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PubMed1998

Supramolecular structure of the Salmonella typhimurium type III protein secretion system.

The type III secretion system of Salmonella typhimurium directs the translocation of proteins into host cells. Evolutionarily related to the flagellar assembly machinery, this system is also present in other pathogenic bacteria, but its organization is unknown. Electron microscopy revealed supramolecular structures spanning the inner and outer membranes of flagellated and nonflagellated strains; such structures were not detected in strains carrying null mutations in components of the type III apparatus. Isolated structures were found to contain at least three proteins of this secretion system. Thus, the type III apparatus of S. typhimurium, and presumably other bacteria, exists as a supramolecular structure in the bacterial envelope.

Bacterial Outer Membrane Proteins↗

PubMed2001

The AprX protein of Pseudomonas aeruginosa: a new substrate for the Apr type I secretion system.

Protein secretion in Pseudomonas aeruginosa involves different mechanisms. The type II and type III secretory pathways control the extracellular release of a wide range of substrates. The type I secretion process, or ABC transporter, was believed to be exclusively involved in alkaline protease secretion. Recently, it was discovered that a P. aeruginosa heme binding protein, HasAp, is also secreted by a type I process. We present here the identification of a third putative type I-dependent protein of P. aeruginosa, AprX. The function of this protein has not yet been elucidated but very interestingly it appears to be linked to the apr cluster, and organized in one single operon together with the aprD, -E and -F genes.

ATP-Binding Cassette Transporters↗

PubMed2006

VirB8: a conserved type IV secretion system assembly factor and drug target.

Type IV secretion systems are used by many gram-negative bacteria for the translocation of macromolecules (proteins, DNA, or DNA-protein complexes) across the cell envelope. Among them are many pathogens for which type IV secretion systems are essential virulence factors. Type IV secretion systems comprise 8-12 conserved proteins, which assemble into a complex spanning the inner and the outer membrane, and many assemble extracellular appendages, such as pili, which initiate contact with host and recipient cells followed by substrate translocation. VirB8 is an essential assembly factor for all type IV secretion systems. Biochemical, cell biological, genetic, and yeast two-hybrid analyses showed that VirB8 undergoes multiple interactions with other type IV secretion system components and that it directs polar assembly of the membrane-spanning complex in the model organism Agrobacterium tumefaciens. The availability of the VirB8 X-ray structure has enabled a detailed structure-function analysis, which identified sites for the binding of VirB4 and VirB10 and for self-interaction. Due to its multiple interactions, VirB8 is an excellent model for the analysis of assembly factors of multiprotein complexes. In addition, VirB8 is a possible target for drugs that target its protein-protein interactions, which would disarm bacteria by depriving them of their essential virulence functions.

Amino Acid Sequence↗

PubMed1999

pH-dependent secretion of SseB, a product of the SPI-2 type III secretion system of Salmonella typhimurium.

The type III secretion system of Salmonella pathogenicity island 2 (SPI-2) is required for bacterial replication inside macrophages. SseB has been considered a putative target of the secretion system on the basis of its similarity with EspA, a protein secreted by the type III secretion system of enteropathogenic Escherichia coli (EPEC). EspA forms a filamentous structure on the bacterial cell surface and is involved in translocation of proteins into the eukaryotic cytosol. In this paper, we show that SseB is a secreted protein that associates with the surface of the bacterial cell and might, therefore, also be required for delivery of SPI-2 effector proteins to the eukaryotic cell cytosol. SseB begins to accumulate inside the bacterial cell when the culture enters early stationary phase. However, SseB is only secreted if the bacteria are grown at low pH or if the pH is shifted after growth from 7.0 to below pH 5.0. The secretion occurs within minutes of acidification and is totally dependent on a functional SPI-2 type III secretion system. As the pH of the Salmonella-containing vacuole inside host cells has been shown to acidify to between pH 4.0 and 5.0, and as SPI-2 gene expression occurs inside host cells, low pH might be a physiological stimulus for SPI-2-mediated secretion in vivo.

