas syringae Hrp type III secretion system and effector proteins." Proceedings of the National Academy of Sciences 97, no. 16 (August 1, 2000): 8770–77. http://dx.doi.org/10.1073/pnas.97.16.8770.

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25

Hansen-Wester, Imke, Bärbel Stecher, and Michael Hensel. "Type III Secretion of Salmonella enterica Serovar Typhimurium Translocated Effectors and SseFG." Infection and Immunity 70, no. 3 (March 2002): 1403–9. http://dx.doi.org/10.1128/iai.70.3.1403-1409.2002.

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ABSTRACT The type III secretion system (TTSS) encoded by Salmonella enterica serovar Typhimurium pathogenicity island 2 (SPI2) is employed by Salmonella enterica for interaction with host cells during the intracellular phase of pathogenesis. This TTSS secretes a set of SPI2-encoded proteins in vitro and translocates Salmonella serovar Typhimurium translocated effectors (STE) that are encoded by genes outside of SPI2 into host cells. Using an epitope-tagging approach, we analyzed secretion of proteins by the TTSS of SPI2 and identified SseF and SseG as further secreted substrate proteins. Three members of the STE family, SifA, SifB, and SseJ, were secreted under conditions that also induce secretion of SPI2-encoded substrate proteins.

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26

Veenendaal, Andreas K. J., Charlotta Sundin, and Ariel J. Blocker. "Small-Molecule Type III Secretion System Inhibitors Block Assembly of the Shigella Type III Secreton." Journal of Bacteriology 191, no. 2 (November 7, 2008): 563–70. http://dx.doi.org/10.1128/jb.01004-08.

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ABSTRACT Type III secretion systems (T3SSs) are essential virulence devices for many gram-negative bacteria that are pathogenic for plants, animals, and humans. They serve to translocate virulence effector proteins directly into eukaryotic host cells. T3SSs are composed of a large cytoplasmic bulb and a transmembrane region into which a needle is embedded, protruding above the bacterial surface. The emerging antibiotic resistance of bacterial pathogens urges the development of novel strategies to fight bacterial infections. Therapeutics that rather than kill bacteria only attenuate their virulence may reduce the frequency or progress of resistance emergence. Recently, a group of salicylidene acylhydrazides were identified as inhibitors of T3SSs in Yersinia, Chlamydia, and Salmonella species. Here we show that these are also effective on the T3SS of Shigella flexneri, where they block all related forms of protein secretion so far known, as well as the epithelial cell invasion and induction of macrophage apoptosis usually demonstrated by this bacterium. Furthermore, we show the first evidence for the detrimental effect of these compounds on T3SS needle assembly, as demonstrated by increased numbers of T3S apparatuses without needles or with shorter needles. Therefore, the compounds generate a phenocopy of T3SS export apparatus mutants but with incomplete penetrance. We discuss why this would be sufficient to almost completely block the later secretion of effector proteins and how this begins to narrow the search for the molecular target of these compounds.

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27

Ghosh, Partho. "Process of Protein Transport by the Type III Secretion System." Microbiology and Molecular Biology Reviews 68, no. 4 (December 2004): 771–95. http://dx.doi.org/10.1128/mmbr.68.4.771-795.2004.

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SUMMARY The type III secretion system (TTSS) of gram-negative bacteria is responsible for delivering bacterial proteins, termed effectors, from the bacterial cytosol directly into the interior of host cells. The TTSS is expressed predominantly by pathogenic bacteria and is usually used to introduce deleterious effectors into host cells. While biochemical activities of effectors vary widely, the TTSS apparatus used to deliver these effectors is conserved and shows functional complementarity for secretion and translocation. This review focuses on proteins that constitute the TTSS apparatus and on mechanisms that guide effectors to the TTSS apparatus for transport. The TTSS apparatus includes predicted integral inner membrane proteins that are conserved widely across TTSSs and in the basal body of the bacterial flagellum. It also includes proteins that are specific to the TTSS and contribute to ring-like structures in the inner membrane and includes secretin family members that form ring-like structures in the outer membrane. Most prominently situated on these coaxial, membrane-embedded rings is a needle-like or pilus-like structure that is implicated as a conduit for effector translocation into host cells. A short region of mRNA sequence or protein sequence in effectors acts as a signal sequence, directing proteins for transport through the TTSS. Additionally, a number of effectors require the action of specific TTSS chaperones for efficient and physiologically meaningful translocation into host cells. Numerous models explaining how effectors are transported into host cells have been proposed, but understanding of this process is incomplete and this topic remains an active area of inquiry.

