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PubMed2006

PsrA is a positive transcriptional regulator of the type III secretion system in Pseudomonas aeruginosa.

The type III secretion system (TTSS) of Pseudomonas aeruginosa is induced in vivo upon contact with eukaryotic cells and in vitro by calcium depletion in culture medium. We have observed a previously identified protein, PsrA, necessary for full activation of TTSS gene expression in P. aeruginosa. Electrophoretic mobility shift assays showed that recombinant PsrA could bind to the exsCEBA promoter region. A mutant with a deletion in the psrA gene was constructed. Using transcriptional fusions, we demonstrated that PsrA is required for the full activation of transcription of the TTSS regulatory operon exsCEBA and effector exoS, although the deletion mutant still responded to calcium depletion, to serum, and to host cell contact. The psrA mutant showed a marked decrease in the secretion of the type III effectors and weak resistance to phagocyte-like PLB-985 cells. The defect in TTSS transcription and secretion in the psrA mutant could be complemented by expression in trans of psrA. PsrA was previously identified as a transcriptional activator of RpoS, a central regulator during stationary phase. We confirmed with our strain that RpoS has a negative effect on TTSS gene expression. Taken altogether, these results suggest that PsrA is a newly identified activator that is involved in the expression of the TTSS by enhancing the exsCEBA transcriptional level.

ADP Ribose Transferases↗

PubMed2003

Protein-peptidoglycan interactions modulate the assembly of the needle complex in the Salmonella invasion-associated type III secretion system.

The invasion-associated type III secretion system of Salmonella enterica assembles as a supra-molecular structure, termed needle complex, which spans the bacterial envelope. Here, we present evidence for protein-peptidoglycan interactions that modulate the assembly of this organelle. The presence of major membrane components of the needle complex (PrgH, PrgK and InvG) and InvH, required for efficient assembly of the organelle, was examined in peptidoglycan purified by extensive boiling of bacteria in 4% SDS. InvH, PrgH and PrgK, but not InvG, were detected in this purified material. InvH was present in the peptidoglycan in higher relative amounts than PrgH or PrgK, and was the only protein efficiently bound to peptidoglycan in cross-linking experiments. Analysis in mutants defective for needle complex proteins showed that the needle proteins PrgI and PrgJ and, to a lesser extent, InvH, sustain the association of PrgH and PrgK with peptidoglycan. In contrast, the association of InvH with peptidoglycan did not necessitate other needle complex proteins. Functional analysis showed that the association of InvH, PrgH and PrgK with peptidoglycan is abolished in live bacteria carrying structural modifications in the peptidoglycan. The loss of these interactions caused a marked reduction in the number of needle complexes and, concomitantly, in protein secretion and bacterial invasion of cultured eukaryotic cells. Altogether, these data provide the first evidence for an association between proteins of the Salmonella needle complex and the peptidoglycan. In addition, we demonstrate that these protein-peptidoglycan interactions are critical for an efficient and correct assembly of this specialized organelle.

Bacterial Proteins↗

PubMed2004

Agrobacterium tumefaciens VirB6 domains direct the ordered export of a DNA substrate through a type IV secretion System.

The Agrobacterium tumefaciens VirB/D4 type IV secretion system (T4SS) translocates DNA and protein substrates across the bacterial cell envelope. Six presumptive channel subunits of this T4SS (VirD4, VirBll, VirB6, VirB8, VirB2, and VirB9) form close contacts with the VirD2-T-strand transfer intermediate during export, as shown recently by a novel transfer DNA immunoprecipitation (TrIP) assay. Here, we characterize the contribution of the hydrophobic channel component VirB6 to substrate translocation. Results of reporter protein fusion and cysteine accessibility studies support a model for VirB6 as a polytopic membrane protein with a periplasmic N terminus, five transmembrane segments, and a cytoplasmic C terminus. TrIP studies aimed at characterizing the effects of VirB6 insertion and deletion mutations on substrate translocation identified several VirB6 functional domains: (i) a central region composed of a large periplasmic loop (P2) (residues 84 to 165) mediates the interaction of VirB6 with the exiting T-strand; (ii) a multi-membrane-spanning region carboxyl-terminal to loop P2 (residues 165 to 245) is required for substrate transfer from VirB6 to the bitopic membrane subunit VirB8; and (iii) the two terminal regions (residues 1 to 64 and 245 to 290) are required for substrate transfer to the periplasmic and outer membrane-associated VirB2 and VirB9 subunits. Our findings support a model whereby the periplasmic loop P2 comprises a portion of the secretion channel and distinct domains of VirB6 participate in channel subunit interactions required for substrate passage to the cell exterior.