Agglutination Tests↗

PubMed2005

The Icm/Dot type-IV secretion systems of Legionella pneumophila and Coxiella burnetii.

Type-IV secretion systems are devices present in a wide range of bacteria (including bacterial pathogens) that deliver macromolecules (proteins and single-strand-DNA) across kingdom barriers (as well as between bacteria and into the surroundings). The type-IV secretion systems were divided into two subgroups and Legionella pneumophila and Coxiella burnetii are the only two bacteria known today to utilize a type-IVB secretion system for pathogenesis. In this review we summarized the available information concerning the icm/dot type-IVB secretion systems by comparing the two bacteria that possess this system, the proteins components of their systems as well as the homology of proteins from type-IVB secretion systems to proteins from type-IVA secretion systems. In addition, the phenotypes associated with mutants in the L. pneumophila icm/dot genes, their relations to properties of specific Icm/Dot proteins as well as the protein substrates delivered by this system are described.

Bacterial Proteins↗

PubMed2004

Lactoferrin disruption of bacterial type III secretion systems.

Many gram-negative bacteria share a closely related mechanism for secretion of virulence proteins. This complex machine, the type III secretion system, secretes virulence proteins in response to sensing the presence of target mammalian cells. We have found that recombinant human lactoferrin impairs the function of this system in two model organisms: Shigella and Enteropathogenic E. coli (EPEC). In the case of Shigella, there is loss and degradation of two proteins secreted by the type III mechanism, invasion plasmid antigens B and C (IpaB and IpaC); these proteins normally form a complex that causes Shigella to be taken up by host mammalian cells. In the case of EPEC, lactoferrin causes loss and degradation of E. coli secreted proteins A, B and D (EspABD) particularly EspB. These proteins are components of type III machinery and are known to be key elements of EPEC pathogenesis. Studies using purified EspB demonstrated that lactoferrin has a direct proteolytic effect on EspB that can be prevented by serine protease inhibitors. A synthetic peptide of the N-terminal 33 amino acids of lactoferrin caused loss of cell associated EspB but, unlike the whole lactoferrin molecule, did not caused degradation of EspB. Thus, in both model systems, brief exposure to lactoferrin causes loss and degradation of type III secretion system virulence proteins.

Bacterial Proteins↗

PubMed1999

Relationships between a new type IV secretion system and the icm/dot virulence system of Legionella pneumophila.

We describe here a Legionella pneumophila type IV secretion system that is distinct from the previously described icm/dot system. This type IV secretion system contains 11 genes (lvh ) homologous to genes of other type IV secretion systems, arranged in a similar manner. The lvh genes were found to be located on a DNA island with a GC content higher than the L. pneumophila chromosome. In contrast to the icm/dot system that was shown to be required for intracellular growth in HL-60-derived human macrophages and Acanthamoeba castellanii, the lvh system was found to be dispensable for intracellular growth in these two hosts. The lvh system was found to be partially required for RSF1010 conjugation, a process that was previously shown to be completely dependent on several icm/dot genes. However, results obtained from analysis of double mutants in the icm/dot genes and the lvh genes revealed that lvh genes can substitute for some components of the icm/dot system for RSF1010 conjugation, but not for intracellular growth. These results indicate that components of the icm/dot system and components of the lvh type IV secretion system are able to interact with one another.

Bacterial Proteins↗

PubMed2005

Activities of Pseudomonas aeruginosa effectors secreted by the Type III secretion system in vitro and during infection.