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Munera, Diana, Valerie F. Crepin, Olivier Marches, and Gad Frankel. "N-Terminal Type III Secretion Signal of Enteropathogenic Escherichia coli Translocator Proteins." Journal of Bacteriology 192, no. 13 (April 16, 2010): 3534–39. http://dx.doi.org/10.1128/jb.00046-10.

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ABSTRACT We report that the N terminus of the type III secretion system translocator proteins EspB, EspD, and EspA mediate protein secretion and translocation from wild-type enteropathogenic Escherichia coli and hypersecretion from sepL and sepD mutants. EspA containing the translocation signal of Map and Tir containing the secretion signal of EspA are biologically active.

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29

Zhou, Xiaohui, Seth D. Nydam, Jeffrey E. Christensen, Michael E. Konkel, Lisa Orfe, Patrick Friel, and Douglas R. Call. "Identification of Potential Type III Secretion Proteins via Heterologous Expression of Vibrio parahaemolyticus DNA." Applied and Environmental Microbiology 78, no. 9 (March 2, 2012): 3492–94. http://dx.doi.org/10.1128/aem.07977-11.

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ABSTRACTWe employed a heterologous secretion assay to identify proteins potentially secreted by type III secretion systems (T3SSs) inVibrio parahaemolyticus. N-terminal sequences from 32 proteins within T3SS genomic islands and seven proteins from elsewhere in the chromosome included proteins that were recognized for export by theYersinia enterocoliticaflagellar T3SS.

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30

Neves, Bianca C., Rosanna Mundy, Liljana Petrovska, Gordon Dougan, Stuart Knutton, and Gad Frankel. "CesD2 of Enteropathogenic Escherichia coli Is a Second Chaperone for the Type III Secretion Translocator Protein EspD." Infection and Immunity 71, no. 4 (April 2003): 2130–41. http://dx.doi.org/10.1128/iai.71.4.2130-2141.2003.

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ABSTRACT Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli are extracellular pathogens that employ a type III secretion system to export translocator and effector proteins, proteins which facilitates colonization of the mucosal surface of the intestine via formation of attaching and effacing (A/E) lesions. The genes encoding the proteins for A/E lesion formation are located on a pathogenicity island, termed the locus of enterocyte effacement (LEE), which contains eae encoding intimin as well as the type III secretion system and effector genes. Many type III secreted proteins are stabilized and maintained in a secretion-competent conformation in the bacterial cytosol by specific chaperone proteins. Three type III chaperones have been described thus far within the EPEC LEE region: CesD, for the translocator proteins EspB and EspD; CesT, for the effector proteins Tir and Map; and CesF, for EspF. In this study we report the characterization of CesD2 (previously Orf27), a second LEE-encoded chaperone for EspD. We show specific CesD2-EspD protein interaction which appears to be necessary for proper EspD secretion in vitro and pathogenesis in vivo as demonstrated in the A/E-lesion-forming mouse pathogen Citrobacter rodentium.

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31

Kato, Junya, Matthew Lefebre, and Jorge E. Galán. "Structural Features Reminiscent of ATP-Driven Protein Translocases Are Essential for the Function of a Type III Secretion-Associated ATPase." Journal of Bacteriology 197, no. 18 (July 13, 2015): 3007–14. http://dx.doi.org/10.1128/jb.00434-15.