Agrobacterium tumefaciens↗

PubMed2002

The Dot/Icm type IV secretion system of Legionella pneumophila is essential for the induction of apoptosis in human macrophages.

We have previously shown that Legionella pneumophila induces caspase 3-dependent apoptosis in mammalian cells during early stages of infection. In this report, we show that nine L. pneumophila strains with mutations in the dotA, dotDCB, icmT, icmGCD, and icmJB loci are completely defective in the induction of apoptosis, in addition to their severe defects in intracellular replication and pore formation-mediated cytotoxicity. Importantly, all nine dot/icm mutants were complemented for all their defective phenotypes with the respective wild-type loci. We show that the role of the Dot/Icm type IV secretion system in the induction of apoptosis is independent of the RtxA toxin, the dot/icm-regulated pore-forming toxin, and the type II secretion system. However, the pore-forming toxin, which is triggered upon entry into the postexponential growth phase, enhances the ability of L. pneumophila to induce apoptosis. Our data provide the first example of the role of a type IV secretion system of a bacterial pathogen in the induction of apoptosis in the host cell.

Apoptosis↗

PubMed2001

The psp locus of Yersinia enterocolitica is required for virulence and for growth in vitro when the Ysc type III secretion system is produced.

The phage shock protein locus (pspFpspABCDE) of Escherichia coli has proved to be something of an enigma since its discovery. The physiological functions of the psp locus, including those of the predicted effector protein PspA, are unknown. In a previous genetic screen, we determined that a Yersinia enterocolitica pspC mutant was severely attenuated for virulence. In this study, the psp locus of Y. enterocolitica was characterized further. The pspC gene of Y. enterocolitica was found to be important for normal growth when the Ysc type III secretion system was expressed in the laboratory. This growth defect was specifically caused by production of the secretin protein, YscC. Expression of the psp genes was induced when the type III secretion system was functional or when only the yscC gene was expressed. This induction of psp gene expression required a functional pspC gene. Most significantly, evidence suggests that the expression of at least one gene that is not part of the psp locus is regulated by Psp proteins. This unidentified gene (or genes) may also be important for growth when the type III secretion system is expressed. These conclusions are supported by the effects of various psp mutations on virulence. This is the first indication that Psp proteins might be involved in the regulation of genes besides the psp locus itself.

Animals↗

PubMed1996

Secretion of extracellular proteins by enterohemorrhagic Escherichia coli via a putative type III secretion system.

Enterohemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC) infections result in attaching and effacing lesions on intestinal epithelial cells. Secretion of extracellular proteins via a type III secretion apparatus is necessary for the formation of attaching and effacing lesions by EPEC. We now show that EHEC also secretes polypeptides via a putative type III secretion system. The secreted EHEC proteins are recognized by rabbit antiserum raised against the proteins secreted from EPEC and by human serum from a patient infected with an EHEC O157:H7 strain.

Animals↗

PubMed2006

A two-partner secretion system is involved in seed and root colonization and iron uptake by Pseudomonas putida KT2440.

We describe the first two-partner secretion system known to play a role in mutualistic plant-bacterial interactions, identified in the soil and rhizosphere-colonizing bacterium Pseudomonas putida KT2440. The genes coding for the two components of the system are organized in an operon, which we have named hlpBA. HlpA is a secreted protein that has similarities with iron-regulated haemolysins, while HlpB would be responsible for the activation and transport of HlpA across the outer membrane. Mutations in this novel two-partner secretion system result in reduced capacity to colonize corn seeds. When introduced in the rhizosphere, hlpA and hlpB mutants show no competitive disadvantage, but the number of cells attached to the root surface is reduced with respect to the wild type, suggesting this protein plays a role directly in the bacterial cell-root surface interaction. Under iron-limiting conditions, the presence of a truncated HlpA causes reduced viability and high levels of siderophore release. These data further strengthen our previous observations indicating the importance of iron acquisition for attachment of P. putida KT2440 to plant surfaces.

Bacterial Adhesion↗

PubMed2005

The Erwinia chrysanthemi type III secretion system is required for multicellular behavior.

