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PubMed2006

The single transmembrane segment drives self-assembly of OutC and the formation of a functional type II secretion system in Erwinia chrysanthemi.

Many pathogenic Gram-negative bacteria secrete toxins and lytic enzymes via a multiprotein complex called the type II secretion system. This system, named Out in Erwinia chrysanthemi, consists of 14 proteins integrated or associated with the two bacterial membranes. OutC, a key player in this process, is probably implicated in the recognition of secreted proteins and signal transduction. OutC possesses a short cytoplasmic sequence, a single transmembrane segment (TMS), and a large periplasmic region carrying a putative PDZ domain. A hydrodynamic study revealed that OutC forms stable dimers of an elongated shape, whereas the PDZ domain adopts a globular shape. Bacterial two-hybrid, cross-linking, and pulldown assays revealed that the self-association of OutC is driven by the TMS, whereas the periplasmic region is dispensable for self-association. Site-directed mutagenesis of the TMS revealed that cooperative interactions between three polar residues located at the same helical face provide adequate stability for OutC self-assembly. An interhelical H-bonding mediated by Gln(29) appears to be the main driving force, and two Arg residues located at the TMS boundaries are essential for the stabilization of OutC oligomers. Stepwise mutagenesis of these residues gradually diminished OutC functionality and self-association ability. The triple OutC mutant R15V/Q29L/R36A became monomeric and nonfunctional. Self-association and functionality of the triple mutant were partially restored by the introduction of a polar residue at an alternative position in the interhelical interface. Thus, the OutC TMS is more than just a membrane anchor; it drives the protein self-association that is essential for formation of a functional secretion system.

Amino Acid Sequence↗

PubMed2000

Effect of vasoactive intestinal polypeptide (VIP) antagonism on rat jejunal fluid and electrolyte secretion induced by cholera and Escherichia coli enterotoxins.

BACKGROUND: The enteric nervous system is important in the pathophysiology of intestinal fluid secretion induced by cholera toxin (CT), Escherichia coli heat labile (LT), and heat stable (STa) toxins. The neurotransmitters involved are not fully elucidated. Vasoactive intestinal polypeptide (VIP), a potent intestinal secretagogue present in the enteric nervous system, is increased after exposure of the cat intestine to CT. Whether VIP is involved in the pathogenesis of cholera and other toxins in not known. AIM: To study in vivo the effect of VIP antagonism on jejunal fluid secretion induced by CT, LT, and STa. METHODS: CT, LT (25 microg), or 0.9% NaCl was instilled in an isolated 25 cm segment of rat jejunum, and the VIP antagonist (VIPa) [4Cl-D-Phe(6), Leu(17)]-VIP (0.2 or 2 microg/kg/min) or 0.9% NaCl was given intravenously. Two hours later, single pass in vivo jejunal perfusion was performed to assess fluid movement. In STa experiments, intravenous VIPa or 0.9% NaCl was given and 30 minutes later the jejunal segment was perfused with a solution containing STa 200 microg/l. RESULTS: VIPa had no effect on basal intestinal fluid absorption. CT induced net fluid secretion (median -68 microl/min/g dry intestinal weight (interquartile range -80 to -56)) which was dose dependently reversed by VIPa (6.2 (-16 to 34) and 29 (17 to 42); p<0.01). Similarly, LT induced secretion (-63 (-73 to -30)) was attenuated by VIPa (0.2 microg/kg/min) (-15 (-24 to -1); p<0.01) and totally reversed to normal levels by VIPa (2 microg/kg/min) (37 (28-56); p<0. 01 compared with LT and not significant compared with normal controls). STa induced secretion (-17 (-19 to -2)) was also reversed by VIPa (12 (9-23) and 14 (0-26); p<0.01). CONCLUSION: VIP plays an important role in CT, LT, and STa induced intestinal secretion and may be the final putative neurotransmitter in the pathophysiology of these toxins.

Animals↗

PubMed2006

Interactions between peptidoglycan and the ExeAB complex during assembly of the type II secretin of Aeromonas hydrophila.