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ABSTRACTMany bacterial pathogens and symbionts utilize type III secretion systems to interact with their hosts. These machines have evolved to deliver bacterial effector proteins into eukaryotic target cells to modulate a variety of cellular functions. One of the most conserved components of these systems is an ATPase, which plays an essential role in the recognition and unfolding of proteins destined for secretion by the type III pathway. Here we show that structural features reminiscent of other ATP-driven protein translocases are essential for the function of InvC, the ATPase associated with aSalmonella entericaserovar Typhimurium type III secretion system. Mutational and functional analyses showed that a two-helix-finger motif and a conserved loop located at the entrance of and within the predicted pore formed by the hexameric ATPase are essential for InvC function. These findings provide mechanistic insight into the function of this highly conserved component of type III secretion machines.IMPORTANCEType III secretion machines are essential for the virulence or symbiotic relationships of many bacteria. These machines have evolved to deliver bacterial effector proteins into host cells to modulate cellular functions, thus facilitating bacterial colonization and replication. An essential component of these machines is a highly conserved ATPase, which is necessary for the recognition and secretion of proteins destined to be delivered by the type III secretion pathway. Using modeling and structure and function analyses, we have identified structural features of one of these ATPases fromSalmonella entericaserovar Typhimurium that help to explain important aspects of its function.

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Ho, Theresa D., and Michael N. Starnbach. "The Salmonella enterica Serovar Typhimurium-Encoded Type III Secretion Systems Can Translocate Chlamydia trachomatis Proteins into the Cytosol of Host Cells." Infection and Immunity 73, no. 2 (February 2005): 905–11. http://dx.doi.org/10.1128/iai.73.2.905-911.2005.

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ABSTRACT Chlamydia trachomatis is an obligate, intracellular pathogen that is a major cause of preventable blindness and infertility worldwide. Although the published genome sequence suggests that C. trachomatis encodes a type III secretion system, the lack of genetic tools for studying Chlamydia has hindered the examination of this potentially important class of virulence genes. We have developed a technique to identify Chlamydia proteins that can be translocated into the host cell cytoplasm by a type III secretion system. We have selected several Chlamydia proteins and tagged them with a multiple peptide motif element called F8M4. Epitopes contained in the F8M4 tag allow us to use tools corresponding to different arms of the adaptive immune system to detect the expression and translocation of these proteins by Salmonella enterica serovar Typhimurium. In particular, CD8+-T-cell reactivity can be used to detect the translocation of F8M4-tagged proteins into the cytoplasm of host cells. We have found that CD8+-T-cell activity assays are sensitive enough to detect translocation of even a small amount of F8M4-tagged protein. We have used CD8+-T-cell activity to show that CopN, a Chlamydia protein previously shown to be translocated by Yersinia type III secretion, can be translocated by the Salmonella pathogenicity island 1 (SPI-1) type III secretion system. Additionally, we demonstrate that CopD and Pkn5, two Chlamydia proteins hypothesized to be substrates of a type III secretion system, are translocated via the SPI-2 type III secretion system of serovar Typhimurium. The epitope tag system described here can be used more generally to examine the expression and subcellular compartmentalization of bacterial proteins deployed during the interaction of pathogens with mammalian cells.

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Chen, Li-Mei, Gabriel Briones, Ruben O. Donis, and Jorge E. Galán. "Optimization of the Delivery of Heterologous Proteins by the Salmonella enterica Serovar Typhimurium Type III Secretion System for Vaccine Development." Infection and Immunity 74, no. 10 (October 2006): 5826–33. http://dx.doi.org/10.1128/iai.00375-06.

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ABSTRACT Type III protein secretion systems, which are organelles with the capacity to deliver bacterial proteins into host cells, have been adapted to deliver heterologous antigens for vaccine development. A limitation of these antigen delivery systems is that some proteins are not amenable to secretion through this pathway. We show here that proteins from the simian and human immunodeficiency viruses that are not permissive for secretion through a Salmonella enterica serovar Typhimurium type III secretion system can be modified to travel this secretion pathway by introduction of discrete mutations. Proteins optimized for secretion were presented more efficiently via the major histocompatibility complex class I pathway and were able to induce a better immune response.

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Ohgita, Takashi, Naoki Hayashi, Naomasa Gotoh, and Kentaro Kogure. "Suppression of type III effector secretion by polymers." Open Biology 3, no. 12 (December 2013): 130133. http://dx.doi.org/10.1098/rsob.130133.

