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PubMed2004

Transcriptional regulation of components of the type III secretion system and effectors in Pseudomonas syringae pv. phaseolicola.

Quantitative real-time polymerase chain reaction was used with specific TaqMan probes to examine transcription of selected hrp and effector genes in Pseudomonas syringae pv. phaseolicola strains 1448A (race 6) and 1449B (race 7). Transcripts examined were from genes encoding the regulators hrpR and hrpL, core structural components of the type III secretion system (TTSS) hrcC, hrcJ, hrcN, hrcU, and hrpA; the first open-reading frame of each hrp operon, including hrpF, hrpJ, hrpP, and hrpY, and also secreted effectors hrpZ, avrPphE, avrPphF, and virPphA. All genes were induced by incubation in a minimal medium and showed patterns of expression indicating regulation by HrpRS and HrpL. Basal mRNA levels and the timing of accumulation of transcripts after induction differed significantly, suggesting the operation of additional regulatory elements. However, no clear transcriptional hierarchy emerged to explain the ordered construction of the TTSS. Quantitative analysis confirmed that the rates and levels of transcript accumulation within the first 2 h after inoculation were considerably higher in planta than in vitro, and indicated that plant cell wall contact may enhance transcription of TTSS and effector genes in P. syringae pv. phaseolicola. The low-abundance hrcU mRNA had a half-life of 16.5 min, whereas other transcripts had half-lives between 3 and 8 min.

Bacterial Proteins↗
PubMed2006

Identification of proteins secreted via Vibrio parahaemolyticus type III secretion system 1.

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.

Amino Acid Sequence↗
PubMed2002

Salmonella effectors within a single pathogenicity island are differentially expressed and translocated by separate type III secretion systems.

Pathogenicity islands (PAIs) are large DNA segments in the genomes of bacterial pathogens that encode virulence factors. Five PAIs have been identified in the Gram-negative bacterium Salmonella enterica. Two of these PAIs, Salmonella pathogenicity island (SPI)-1 and SPI-2, encode type III secretion systems (TTSS), which are essential virulence determinants. These 'molecular syringes' inject effectors directly into the host cell, whereupon they manipulate host cell functions. These effectors are either encoded with their respective TTSS or scattered elsewhere on the Salmonella chromosome. Importantly, SPI-1 and SPI-2 are expressed under distinct environmental conditions: SPI-1 is induced upon initial contact with the host cell, whereas SPI-2 is induced intracellularly. Here, we demonstrate that a single PAI, in this case SPI-5, can encode effectors that are induced by distinct regulatory cues and targeted to different TTSS. SPI-5 encodes the SPI-1 TTSS translocated effector, SigD/SopB. In contrast, we report that the adjacently encoded effector PipB is part of the SPI-2 regulon. PipB is translocated by the SPI-2 TTSS to the Salmonella-containing vacuole and Salmonella-induced filaments. We also show that regions of SPI-5 are not conserved in all Salmonella spp. Although sigD/sopB is present in all Salmonella spp., pipB is not found in Salmonella bongori, which also lacks a functional SPI-2 TTSS. Thus, we demonstrate a functional and regulatory cross-talk between three chromosomal PAIs, SPI-1, SPI-2 and SPI-5, which has significant implications for the evolution and role of PAIs in bacterial pathogenesis.

Animals↗
PubMed2004

A type III secretion system is required for Aeromonas hydrophila AH-1 pathogenesis.

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.

Aeromonas hydrophila↗
PubMed2005

Basal resistance against bacteria in Nicotiana benthamiana leaves is accompanied by reduced vascular staining and suppressed by multiple Pseudomonas syringae type III secretion system effector proteins.