Aeromonas hydrophila transports extracellular protein toxins via the type II secretion system, an export mechanism comprised of numerous proteins that spans both the inner and outer membranes. Two components of this secretion system, ExeA and ExeB, form a complex in the inner membrane that functions to locate and/or assemble the ExeD secretin in the outer membrane. In the studies reported here, two-codon insertion mutagenesis of exeA revealed that an insertion at amino acid 495 in the C-terminal region of ExeA did not alter ExeAB complex formation yet completely abrogated its involvement in ExeD secretin assembly and thus rendered the bacteria secretion negative. In silico analysis of protein motifs with similar amino acid profiles revealed that this amino acid is located within a putative peptidoglycan (PG) binding motif in the periplasmic domain of ExeA. Substitution mutations of three highly conserved amino acids in the motif were constructed. In cells expressing each of these mutants, the ability to assemble the ExeD secretin or secrete aerolysin was lost, while ExeA retained the ability to form a complex with ExeB. In in vivo cross-linking experiments, wild-type ExeA could be cross-linked to PG, whereas the three substitution mutants of ExeA could not. These data indicate that PG binding and/or remodelling plays a role in the function of the ExeAB complex during assembly of the ExeD secretin.

Aeromonas hydrophila↗

PubMed2002

Structure and role of coupling proteins in conjugal DNA transfer.

Type IV secretory systems are transmembrane bacterial multiprotein complexes. They are pivotal for conjugation, bacterial-induced plant tumour formation, toxin secretion and mammalian pathogen intracellular activity. These systems are involved in the spread of antibiotic resistance genes among bacteria by enabling conjugative DNA transfer. When such translocons transport DNA, they require the assistance of multimeric integral inner membrane proteins, the type IV coupling proteins. Its structural prototype is plasmid R388 TrwB protein, responsible for coupling the relaxosome with the DNA transport apparatus during bacterial conjugation. Its monomeric molecular structure is reminiscent of ring helicases and AAA ATPases. The quaternary structure is made up by six equivalent protomers featuring a flattened sphere resembling F1-ATPase, with a central channel traversing the particle, thus connecting cytoplasm and periplasm.

Bacteria↗

PubMed2000

Legionella pneumophila pathogesesis: a fateful journey from amoebae to macrophages.

Legionella pneumophila first commanded attention in 1976, when investigators from the Centers for Disease Control and Prevention identified it as the culprit in a massive outbreak of pneumonia that struck individuals attending an American Legion convention (). It is now clear that this gram-negative bacterium flourishes naturally in fresh water as a parasite of amoebae, but it can also replicate within alveolar macrophages. L. pneumophila pathogenesis is discussed using the following model as a framework. When ingested by phagocytes, stationary-phase L. pneumophila bacteria establish phagosomes which are completely isolated from the endosomal pathway but are surrounded by endoplasmic reticulum. Within this protected vacuole, L. pneumophila converts to a replicative form that is acid tolerant but no longer expresses several virulence traits, including factors that block membrane fusion. As a consequence, the pathogen vacuoles merge with lysosomes, which provide a nutrient-rich replication niche. Once the amino acid supply is depleted, progeny accumulate the second messenger guanosine 3',5'-bispyrophosphate (ppGpp), which coordinates entry into the stationary phase with expression of traits that promote transmission to a new phagocyte. A number of factors contribute to L. pneumophila virulence, including type II and type IV secretion systems, a pore-forming toxin, type IV pili, flagella, and numerous other factors currently under investigation. Because of its resemblance to certain aspects of Mycobacterium, Toxoplasma, Leishmania, and Coxiella pathogenesis, a detailed description of the mechanism used by L. pneumophila to manipulate and exploit phagocyte membrane traffic may suggest novel strategies for treating a variety of infectious diseases. Knowledge of L. pneumophila ecology may also inform efforts to combat the emergence of new opportunistic macrophage pathogens.

Amoeba↗

PubMed1999

Pathogenesis of septic shock in Pseudomonas aeruginosa pneumonia.

The pathogenesis of septic shock occurring after Pseudomonas aeruginosa pneumonia was studied in a rabbit model. The airspace instillation of the cytotoxic P. aeruginosa strain PA103 into the rabbit caused a consistent alveolar epithelial injury, progressive bacteremia, and septic shock. The lung instillation of a noncytotoxic, isogenic mutant strain (PA103DeltaUT), which is defective for production of type III secreted toxins, did not cause either systemic inflammatory response or septic shock, despite a potent inflammatory response in the lung. The intravenous injection of PA103 did not cause shock or an increase in TNF-alpha, despite the fact that the animals were bacteremic. The systemic administration of either anti-TNF-alpha serum or recombinant human IL-10 improved both septic shock and bacteremia in the animals that were instilled with PA103. Radiolabeled TNF-alpha instilled in the lung significantly leaked into the circulation only in the presence of alveolar epithelial injury. We conclude that injury to the alveolar epithelium allows the release of proinflammatory mediators into the circulation that are primarily responsible for septic shock. Our results demonstrate the importance of compartmentalization of inflammatory mediators in the lung, and the crucial role of bacterial cytotoxins in causing alveolar epithelial damage in the pathogenesis of acute septic shock in P. aeruginosa pneumonia.