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Bacteria secrete effector proteins required for successful infection and expression of toxicity into host cells. The type III secretion apparatus is involved in these processes. Previously, we showed that the viscous polymer polyethylene glycol (PEG) 8000 suppressed effector secretion by Pseudomonas aeruginosa . We thus considered that other viscous polymers might also suppress secretion. We initially showed that PEG200 (formed from the same monomer (ethylene glycol) as PEG8000, but which forms solutions of lower viscosity than the latter compound) did not decrease effector secretion. By contrast, alginate, a high-viscous polymer formed from mannuronic and guluronic acid, unlike PEG8000, effectively inhibited secretion. The effectiveness of PEG8000 and alginate in this regard was closely associated with polymer viscosity, but the nature of viscosity dependence differed between the two polymers. Moreover, not only the natural polymer alginate, but also mucin, which protects against infection, suppressed secretion. We thus confirmed that polymer viscosity contributes to the suppression of effector secretion, but other factors (e.g. electrostatic interaction) may also be involved. Moreover, the results suggest that regulation of bacterial secretion by polymers may occur naturally via the action of components of biofilm or mucin layer.

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Yang, Hongjing, Zhiying Shan, Jaewha Kim, Weihui Wu, Wei Lian, Lin Zeng, Laijun Xing, and Shouguang Jin. "Regulatory Role of PopN and Its Interacting Partners in Type III Secretion of Pseudomonas aeruginosa." Journal of Bacteriology 189, no. 7 (January 19, 2007): 2599–609. http://dx.doi.org/10.1128/jb.01680-06.

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ABSTRACT The type III secretion system (T3SS) of Pseudomonas aeruginosa plays a significant role in pathogenesis. We have previously identified type III secretion factor (TSF), which is required for effective secretion of the type III effector molecules, in addition to the low calcium signal. TSF includes many low-affinity high-capacity calcium binding proteins, such as serum albumin and casein. A search for the TSF binding targets on the bacterial outer membrane resulted in identification of PopN, a component of the T3SS that is readily detectable on the bacterial cell surface. PopN specifically interacts with Pcr1, and both popN and pcr1 mutants have a constitutive type III secretion phenotype, suggesting that the two proteins form a complex that functions as a T3SS repressor. Further analysis of the popN operon genes resulted in identification of protein-protein interactions between Pcr1 and Pcr4 and between Pcr4 and Pcr3, as well as between PopN and Pcr2 in the presence of PscB. Unlike popN and pcr1 mutants, pcr3 and pcr4 mutants are totally defective in type III secretion, while a pcr2 mutant exhibits reduced type III secretion. Interestingly, PopN, Pcr1, Pcr2, and Pcr4 are all secreted in a type III secretion machinery-dependent manner, while Pcr3 is not. These findings imply that these components have important regulatory roles in controlling type III secretion.

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Briones, Gabriel, Dirk Hofreuter, and Jorge E. Galán. "Cre Reporter System To Monitor the Translocation of Type III Secreted Proteins into Host Cells." Infection and Immunity 74, no. 2 (February 2006): 1084–90. http://dx.doi.org/10.1128/iai.74.2.1084-1090.2006.

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ABSTRACT Central to the study of type III secretion systems is the availability of reporter systems to monitor bacterial protein translocation into host cells. We report here the development of a bacteriophage P1 Cre recombinase-based system to monitor the translocation of bacterial proteins into mammalian cells. Bacteriophage P1 Cre recombinase fused to the secretion and translocation signals of Salmonella enterica serovar Typhimurium of the type III secreted protein SopE was secreted in a type III secretion system-dependent fashion. More importantly, the SopE-Cre chimera was translocated into host cells via the type III secretion system and activated the expression of luciferase and green fluorescent protein reporters of Cre recombinase activity.

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37

Stone, Chris B., David C. Bulir, Jodi D. Gilchrist, Raman K. Toor, and James B. Mahony. "Interactions between flagellar and type III secretion proteins in Chlamydia pneumoniae." BMC Microbiology 10, no. 1 (2010): 18. http://dx.doi.org/10.1186/1471-2180-10-18.

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38

Pallen, Mark J., Gordon Dougan, and Gad Frankel. "Coiled-coil domains in proteins secreted by type III secretion systems." Molecular Microbiology 25, no. 02 (July 1997): 423–25. http://dx.doi.org/10.1046/j.1365-2958.1997.4901850.x.