Pseudomonas aeruginosa utilizes a number of distinct pathways to secrete proteins that play various roles during infection. These include the type II secretion system, which is responsible for the secretion of the majority of exoproducts into the surrounding environment, including toxins and degradative enzymes. In contrast, the type III secretion system mediates the delivery of protein effectors directly into the cytoplasm of the host cell. Using tissue culture assays and a mouse acute-pneumonia model, we have determined the contribution of each of the type III effectors during infection. In strain PAK, ExoS is the major cytotoxin required for colonization and dissemination during infection. ExoT confers protection of tissue culture cells from type III-dependent lysis, while ExoY seemed to have little effect on cytotoxicity. ExoU is over 100-fold more cytotoxic than ExoS. The cytotoxicity of type II secretion was determined following deletion of the genes for the more toxic type III secretion system. The participation of these secretion systems during lifelong colonization of cystic fibrosis (CF) patients is unclear. By comparing clonal strains from the same patient isolated at the initial onset of P. aeruginosa infection and more than a decade later, after chronic colonization has been established, we show that initial strains are more cytotoxic than chronic strains that have evolved to reduce type III secretion. Constitutive expression of genes for the type III secretion system restored ExoS secretion but did not always reestablish cytotoxicity, suggesting that CF strains accumulate a number of mutations to reduce bacterial toxicity to the host.

ADP Ribose Transferases↗

PubMed2000

Phylogenetic analyses of the constituents of Type III protein secretion systems.

Multicomponent Type III protein secretion systems transfer gram-negative bacterial virulence factors directly from the bacterial cytoplasm to the cytoplasm of a host eukaryotic cell in a process that may involve a single energy-coupled step. Extensive evidence supports the conclusion that the genetic apparatuses that encode these systems have been acquired independently by different gram-negative bacteria, presumably by lateral transfer. In this paper we conduct phylogenetic analyses of currently sequenced constituents of these systems and their homologues. The results reveal the relative relatedness of these systems and show that they evolved with little or no exchange of constituents between systems. This fact suggests that horizontal transmission of the genes encoding these systems always occurred as a unit without the formation of hybrid gene clusters. Moreover, homologous flagellar proteins show phylogenetic clustering that suggests that the flagellar systems and Type III protein secretory systems diverged from each other following very early duplication of a gene cluster sharing many (but not all) genes. Phylogenies of most or all of the flagellar proteins follow those of the source organisms with little or no lateral gene transfer suggesting that homologous flagellar proteins are true orthologues. We suggest that the flagellar apparatus was the evolutionary precursor of Type III protein secretion systems.

Bacterial Proteins↗

PubMed2003

SseA is a chaperone for the SseB and SseD translocon components of the Salmonella pathogenicity-island-2-encoded type III secretion system.

The type III secretion system (TTSS) encoded by the Salmonella pathogenicity island 2 (SPI-2) is required for bacterial replication inside macrophages and for systemic infection in mice. Many TTSS secreted proteins, including effectors and components of the translocon, require chaperones which promote their stability, prevent their premature interactions or facilitate their secretion. In this study, the function of the first gene (sseA) of one of the SPI-2 operons (sseA-G) was investigated. This operon includes genes that encode translocon components (SseB, SseC and SseD), translocated proteins (SseF and SseG) and putative chaperones (SscA and SscB). sseA encodes a 12.5 kDa protein with a C-terminal region with the potential to form a coiled-coil structure, but no sequence similarity to other proteins. Mutation of sseA results in severe virulence attenuation and an intracellular replication defect. It is shown here that SseA is not a secreted protein, but is required for SPI-2-dependent translocation of two effector proteins (SifA and PipB). Furthermore, the translocon components SseB and SseD were not detected in an sseA mutant strain. By using a yeast two-hybrid assay and column binding experiments, it is demonstrated that SseA interacts directly with SseB and SseD. These results indicate that SseA is a chaperone for SseB and SseD. The inability of an sseA mutant to assemble the SPI-2 TTSS translocon accounts for its high level of virulence attenuation in vivo. To the authors' knowledge, this is the first chaperone described for the SPI-2 TTSS.

Animals↗

PubMed2004

The autotransporter secretion system.

The type V secretion system includes the autotransporter family, the two-partner system and the Oca family. The autotransporter secretion process involving first the translocation of the precursor through the inner membrane and then its translocation through the outer membrane via a pore formed by a beta-barrel is reviewed.