Basal resistance in plants is induced by flagellin and several other common bacterial molecules and is implicated in the immunity of plants to most bacteria and other microbes. However, basal resistance can be suppressed by effector proteins that are injected by the type III secretion system (TTSS) of pathogens such as Pseudomonas syringae. This study demonstrates that basal resistance in the leaves of Nicotiana benthamiana is accompanied by reduced vascular flow into minor veins. Reduced vascular flow was assayed by feeding leaves, via freshly excised petioles, with 1% (weight in volume, w/v) neutral red (NR) and then observing differential staining of minor veins or altered levels of extractable dye in excised leaf samples. The reduced vascular staining was localized to tissues expressing basal resistance and was observable when resistance was induced by either the non-pathogen Pseudomonas fluorescens, a TTSS-deficient mutant of P. syringae pv. tabaci, or flg22 (a flagellin-derived peptide elicitor of basal resistance). Nicotiana benthamiana leaf areas expressing basal resistance no longer elicited the hypersensitive response when challenge inoculated with P. syringae pv. tomato DC3000. The reduced vascular staining effect was suppressed by wild-type P. syringae pv. tabaci and P. fluorescens heterologously expressing a P. syringae TTSS and AvrPto1(PtoJL1065). TTSS-proficient P. fluorescens was used to test the ability of several P. syringae pv. tomato DC3000 effectors for their ability to suppress the basal resistance-associated reduced vascular staining effect. AvrE(PtoDC3000), HopM1(PtoDC3000) (formerly known as HopPtoM), HopF2(PtoDC3000) (HopPtoF) and HopG1(PtoDC3000) (HopPtoG) suppressed basal resistance by this test, whereas HopC1(PtoDC3000) (HopPtoC) did not. In summary, basal resistance locally alters vascular function and the vascular dye uptake assay should be a useful tool for characterizing effectors that suppress basal resistance.

Bacterial Proteins↗
PubMed2002

Role and activation of type III secretion system genes in Pseudomonas aeruginosa-induced Drosophila killing.

Pseudomonas aeruginosa strains PAO1 and CHA showing type III system-dependent cytotoxicity towards macrophages ex vivo are able to induce rapid death of adult fly Drosophila melanogaster accompanied by bacterial multiplication to high-titers. The role of P. aeruginosa type III secretion system in rapid fly killing was demonstrated here by using several isogenic CHA mutants, selectively affected in this system. The activation of P. aeruginosa pexsCBA, the regulatory operon of the type III system, and the activation of the Drosophila gene diptericin, showed the host-pathogen recognition during infection process.

Animals↗
PubMed1999

Cell death of human polymorphonuclear neutrophils induced by a Pseudomonas aeruginosa cystic fibrosis isolate requires a functional type III secretion system.

With a coincubation model incorporating Pseudomonas aeruginosa and human polymorphonuclear neutrophils (PMNs), a cystic fibrosis (CF) P. aeruginosa isolate has been shown to resist the bactericidal action of PMNs and to induce their cellular death. An isogenic mutant of this CF isolate in which the type III secretion system was rendered nonfunctional was unable to induce cellular death of PMNs.

Blood Bactericidal Activity↗
PubMed2005

Site-specific antiphagocytic function of the Photorhabdus luminescens type III secretion system during insect colonization.

Photorhabdus is an entomopathogenic bacterium belonging to the Enterobacteriaceae. The genome of the TT01 strain of Photorhabdus luminescens was recently sequenced and a large number of toxin-encoding genes were found. Genomic analysis predicted the presence on the chromosome of genes encoding a type three secretion system (TTSS), the main role of which is the delivery of effector proteins directly into eukaryotic host cells. We report here the functional characterization of the TTSS. The locus identified encodes the secretion/translocation apparatus, gene expression regulators and an effector protein - LopT - homologous to the Yersinia cysteine protease cytotoxin YopT. Heterologous expression in Yersinia demonstrated that LopT was translocated into mammal cells in an active form, as shown by the appearance of a form of the RhoA GTPase with modified electrophoretic mobility. In vitro study showed that recombinant LopT was able to release RhoA and Rac from human and insect cell membrane. In vivo assays of infection of the cutworm Spodoptera littoralis and the locust Locusta migratoria with a TT01 strain carrying a translational fusion of the lopT gene with the gfp reporter gene revealed that the lopT gene was switched on only at sites of cellular defence reactions, such as nodulation, in insects. TTSS-mutant did not induce nodule formation and underwent phagocytosis by insect macrophage cells, suggesting that the LopT effector plays an essential role in preventing phagocytosis and indicating an unexpected link between TTSS expression and the nodule reaction in insects.

Amino Acid Sequence↗
PubMed2006

The posttranscriptional regulator RsmA plays a role in the interaction between Pseudomonas aeruginosa and human airway epithelial cells by positively regulating the type III secretion system.

Posttranscriptional regulation of certain virulence-related genes in Pseudomonas aeruginosa is brought about by RsmA, a small RNA-binding protein. During interaction with airway epithelial cells, RsmA promoted actin depolymerization, cytotoxicity, and anti-internalization of P. aeruginosa by positively regulating the virulence-associated type III secretion system.

Actins↗
PubMed2001

The Type III secretion system of Gram-negative bacteria: a potential therapeutic target?