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39

Martinez-Becerra, Francisco J., Julian M. Kissmann, Jovita Diaz-McNair, Shyamal P. Choudhari, Amy M. Quick, Gabriela Mellado-Sanchez, John D. Clements, Marcela F. Pasetti, and Wendy L. Picking. "Broadly Protective Shigella Vaccine Based on Type III Secretion Apparatus Proteins." Infection and Immunity 80, no. 3 (December 27, 2011): 1222–31. http://dx.doi.org/10.1128/iai.06174-11.

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Shigellaspp. are food- and waterborne pathogens that cause severe diarrheal and dysenteric disease associated with high morbidity and mortality. Individuals most often affected are children under 5 years of age in the developing world. The existence of multipleShigellaserotypes and the heterogenic distribution of pathogenic strains, as well as emerging antibiotic resistance, require the development of a broadly protective vaccine. AllShigellaspp. utilize a type III secretion system (TTSS) to initiate infection. The type III secretion apparatus (TTSA) is the molecular needle and syringe that form the energized conduit between the bacterial cytoplasm and the host cell to transport effector proteins that manipulate cellular processes to benefit the pathogen. IpaB and IpaD form a tip complex atop the TTSA needle and are required for pathogenesis. Because they are common to all virulentShigellaspp., they are ideal candidate antigens for a subunit-based, broad-spectrum vaccine. We examined the immunogenicity and protective efficacy of IpaB and IpaD, alone or combined, coadministered with a double mutant heat-labile toxin (dmLT) fromEscherichia coli, used as a mucosal adjuvant, in a mouse model of intranasal immunization and pulmonary challenge. Robust systemic and mucosal antibody- and T cell-mediated immunities were induced against both proteins, particularly IpaB. Mice immunized in the presence of dmLT with IpaB alone or IpaB combined with IpaD were fully protected against lethal pulmonary infection withShigella flexneriandShigella sonnei. We provide the first demonstration that theShigellaTTSAs IpaB and IpaD are promising antigens for the development of a cross-protectiveShigellavaccine.

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Ide, Tina, Silke Michgehl, Sabine Knappstein, Gerhard Heusipp, and M. Alexander Schmidt. "Differential Modulation by Ca2+ of Type III Secretion of Diffusely Adhering Enteropathogenic Escherichia coli." Infection and Immunity 71, no. 4 (April 2003): 1725–32. http://dx.doi.org/10.1128/iai.71.4.1725-1732.2003.

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ABSTRACT Enteropathogenic Escherichia coli (EPEC) strains are a common cause of persistent diarrhea among infants, primarily in developing countries. The pathogenicity of EPEC is associated with the expression and secretion of bacterial proteins encoded by the chromosomal locus of enterocyte effacement (LEE). The LEE-encoded type III-secreted proteins EspA, EspB, and EspD are part of a molecular syringe, which is used by EPEC to translocate effector proteins directly into the cytoplasm of host cells. The type III-secreted translocated intimin receptor (Tir) protein is thought to be delivered by an Esp-dependent mechanism into the host cell, and this is followed by insertion into the host plasma membrane, where the protein serves as the receptor for intimin, an afimbrial bacterial adhesin. Type III secretion is subject to environmental regulation, and secretion can be induced in vitro by growing bacteria in cell culture medium. In this study we found that Ca2+ is involved in the regulation of type III secretion both in classical locally adherent EPEC and in atypical diffusely adherent EPEC. Interestingly, we observed contrasting secretion of Esp proteins and Tir in response to Ca2+. While the secretion of Tir is clearly enhanced and the protein is integrated into HeLa membranes under calcium chelation conditions, Esp secretion is strongly reduced under these conditions. These data suggest that under Ca2+-depleted conditions Tir might be secreted into the medium and integrated into host membranes by an Esp-independent mechanism, without the need for a functional type III translocation machinery.

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41

Klein, Joanna R., and Bradley D. Jones. "Salmonella Pathogenicity Island 2-Encoded Proteins SseC and SseD Are Essential for Virulence and Are Substrates of the Type III Secretion System." Infection and Immunity 69, no. 2 (February 1, 2001): 737–43. http://dx.doi.org/10.1128/iai.69.2.737-743.2001.