Enterobacterial animal pathogens exhibit aggregative multicellular behavior, which is manifested as pellicles on the culture surface and biofilms at the surface-liquid-air interface. Pellicle formation behavior requires production of extracellular polysaccharide, cellulose, and protein filaments, known as curli. Protein filaments analogous to curli are formed by many protein secretion systems, including the type III secretion system (TTSS). Here, we demonstrate that Erwinia chrysanthemi, which does not carry curli genes, requires the TTSS for pellicle formation. These data support a model where cellulose and generic protein filaments, which consist of either curli or TTSS-secreted proteins, are required for enterobacterial aggregative multicellular behavior. Using this assay, we found that hrpY, which encodes a two-component system response regulator homolog, is required for activity of hrpS, which encodes a sigma54-dependent enhancer-binding protein homolog. In turn, hrpS is required for activity of the sigma factor homolog hrpL, which activates genes encoding TTSS structural and secreted proteins. Pellicle formation was temperature dependent and pellicles did not form at 36 degrees C, even though TTSS genes were expressed at this temperature. We found that cellulose is a component of the E. chrysanthemi pellicle but that pellicle formation still occurs in a strain with an insertion in a cellulose synthase subunit homolog. Since the TTSS, but not the cellulose synthase subunit, is required for E. chrysanthemi pellicle formation, this inexpensive assay can be used as a high throughput screen for TTSS mutants or inhibitors.

Adaptation, Physiological↗

PubMed2002

MxiE regulates intracellular expression of factors secreted by the Shigella flexneri 2a type III secretion system.

The mxi-spa locus on the virulence plasmid of Shigella flexneri encodes components of the type III secretion system. mxiE, a gene within this locus, encodes a protein that is homologous to the AraC/XylS family of transcriptional regulators, but currently its role in pathogenesis remains undefined. We characterized the virulence phenotype of a nonpolar mxiE mutant and found that this mutant retained the ability to invade mammalian cells in tissue culture and secrete Ipas (type III effectors required for host cell invasion), although it was less efficient than wild-type Shigella at cell-to-cell spread. Despite its invasive properties in culture, the mxiE mutant was completely avirulent in an animal model. Potential targets for MxiE activation were identified by using promoter-green fluorescent protein fusions, and gene expression was examined under various growth conditions. Six MxiE-regulated genes were discovered: ospB, ospC1, ospE2, ospF, virA, and ipaH(9.8). Notably, activation of these genes only occurred within the intracellular environment of the host and not during growth at 37 degrees C in liquid culture. Interestingly, all of the MxiE-regulated proteins previously have been shown to be secreted through the type III secretion system and are putative virulence factors. Our findings suggest that some of these Osp proteins may be involved in postinvasion events related to virulence. Since bacterial pathogens adapt to multiple environments during the course of infecting a host, we propose that Shigella evolved a mechanism to take advantage of a unique intracellular cue, which is mediated through MxiE, to express proteins when the organism reaches the eukaryotic cytosol.

Antigens, Bacterial↗

PubMed2000

Genes for the type IV secretion system in an intracellular symbiont, Wolbachia, a causative agent of various sexual alterations in arthropods.

Wolbachia species are intracellular bacteria known to cause reproductive abnormalities in their hosts. In this study, we identified Wolbachia genes encoding homologs to the type IV secretion system by which many pathogenic bacteria secrete macromolecules. The genes identified encoded most of the essential components of the secretion system and were cotranscribed as an operon.

Animals↗

PubMed2003

TraG-like proteins of type IV secretion systems: functional dissection of the multiple activities of TraG (RP4) and TrwB (R388).