Bacteria↗

PubMed2003

Channel-tunnels: outer membrane components of type I secretion systems and multidrug efflux pumps of Gram-negative bacteria.

For translocation across the cell envelope of Gram-negative bacteria, substances have to overcome two permeability barriers, the inner and outer membrane. Channel-tunnels are outer membrane proteins, which are central to two distinct export systems: the type I secretion system exporting proteins such as toxins or proteases, and efflux pumps discharging antibiotics, dyes, or heavy metals and thus mediating drug resistance. Protein secretion is driven by an inner membrane ATP-binding cassette (ABC) transporter while drug efflux occurs via an inner membrane proton antiporter. Both inner membrane transporters are associated with a periplasmic accessory protein that recruits an outer membrane channel-tunnel to form a functional export complex. Prototypes of these export systems are the hemolysin secretion system and the AcrAB/TolC drug efflux pump of Escherichia coli, which both employ TolC as an outer membrane component. Its remarkable conduit-like structure, protruding 100 A into the periplasmic space, reveals how both systems are capable of transporting substrates across both membranes directly from the cytosol into the external environment. Proteins of the channel-tunnel family are widespread within Gram-negative bacteria. Their involvement in drug resistance and in secretion of pathogenic factors makes them an interesting system for further studies. Understanding the mechanism of the different export apparatus could help to develop new drugs, which block the efflux pumps or the secretion system.

Bacterial Outer Membrane Proteins↗

PubMed2026

A novel reverse lipase toxin substrate of the Staphylococcus aureus type VII secretion system.

The type VII secretion system (T7SS) is found in many Gram-positive bacteria and secretes toxins with antibacterial activity. Most characterized substrates have an N-terminal LXG domain that interacts with other helical partner proteins to form a composite T7SS targeting signal. Here we describe only the second substrate family to have a reverse domain arrangement. We show that TslM has a C-terminal LXG-like domain and an N-terminal lipase domain that has phospholipase activity. Secretion of TslM requires a single helical partner protein that binds to the TslM C-terminus, and its toxic activity is neutralized by a distinct family of membrane proteins. Genome analysis reveals that Staphylococcus aureus strains have the capacity to encode up to seven paralogous copies of this toxin family. Taken together our findings show that lipases are an important component of the staphylococcal T7SS toxin arsenal, and that toxins with a reverse domain arrangement are more widespread than previously appreciated.

Staphylococcus aureus↗

PubMed1998

Type III protein secretion systems in bacterial pathogens of animals and plants.

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.

Animals↗

PubMed2003

YscP and YscU regulate substrate specificity of the Yersinia type III secretion system.

Pathogenic Yersinia species use a type III secretion system to inhibit phagocytosis by eukaryotic cells. At 37 degrees C, the secretion system is assembled, forming a needle-like structure on the bacterial cell surface. Upon eukaryotic cell contact, six effector proteins, called Yops, are translocated into the eukaryotic cell cytosol. Here, we show that a yscP mutant exports an increased amount of the needle component YscF to the bacterial cell surface but is unable to efficiently secrete effector Yops. Mutations in the cytoplasmic domain of the inner membrane protein YscU suppress the yscP phenotype by reducing the level of YscF secretion and increasing the level of Yop secretion. These results suggest that YscP and YscU coordinately regulate the substrate specificity of the Yersinia type III secretion system. Furthermore, we show that YscP and YscU act upstream of the cell contact sensor YopN as well as the inner gatekeeper LcrG in the pathway of substrate export regulation. These results further strengthen the strong evolutionary link between flagellar biosynthesis and type III synthesis.

Amino Acid Sequence↗

PubMed2005

Structural characterization of the molecular platform for type III secretion system assembly.