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ABSTRACT Survival of Salmonella enterica serovar Typhimurium within host phagocytic cells is a critical step in establishing systemic infection in mice. Genes within Salmonellapathogenicity island 2 (SPI-2) encode a type III secretion system that is required for establishment of systemic infection. Several proteins encoded by SPI-2 have homology to type III secreted proteins from enteropathogenic Escherichia coli and Yersiniaand, based on that homology, are predicted to be secreted through the SPI-2 type III secretion system. We have investigated the roles of two of these proteins, SseC and SseD. We demonstrate here that the SseD protein is required for systemic Salmonella infection of the mouse, and we confirmed the virulence requirement for the SseC protein. Experiments were performed, using cellular fractionation and immunoblotting, to identify the subcellular location of the SseC and SseD proteins. Both proteins were found to localize predominantly to the bacterial cell membrane. In addition, our work revealed that SseC and SseD are exposed to the extracellular environment and are loosely associated with the bacterial membrane. Furthermore, localization of SseC and SseD to the bacterial membrane was found to require a functional SPI-2 type III secretion system. Collectively, these results indicate that the SseC and SseD proteins are secreted by the SPI-2 type III secretion system to the bacterial membrane in order to perform their virulence functions.

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Furutani, Ayako, Minako Takaoka, Harumi Sanada, Yukari Noguchi, Takashi Oku, Kazunori Tsuno, Hirokazu Ochiai, and Seiji Tsuge. "Identification of Novel Type III Secretion Effectors in Xanthomonas oryzae pv. oryzae." Molecular Plant-Microbe Interactions® 22, no. 1 (January 2009): 96–106. http://dx.doi.org/10.1094/mpmi-22-1-0096.

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Many gram-negative bacteria secrete so-called effector proteins via a type III secretion (T3S) system. Through genome screening for genes encoding potential T3S effectors, 60 candidates were selected from rice pathogen Xanthomonas oryzae pv. oryzae MAFF311018 using these criteria: i) homologs of known T3S effectors in plant-pathogenic bacteria, ii) genes with expression regulated by hrp regulatory protein HrpX, or iii) proteins with N-terminal amino acid patterns associated with T3S substrates of Pseudomonas syringae. Of effector candidates tested with the Bordetella pertussis calmodulin-dependent adenylate cyclase reporter for translocation into plant cells, 16 proteins were translocated in a T3S system-dependent manner. Of these 16 proteins, nine were homologs of known effectors in other plant-pathogenic bacteria and seven were not. Most of the effectors were widely conserved in Xanthomonas spp.; however, some were specific to X. oryzae. Interestingly, all these effectors were expressed in an HrpX-dependent manner, suggesting coregulation of effectors and the T3S system. In X. campestris pv. vesicatoria, HpaB and HpaC (HpaP in X. oryzae pv. oryzae) have a central role in recruiting T3S substrates to the secretion apparatus. Secretion of all but one effector was reduced in both HpaB– and HpaP– mutant strains, indicating that HpaB and HpaP are widely involved in efficient secretion of the effectors.

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Zahrl, Doris, Maria Wagner, Karin Bischof, Michaela Bayer, Barbara Zavecz, Andreas Beranek, Christoph Ruckenstuhl, Gernot E. Zarfel, and Günther Koraimann. "Peptidoglycan degradation by specialized lytic transglycosylases associated with type III and type IV secretion systems." Microbiology 151, no. 11 (November 1, 2005): 3455–67. http://dx.doi.org/10.1099/mic.0.28141-0.

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Specialized lytic transglycosylases are muramidases capable of locally degrading the peptidoglycan meshwork of Gram-negative bacteria. Specialized lytic transglycosylase genes are present in clusters encoding diverse macromolecular transport systems. This paper reports the analysis of selected members of the specialized lytic transglycosylase family from type III and type IV secretion systems. These proteins were analysed in vivo by assaying their ability to complement the DNA transfer defect of the conjugative F-like plasmid R1-16 lacking a functional P19 protein, the specialized lytic transglycosylase of this type IV secretion system. Heterologous complementation was accomplished using IpgF from the plasmid-encoded type III secretion system of Shigella sonnei and TrbN from the type IV secretion system of the conjugative plasmid RP4. In contrast, neither VirB1 proteins (Agrobacterium tumefaciens, Brucella suis) nor IagB (Salmonella enterica) could functionally replace P19. In vitro, IpgF, IagB, both VirB1 proteins, HP0523 (Helicobacter pylori) and P19 displayed peptidoglycanase activity in zymogram analyses. Using an established test system and a newly developed assay it was shown that IpgF degraded peptidoglycan in solution. IpgF was active only after removal of the chaperonin GroEL, which co-purified with IpgF and inhibited its enzymic activity. A mutant IpgF protein in which the predicted catalytic amino acid, Glu42, was replaced by Gln, was completely inactive. IpgF-catalysed peptidoglycan degradation was optimal at pH 6 and was inhibited by the lytic transglycosylase inhibitors hexa-N-acetylchitohexaose and bulgecin A.