Animals↗

PubMed2024

Yersinia entomophaga Tc toxin is released by T10SS-dependent lysis of specialized cell subpopulations.

Disease-causing bacteria secrete numerous toxins to invade and subjugate their hosts. Unlike many smaller toxins, the secretion machinery of most large toxins remains enigmatic. By combining genomic editing, proteomic profiling and cryo-electron tomography of the insect pathogen Yersinia entomophaga, we demonstrate that a specialized subset of these cells produces a complex toxin cocktail, including the nearly ribosome-sized Tc toxin YenTc, which is subsequently exported by controlled cell lysis using a transcriptionally coupled, pH-dependent type 10 secretion system (T10SS). Our results dissect the Tc toxin export process by a T10SS, identifying that T10SSs operate via a previously unknown lytic mode of action and establishing them as crucial players in the size-insensitive release of cytoplasmically folded toxins. With T10SSs directly embedded in Tc toxin operons of major pathogens, we anticipate that our findings may model an important aspect of pathogenesis in bacteria with substantial impact on agriculture and healthcare.

Proteomics↗

PubMed2006

Interaction with CagF is required for translocation of CagA into the host via the Helicobacter pylori type IV secretion system.

Development of severe gastric diseases is strongly associated with those strains of Helicobacter pylori that contain the cag pathogenicity island (PAI) inserted into the chromosome. The cag PAI encodes a type IV secretion system that translocates the major disease-associated virulence protein, CagA, into the host epithelial cell. CagA then affects host signaling pathways, leading to cell elongations and inflammation. Since the precise mechanism by which the CagA toxin is translocated by the type IV secretion system remained elusive, we used fusion proteins and immunoprecipitation studies to identify CagA-interacting secretion components. Here we demonstrate that CagA, in addition to other yet-unidentified proteins, interacts with CagF, presumably at the inner bacterial membrane. This interaction is required for CagA translocation, since an isogenic nonpolar cagF mutant was translocation deficient. Our results suggest that CagF may be a protein with unique chaperone-like function that is involved in the early steps of CagA recognition and delivery into the type IV secretion channel.

Antigens, Bacterial↗

PubMed1999

Proabsorptive and prosecretory roles for nitric oxide in cholera toxin induced secretion.

BACKGROUND: Cholera toxin causes small intestinal hypersecretion by inducing a coordinated response from enterocytes, enterochromaffin cells, enteric neurones, and the vascular supply. Nitric oxide has been implicated in the function of these separate components. AIMS: To explore the role of nitric oxide in the totality of cholera toxin induced secretion in vivo. METHODS: One group of adult male Wistar rats was treated with the nitric oxide synthase inhibitors NG-nitro-L-arginine methyl ester (L-NAME; subcutaneously or intraluminally), NG-methyl-L-arginine (L-NMA), or 7-nitroindazole. A second group of rats was treated with L-arginine (intraperitoneally or intraluminally) or D-arginine. The small intestine was isolated between two cannulae and instilled with 75 microg cholera toxin or saline for two hours. Small intestinal perfusion of a plasma electrolyte solution containing [14C]-PEG was undertaken to determine net water and electrolyte movement. After the experiment macroscopic and microscopic intestinal appearances were noted and jejunal 5-hydroxytryptamine concentrations were determined. RESULTS: Both L-arginine and L-NAME induced secretion in the basal state, but only when administered intraluminally. Systemically applied L-NAME caused a dose dependent reduction in cholera toxin induced secretion. This was paralleled by L-NMA but not by 7-nitroindazole or by intraluminally applied L-NAME. Systemically applied L-NAME caused notable cyanosis of the intestine, consistent with mesenteric ischaemia, but no microscopic abnormalities. Systemically applied L-arginine but not D-arginine also reduced cholera toxin induced secretion and inhibited 5-hydroxytryptamine release. CONCLUSION: Nitric oxide has a duality of roles in cholera toxin induced secretion, acting both as an absorbagogue and a secretagogue. Its mechanisms of action include the maintenance of mucosal perfusion and enterochromaffin cell stabilisation.

Animals↗

PubMed2005

Combined sacB-based negative selection and cre-lox antibiotic marker recycling for efficient gene deletion in pseudomonas aeruginosa.