Type III secretion systems (TTSSs) are multi-protein macromolecular 'machines' that have a central function in the virulence of many Gram-negative pathogens by directly mediating the secretion and translocation of bacterial proteins (termed effectors) into the cytoplasm of eukaryotic cells. Most of the 20 unique structural components constituting this secretion apparatus are highly conserved among animal and plant pathogens and are also evolutionarily related to proteins in the flagellar-specific export system. Recent electron microscopy experiments have revealed the gross 'needle-shaped' morphology of the TTSS, yet a detailed understanding of the structural characteristics and organization of these protein components within the bacterial membranes is lacking. Here we report the 1.8-A crystal structure of EscJ from enteropathogenic Escherichia coli (EPEC), a member of the YscJ/PrgK family whose oligomerization represents one of the earliest events in TTSS assembly. Crystal packing analysis and molecular modelling indicate that EscJ could form a large 24-subunit 'ring' superstructure with extensive grooves, ridges and electrostatic features. Electron microscopy, labelling and mass spectrometry studies on the orthologous Salmonella typhimurium PrgK within the context of the assembled TTSS support the stoichiometry, membrane association and surface accessibility of the modelled ring. We propose that the YscJ/PrgK protein family functions as an essential molecular platform for TTSS assembly.

Amino Acid Sequence↗

PubMed2000

The Sinorhizobium meliloti ExpE1 protein secreted by a type I secretion system involving ExpD1 and ExpD2 is required for biosynthesis or secretion of the exopolysaccharide galactoglucan.

In Sinorhizobium meliloti the biosynthesis of the exopolysaccharide galactoglucan (EPS II) is directed by the exp genes. The expD1 and expD2 gene products are homologous to components of type I secretion systems. ExpE1, the gene of which is located adjacent to expD1 and expD2, was detected in S. meliloti cells and culture supernatants. ExpD1 and ExpD2 were required for the secretion of ExpE1, indicating that ExpE1 is secreted by a type I secretion system involving ExpD1 and ExpD2. ExpE1 contains 15 aspartate- and glycine-rich nonapeptide repeats that were suggested to bind Ca(2+). The ability to bind Ca(2+) was demonstrated for a recombinant ExpE1 protein. Extracellular EPS II was not detected in cultures of non-polar expD1, expD2 and expE1 deletion mutants implying that these three genes are required for biosynthesis or secretion of galactoglucan.

Animals↗

PubMed2003

The roles of SsrA-SsrB and OmpR-EnvZ in the regulation of genes encoding the Salmonella typhimurium SPI-2 type III secretion system.

The type III secretion system (TTSS) encoded by Salmonella typhimurium pathogenicity island 2 (SPI-2) is expressed after bacterial entry into host cells. The SPI-2 TTSS secretes the translocon components SseBCD, which translocate across the vacuolar membrane a number of effector proteins whose action is required for intracellular bacterial replication. Several of these effectors, including SifA and SifB, are encoded outside SPI-2. The two-component regulatory system SsrA-SsrB, encoded within SPI-2, controls the expression of components of the SPI-2 TTSS apparatus as well as its translocated effectors. The expression of SsrA-B is in turn regulated by the OmpR-EnvZ two-component system, by direct binding of OmpR to the ssrAB promoter. Several environmental signals have been shown to induce in vitro expression of genes regulated by the SsrA-B or OmpR-EnvZ systems. In this work, immunoblotting and flow cytometry were used to analyse the roles of SsrA-B and OmpR-EnvZ in coupling different environmental signals to changes in expression of a SPI-2 TTSS translocon component (SseB) and two effector genes (sifA and sifB). Using single and double mutant strains the relative contribution of each regulatory system to the response generated by low osmolarity, acidic pH or the absence of Ca2+ was determined. SsrA-B was found to be essential for the induction of SPI-2 gene expression in response to each of these individual signals. OmpR-EnvZ was found to play a minor role in sensing these signals and to require a functional SsrA-B system to mediate their effect on SPI-2 TTSS gene expression.

Bacterial Proteins↗