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Blaylock, Bill, Kelly E. Riordan, Dominique M. Missiakas, and Olaf Schneewind. "Characterization of the Yersinia enterocolitica Type III Secretion ATPase YscN and Its Regulator, YscL." Journal of Bacteriology 188, no. 10 (May 15, 2006): 3525–34. http://dx.doi.org/10.1128/jb.188.10.3525-3534.2006.

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ABSTRACT Type III secretion is a mechanism used by a broad range of gram-negative bacteria to neutralize eukaryotic defenses by enabling translocation of bacterial proteins directly into the cytoplasm of host cells. The bacterial energy source for secretion is ATP, which is consumed by an ATPase that couples ATP hydrolysis to the unfolding of secreted proteins and the dissociation of their chaperones just prior to secretion. By studying the biochemical properties of YscN and YscL of Yersinia enterocolitica, we have characterized them as the ATPase and ATPase regulator, respectively, of the type III secretion system of this organism. In vivo, YscL and YscN interact with each other, and the overexpression of glutathione S-transferase-YscL abolishes secretion and down-regulates the expression of secretion apparatus components.

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45

Ono, Takahiro, Kwon-Sam Park, Mayumi Ueta, Tetsuya Iida, and Takeshi Honda. "Identification of Proteins Secreted via Vibrio parahaemolyticus Type III Secretion System 1." Infection and Immunity 74, no. 2 (February 2006): 1032–42. http://dx.doi.org/10.1128/iai.74.2.1032-1042.2006.

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ABSTRACT Vibrio parahaemolyticus, a gram-negative marine bacterium, is an important pathogen causing food-borne gastroenteritis or septicemia. Recent genome sequencing of the RIMD2210633 strain (a Kanagawa phenomenon-positive clinical isolate of serotype O3:K6) revealed that the strain has two sets of gene clusters that encode the type III secretion system (TTSS) apparatus. The first cluster, TTSS1, is located on the large chromosome, and the second, TTSS2, is on the small chromosome. Previously, we reported that TTSS1 is involved in the cytotoxicity of the RIMD2210633 strain against HeLa cells. Here, we analyzed proteins secreted via the TTSS apparatus encoded by TTSS1 by using two-dimensional gel electrophoresis and identified the proteins encoded by genes VP1680, VP1686, and VPA450. To investigate the roles of those secreted proteins, we constructed and analyzed a series of deletion mutants. Flow cytometry analysis using fluorescence-activated cell sorting with fluorescein isothiocyanate-labeled annexin V demonstrated that the TTSS1-dependent cell death was by apoptosis. The cytotoxicity to HeLa cells was related to one of the newly identified secreted proteins encoded by VP1680. Adenylate cyclase fusion protein studies proved that the newly identified secreted proteins were translocated into HeLa cells. Thus, these appear to be the TTSS effector proteins in V. parahaemolyticus.

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46

Alam, Ashfaqul, Kelly A. Miller, Mudit Chaand, J. Scott Butler, and Michelle Dziejman. "Identification ofVibrio choleraeType III Secretion System Effector Proteins." Infection and Immunity 79, no. 4 (January 31, 2011): 1728–40. http://dx.doi.org/10.1128/iai.01194-10.