TraG-like proteins are essential components of type IV secretion systems. During secretion, TraG is thought to translocate defined substrates through the inner cell membrane. The energy for this transport is presumably delivered by its potential nucleotide hydrolase (NTPase) activity. TraG of conjugative plasmid RP4 is a membrane-anchored oligomer that binds RP4 relaxase and DNA. TrwB (R388) is a hexameric TraG-like protein that binds ATP. Both proteins, however, lack NTPase activity under in vitro conditions. We characterized derivatives of TraG and TrwB truncated by the N-terminal membrane anchor (TraGdelta2 and TrwBdelta1) and/or containing a point mutation at the putative nucleotide-binding site (TraGdelta2K187T and TraGK187T). Unlike TraG and TrwB, truncated derivatives behaved as monomers without the tendency to form oligomers or aggregates. Surface plasmon resonance analysis with immobilized relaxase showed that mutant TraGK187T was as good a binding partner as the wild-type protein, whereas truncated TraG monomers were unable to bind relaxase. TraGdelta2 and TrwBdelta1 bound ATP and, with similar affinity, ADP. Binding of ATP and ADP was strongly inhibited by the presence of Mg(2+) or single-stranded DNA and was competed for by other nucleotides. Compared to the activity of TraGdelta2, the ATP- and ADP-binding activity of the point mutation derivative TraGdelta2K187T was significantly reduced. Each TraG derivative bound DNA with an affinity similar to that of the native protein. DNA binding was inhibited or competed for by ATP, ADP, and, most prominently, Mg(2+). Thus, both nucleotide binding and DNA binding were sensitive to Mg(2+) and were competitive with respect to each other.

Adenosine Diphosphate↗

PubMed1996

Long-lasting immune response induced by recombinant bacillus Calmette-Guérin (BCG) secretion system.

The recombinant bacillus Calmette-Guérin (rBCG) secretion system utilizing an extracellular alpha antigen of Mycobacterium kansasii (alpha-K) was characterized biochemically and immunologically. The human immunodeficiency virus type 1 (HIV-1) p17gag B cell epitope fused to alpha-K was secreted in extremely large amounts. At least three mice out of seven inoculated with rBCG generated high titres of antibody to the epitope. The long-lasting antibody production persisted more than 14 months.

Amino Acid Sequence↗

PubMed2002

Coiled-coil proteins associated with type III secretion systems: a versatile domain revisited.

The pathogenic potential of many Gram-negative bacteria is indicated by the possession of a specialized type III secretion system that is used to deliver virulence effector proteins directly into the cellular environment of the eukaryotic host. Extracellular assemblies of secreted proteins contrive a physical link between the pathogen and host cytosol and enable the translocated effectors to bypass the bacterial and host membranes in a single step. Subsequent interactions of some effector proteins with host cytoskeletal and signalling proteins result in modulation of the cytoskeletal architecture of the aggressed cell and facilitate entry, survival and dissemination of the pathogen. Although the secreted components of type III secretion systems are diverse, many are predicted to share a common coiled-coil structural feature. Coiled-coils are ubiquitous and highly versatile assembly motifs found in a wide range of structural and regulatory proteins. The prevalence of these domains in secreted virulence effector proteins suggests a fundamental contribution to multiple aspects of their function, and evidence accumulating from functional studies suggests an intrinsic involvement of coiled-coils in subunit assembly, translocation and flexible interactions with multiple bacterial and host proteins. The known functional flexibility that coiled-coil domains confer upon proteins provides insights into some of the pathogenic mechanisms used during interaction with the host.

Bacteria↗

PubMed1999

A homologue of the Agrobacterium tumefaciens VirB and Bordetella pertussis Ptl type IV secretion systems is essential for intracellular survival of Brucella suis.

Analysis of a TnblaM mutant of Brucella suis 1330, identified as being unable to multiply in Hela cells, allowed us to identify a 11 860 bp region of the B. suis genome encoding a type IV secretion system, homologous to the VirB system of Agrobacterium tumefaciens and the Ptl system of Bordetella pertussis. DNA sequence revealed 12 open reading frames (ORFs) encoding homologues of the 11 VirB proteins present in the pTi plasmid of Agrobacterium with a similar genetic organization, and a twelfth ORF encoding a putative lipoprotein, homologous to a protein involved in mating pair formation during bacterial conjugation and to adhesins used by Pseudomonas species to bind to plant roots. Phylogenetic trees based on the sequences of VirB4 and VirB9 protein homologues suggest that evolution of the systems from DNA transfer towards protein secretion did not stem from a single event but that the protein secretion systems have evolved independently. Four independent mutants in virB5, virB9 or virB10 were highly attenuated in an in vitro infection model with human macrophages. The virulence was restored by complementation with a plasmid containing the full virB region. The virB region appears to be essential for the intracellular survival and multiplication of B. suis.

Agrobacterium tumefaciens↗

PubMed2004

[Type III bacterial secretion systems and their relation to virulence].

Newer data on type III secretion systems (SST systems) associated with virulence in gramnegative bacteria are summarized. The focus is on substrates of SST systems (effector proteins), recognition of type III secretion signals, structural components of SST systems and their genetic determination.