Several pathogenic Gram-negative bacteria, including Salmonella, Shigella, Yersinia, Pseudomonas aeruginosa and enteropathogenic Escherichia coli harbour a complex attack system called 'Type III secretion' which is, in every case, an essential virulence determinant. This system, activated by contact with an eukaryotic cell membrane, allows bacteria to inject bacterial proteins across the two bacterial membranes and the eukaryotic cell membrane, to reach the cell's cytosol and destroy or subvert the host cell. The Type III virulence mechanism consists of a secretion apparatus, made up of about 25 proteins, and a set of effector proteins released by this apparatus. The mechanism of protein secretion is highly conserved among the different bacteria, although they cause a variety of diseases with different symptoms and severities, from fatal septicaemia to mild diarrhoea or from fulgurant diarrhoea to chronic infection of the lung. This review focuses on the proteins that make up the secretion machinery and examine if it could be a potential target for novel antimicrobials.

Journal Article↗
PubMed2000

Fed-batch production of recombinant human calcitonin precursor fusion protein using Staphylococcus carnosus as an expression-secretion system.

A pH-auxostatic fed-batch process was developed for the secretory production of a fusion protein consisting of the pro-part of Staphylococcus hyicus lipase and two synthetic human calcitonin (hCT) precursor repeats under the control of a xylose-inducible promotor from Staphylococcus xylosus. Using glycerol as the energy source and pH-controlled addition of yeast extract resulted in the production of 2000 mg 1(-1) of the fusion protein (420 mg 1(-1) of the recombinant hCT precursor) within 14 h, reaching 45 g 1(-1) cell dry mass with Staphylococcus carnosus in a stirred-tank reactor. Product titer and space-time yield (30 mg calcitonin precursor 1(-1) h(-1)) were thus improved by a factor of 2, and 4.5, respectively, compared to Escherichia coli expression-secretion systems for the production of calcitonin precursors. Two hundred grams of the fusion protein was secreted by the recombinant S. carnosus on a 150-1 scale (scale-up factor of 50) with a minimum use of technical-grade yeast extract (40 mg fusion protein g(-1) yeast extract).

Amino Acids↗
PubMed2006

The animal pathogen-like type III secretion system is required for the intracellular survival of Burkholderia mallei within J774.2 macrophages.

Burkholderia mallei is a highly infectious gram-negative pathogen and is the causative agent of human and animal glanders. By generating polar mutations (disruption of bsaQ and bsaZ) in the B. mallei ATCC 23344 animal pathogen-like type III secretion system (TTS), we demonstrate that this bacterial protein delivery system is required for intracellular growth of B. mallei in J774.2 cells, formation of macrophage membrane protrusions, actin polymerization, and phagosomal escape. These findings suggest that TTS plays a role in the intracellular trafficking of B. mallei and may facilitate cell-to-cell spread via actin-based motility.

Animals↗
PubMed2006

The Bordetella bronchiseptica type III secretion system inhibits gamma interferon production that is required for efficient antibody-mediated bacterial clearance.

Several species of pathogenic microorganisms have developed strategies to survive and persist in vital organs which are normally maintained as sterile by the generation of strong immune responses. Here, we report an immunomodulation involving the Bordetella bronchiseptica type III secretion system (TTSS) which contributes to bacterial survival in the lower respiratory tract of the host. The prolonged persistence of B. bronchiseptica that was observed in gamma interferon (IFN-gamma)-/- mice indicates that the efficient clearance of bacteria from the lower respiratory tract requires not only B cells and antibodies but also IFN-gamma production. Our data also suggest that interleukin-10 (IL-10)-producing splenocytes are generated early during infection and that IL-10 inhibits IFN-gamma-producing cells and delays the clearance of B. bronchiseptica from the lungs. The TTSS of B. bronchiseptica inhibits the generation of IFN-gamma-producing splenocytes and is required for long-term bacterial persistence in the lower respiratory tract in wild-type mice. This suggests that a mechanism involving the modulation of IFN-gamma production by the TTSS facilitates B. bronchiseptica survival in the lower respiratory tract.

Animals↗
PubMed2005

Control of the Ralstonia solanacearum Type III secretion system (Hrp) genes by the global virulence regulator PhcA.