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ABSTRACTAM-19226 is a pathogenic O39 serogroupVibrio choleraestrain that lacks the typical virulence factors for colonization (toxin-coregulated pilus [TCP]) and toxin production (cholera toxin [CT]) and instead encodes a type III secretion system (T3SS). The mechanism of pathogenesis is unknown, and few effector proteins have been identified. We therefore undertook a survey of the open reading frames (ORFs) within the ∼49.7-kb T3SS genomic island to identify potential effector proteins. We identified 15 ORFs for their ability to inhibit growth when expressed in yeast and then used a β-lactamase (TEM1) fusion reporter system to demonstrate that 11 proteins werebona fideeffectors translocated into HeLa cellsin vitroin a T3SS-dependent manner. One effector, which we named VopX (A33_1663), is conserved only inV. choleraeandVibrio parahaemolyticusT3SS-positive strains and has not been previously studied. AvopXdeletion reduces the ability of strain AM-19226 to colonizein vivo, and the bile-induced expression of avopX-lacZtranscriptional fusionin vitrois regulated by the T3SS-encoded transcriptional regulators VttRAand VttRB. AnRLM1yeast deletion strain rescued the growth inhibition induced by VopX expression, suggesting that VopX interacts with components of the cell wall integrity mitogen-activated protein kinase (MAPK) pathway. The collective results show that theV. choleraeT3SS encodes multiple effector proteins, one of which likely has novel activities that contribute to disease via interference with eukaryotic signaling pathways.

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47

Yssel, H., M. C. Shanafelt, C. Soderberg, P. V. Schneider, J. Anzola, and G. Peltz. "Borrelia burgdorferi activates a T helper type 1-like T cell subset in Lyme arthritis." Journal of Experimental Medicine 174, no. 3 (September 1, 1991): 593–601. http://dx.doi.org/10.1084/jem.174.3.593.

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18 cloned T cell lines reactive with Borrelia burgdorferi proteins, all CD3+4+8-TCR-alpha/beta+ and restricted by HLA class II proteins, were isolated from four patients with chronic Lyme arthritis. Analysis of these T cell clones indicated that the T cell response to the Lyme disease spirochete is not oligoclonally restricted; yet all produced the same pattern of lymphokines, resembling that of murine type 1 T helper cells, after antigen-specific or nonspecific stimulation. Therefore, a subset of human CD4+ T cells, with a distinct profile of lymphokine secretion, is selectively activated by the pathogen inciting this chronic inflammatory disease.

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48

Burr, Sarah E., Katja Stuber, Thomas Wahli, and Joachim Frey. "Evidence for a Type III Secretion System in Aeromonas salmonicida subsp. salmonicida." Journal of Bacteriology 184, no. 21 (November 1, 2002): 5966–70. http://dx.doi.org/10.1128/jb.184.21.5966-5970.2002.

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ABSTRACT Aeromonas salmonicida subsp. salmonicida, the etiological agent of furunculosis, is an important fish pathogen. We have screened this bacterium with a broad-host-range probe directed against yscV, the gene that encodes the archetype of a highly conserved family of inner membrane proteins found in every known type III secretion system. This has led to the identification of seven open reading frames that encode homologues to proteins functioning within the type III secretion systems of Yersinia species. Six of these proteins are encoded by genes comprising a virA operon. The A. salmonicida subsp. salmonicida yscV homologue, ascV, was inactivated by marker replacement mutagenesis and used to generate an isogenic ascV mutant. Comparison of the extracellular protein profiles from the ascV mutant and the wild-type strain indicates that A. salmonicida subsp. salmonicida secretes proteins via a type III secretion system. The recently identified ADP-ribosylating toxin AexT was identified as one such protein. Finally, we have compared the toxicities of the wild-type A. salmonicida subsp. salmonicida strain and the ascV mutant against RTG-2 rainbow trout gonad cells. While infection with the wild-type strain results in significant morphological changes, including cell rounding, infection with the ascV mutant has no toxic effect, indicating that the type III secretion system we have identified plays an important role in the virulence of this pathogen.

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49

Jarvis, K. G., and J. B. Kaper. "Secretion of extracellular proteins by enterohemorrhagic Escherichia coli via a putative type III secretion system." Infection and immunity 64, no. 11 (1996): 4826–29. http://dx.doi.org/10.1128/iai.64.11.4826-4829.1996.

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Yu, Xiu-Jun, Javier Ruiz-Albert, Kate E. Unsworth, Steven Garvis, Mei Liu, and David W. Holden. "SpiC is required for secretion of Salmonella Pathogenicity Island 2 type III secretion system proteins." Cellular Microbiology 4, no. 8 (August 2002): 531–40. http://dx.doi.org/10.1046/j.1462-5822.2002.00211.x.

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