Bacterial Proteins↗

PubMed1993

Efficient production of a functional single-chain antidigoxin antibody via an engineered Bacillus subtilis expression-secretion system.

We have applied a Bacillus subtilis expression-secretion system to produce a functional antidigoxin SCA (single-chain antibody consisting of VL-linker-VH) and the individual variable domains of light (VL) and heavy (VH) chains. The secreted antidigoxin SCA can be affinity purified in one step by applying the culture supernatant directly to a ouabain-Sepharose column. N-terminal sequence determination indicated that the protein has the expected N-terminus with the signal peptide properly processed. Affinity and ligand specificity studies demonstrated that the engineered antidigoxin SCA has almost identical properties as those of the parental monoclonal antibody. The use of B. subtilis WB600, an engineered, six-extracellular protease-deficient strain, is vital for the production of antidigoxin SCA in high quality and quantity (5 mg/liter in a shake flask culture). All the secreted SCAs are biologically active. The ability to produce secreted SCAs by the B. subtilis expression system provides a simple and efficient means to analyze the binding properties of engineered antibodies generated through rational design or site-directed mutagenesis.

Amino Acid Sequence↗

PubMed2005

The X-ray structure of the type II secretion system complex formed by the N-terminal domain of EpsE and the cytoplasmic domain of EpsL of Vibrio cholerae.

Gram-negative bacteria use type II secretion systems for the transport of virulence factors and hydrolytic enzymes through the outer membrane. These sophisticated multi-protein complexes reach from the pore in the outer membrane via the pseudopilins in the periplasm and a multi-protein inner-membrane sub-complex, to an ATPase in the cytoplasm. The human pathogen Vibrio cholerae uses such a secretion machinery, called the Eps-system, for the export of its major virulence factor cholera toxin into the intestinal tract of the human host. Here, we describe the 2.4 A structure of the hetero-tetrameric complex of the N-terminal domain of the ATPase EpsE and the cytoplasmic domain of the inner membrane protein EpsL, which constitute the major cytoplasmic components of the Eps-system. A stable fragment of EpsE in complex with the cytoplasmic domain of EpsL was identified via limited proteolysis and facilitated the crystallization of the complex. This first structure of a complex between two different proteins of the type II secretion system reveals that the N-terminal domain of EpsE and the cytoplasmic domain of EpsL form a hetero-tetramer, in which EpsL is the central dimer and EpsE binds on the periphery. The dimer of EpsL in this complex is very similar to the dimer seen in the crystal structure of the native cytoplasmic domain of EpsL, suggesting a possible physiological relevance despite a relatively small 675 A2 buried solvent accessible surface. The N-terminal domain of EpsE, which forms a compact domain with an alpha+beta-fold, places its helix alpha2 in a mostly hydrophobic cleft between domains II and III of EpsL burying 1700 A2 solvent accessible surface. This extensive interface involves several residues whose hydrophobic or charged nature is well conserved and is therefore likely to be of general importance in type II secretion systems.

Amino Acid Sequence↗

PubMed2004

Use of the alpha-hemolysin secretion system of Escherichia coli for antigen delivery in the Salmonella typhi Ty21a vaccine strain.

This study examined the suitability of the hemolysin secretion system of Escherichia coli for expression and delivery of alpha-hemolysin (HlyA) by the S. typhi Ty21a strain, the only live oral Salmonella vaccine strain licensed for human use, under in vitro and in vivo conditions. For this purpose, two plasmid vectors encoding either the whole alpha-hemolysin of E. coli (pANN202-812/pMOhly2) or the hemolysin secretion signal (pMOhly1) were transferred into S. typhi Ty21a. S. typhi Ty21a carrying pANN202-812/pMOhly2 revealed efficient secretion of hemolysin in vitro. After formulation according to a process suitable for commercial production of Salmonella-based live bacterial vaccines, plasmids were shown to be stable in Ty21a and hemolysin secretion was demonstrated even after storage of the strains under real-time and stress conditions. After intranasal immunization of mice with S. typhi Ty21a/pANN202-812 plasmids are stable in vivo, and immunization induced a profound immune response against the heterologous HlyA antigen. Therefore, the combination of the hemolysin secretion system and S. typhi Ty21a could form the basis for a new generation of live bacterial vaccines.

Animals↗