Expression of several virulence factors in the plant pathogen bacterium Ralstonia solanacearum is controlled by a complex regulatory network, at the center of which is PhcA. We provide genetic evidence that PhcA also represses the expression of hrp genes that code for the Type III protein secretion system, a major pathogenicity determinant in this bacterium. The repression of hrp genes in complete medium is relieved in a phcA mutant and two distinct signals, a quorum-sensing signal and complex nitrogen sources, appear to trigger this PhcA-dependent repression. This control of hrp gene expression by PhcA is realized at the level of the HrpG regulatory protein.

Bacterial Proteins↗
PubMed2002

Type III secretion systems and the evolution of mutualistic endosymbiosis.

The view that parasites can develop cooperative symbiotic relationships with their hosts is both appealing and widely held; however, there is no molecular genetic evidence of such a transition. Here we demonstrate that a mutualistic bacterial endosymbiont of grain weevils maintains and expresses inv/spa genes encoding a type III secretion system homologous to that used for invasion by bacterial pathogens. Phylogenetic analyses indicate that inv/spa genes were present in presymbiotic ancestors of the weevil endosymbionts, occurring at least 50 million years ago. The function of inv/spa genes in maintaining symbiosis is demonstrated by the up-regulation of their expression under both in vivo and in vitro conditions that coincide with host cell invasion.

Adhesins, Bacterial↗
PubMed2006

A putative multicopper protein secreted by an atypical type II secretion system involved in the reduction of insoluble electron acceptors in Geobacter sulfurreducens.

Extracellular electron transfer onto Fe(III) oxides in Geobacter sulfurreducens is considered to require proteins that must be exported to the outer surface of the cell. In order to investigate this, the putative gene for OxpG, the pseudopilin involved in a type II general secretion pathway of Gram-negative bacteria, was deleted. The mutant was unable to grow with insoluble Fe(III) oxide as the electron acceptor. Growth on soluble Fe(III) was not affected. An analysis of proteins that accumulated in the periplasm of the oxpG mutant, but not in the wild-type, led to the identification of a secreted protein, OmpB. OmpB is predicted to be a multicopper protein, with highest homology to the manganese oxidase, MofA, from Leptothrix discophora. OmpB contains a potential Fe(III)-binding site and a fibronectin type III domain, suggesting a possible role for this protein in accessing Fe(III) oxides. OmpB was localized to the membrane fraction of G. sulfurreducens and in the supernatant of growing cultures, consistent with the type II secretion system exporting OmpB. A mutant in which ompB was deleted had the same phenotype as the oxpG mutant, suggesting that the failure to export OmpB was responsible for the inability of the oxpG-deficient mutant to reduce Fe(III) oxide. This is the first report that proposes a role for a multicopper oxidase-like protein in an anaerobic organism. These results further emphasize the importance of outer-membrane proteins in Fe(III) oxide reduction and suggest that outer-membrane proteins other than c-type cytochromes are required for Fe(III) oxide reduction in Geobacter species.

Bacterial Outer Membrane Proteins↗
PubMed2003

Use of the Galleria mellonella caterpillar as a model host to study the role of the type III secretion system in Pseudomonas aeruginosa pathogenesis.

Nonvertebrate model hosts represent valuable tools for the study of host-pathogen interactions because they facilitate the identification of bacterial virulence factors and allow the discovery of novel components involved in host innate immune responses. In this report, we determined that the greater wax moth caterpillar Galleria mellonella is a convenient nonmammalian model host for study of the role of the type III secretion system (TTSS) in Pseudomonas aeruginosa pathogenesis. Based on the observation that a mutation in the TTSS pscD gene of P. aeruginosa strain PA14 resulted in a highly attenuated virulence phenotype in G. mellonella, we examined the roles of the four known effector proteins of P. aeruginosa (ExoS, ExoT, ExoU, and ExoY) in wax moth killing. We determined that in P. aeruginosa strain PA14, only ExoT and ExoU play a significant role in G. mellonella killing. Strain PA14 lacks the coding sequence for the ExoS effector protein and does not seem to express ExoY. Moreover, using Delta exoU Delta exoY, Delta exoT Delta exoY, and Delta exoT Delta exoU double mutants, we determined that individual translocation of either ExoT or ExoU is sufficient to obtain nearly wild-type levels of G. mellonella killing. On the other hand, data obtained with a Delta exoT Delta exoU Delta exoY triple mutant and a Delta pscD mutant suggested that additional, as-yet-unidentified P. aeruginosa components of type III secretion are involved in virulence in G. mellonella. A high level of correlation between the results obtained in the G. mellonella model and the results of cytopathology assays performed with a mammalian tissue culture system validated the use of G. mellonella for the study of the P. aeruginosa TTSS.

Animals↗