The complete genome of the bacterial pathogen Pseudomonas aeruginosa has now been sequenced, allowing gene deletion, one of the most frequently used methods in gene function study, to be fully exploited. In this study, we combine the sacB-based negative selection system with a cre-lox antibiotic marker recycling method. This methodology allows allelic exchange between a target gene and a gentamicin cassette flanked by the two lox sequences. A tetracycline plasmid expressing the cre recombinase is then introduced in the mutant strain to catalyze the excision of the lox-flanked resistance marker. We demonstrate here the efficiency of the combination of these two methods in P. aeruginosa by successively deleting ExoS and ExoT, which are two genetically independent toxins of the type-three secretion system (TTSS). This functional cre-lox recycling antibiotic marker system can create P. aeruginosa strains with multiple mutations without modifying the antibiotic resistance profile when compared to the parental strain.

Base Sequence↗

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↗

PubMed2003

Molecular pathogenicity of the oral opportunistic pathogen Actinobacillus actinomycetemcomitans.

Periodontitis is mankind's most common chronic inflammatory disease. One severe form of periodontitis is localized aggressive periodontitis (LAP), a condition to which individuals of African origin demonstrate an increased susceptibility. The main causative organism of this disease is Actinobacillus actinomycetemcomitans. A member of the Pasteurellaceae, A. actinomycetemcomitans produces a number of interesting putative virulence factors including (a) an RTX leukotoxin that targets only neutrophils and monocytes and whose action is influenced by a novel type IV secretion system involved in bacterial adhesion; (b) the newly discovered toxin, cytolethal distending toxin (CDT); and (c) a secreted chaperonin 60 with potent leukocyte-activating and bone resorbing activities. This organism also produces a plethora of proteins able to inhibit eukaryotic cell cycle progression and proteins and peptides that can induce distinct forms of proinflammatory cytokine networks. A range of other proteins interacting with the host is currently being uncovered. In addition to these secreted factors, A. actinomycetemcomitans is invasive with an unusual mechanism for entering, and traveling within, eukaryotic cells. This review focuses on recent advances in our understanding of the molecular and cellular pathogenicity of this fascinating oral bacterium.

Actinobacillus Infections↗

PubMed1997

Interactions of dedicated export membrane proteins of the colicin V secretion system: CvaA, a member of the membrane fusion protein family, interacts with CvaB and TolC.

The antibacterial peptide toxin colicin V uses a dedicated signal sequence-independent system for its secretion in Escherichia coli and requires the products of three genes, cvaA, cvaB, and tolC. As a member of the membrane fusion protein family, CvaA is supposed to form a bridge that connects the inner and outer membranes via interaction with CvaB and TolC, respectively. In this study, we investigated the possible interaction of these proteins. When CvaA or CvaB was absent, the corresponding amount of CvaB or CvaA, respectively, was decreased, and the amounts of both proteins were reduced when TolC was depleted. Translational lacZ fusions showed that TolC did not affect the synthesis of either CvaA-beta-galactosidase or CvaB-beta-galactosidase, and CvaA or CvaB did not affect the synthesis of CvaB-beta-galactosidase or CvaA-beta-galactosidase, respectively. However, the stabilities of CvaA and CvaB proteins were affected by the absence of one another and by that of TolC. The instability of CvaA was more severe in TolC-depleted cells than in CvaB-depleted cells. On the other hand, CvaB was less stable in the absence of CvaA than in the absence of TolC. In addition, using a cross-linking reagent, we showed that CvaA directly interacts with both CvaB and TolC proteins. Taken together, these data support the hypothesized structural role of CvaA in connecting CvaB and TolC.

ATP-Binding Cassette Transporters↗

PubMed1999

Involvement of serotonin and calcium channels in the intestinal fluid secretion evoked by bile salt and cholera toxin.

1. The enteric nervous system (ENS) is activated when exposing the intestinal mucosa to cholera toxin or certain bile salts. Cholera toxin stimulates ENS, at least in part, by the release of 5-hydroxytryptamine (5-HT) from the enterochromaffin cells. Calcium channel blockers of the L-type markedly attenuate the fluid secretion and the luminal release of 5-HT caused by cholera toxin. 2. The objective of the present study was to elucidate if sodium deoxycholate activated ENS in a similar manner as cholera toxin. Furthermore, the effect of several calcium channel blockers was tested on the fluid secretion caused by cholera toxin or bile salt. 3. Sodium deoxycholate (4 mM) caused a release of 5-HT into the intestinal lumen, which was inhibited by calcium channel blockade. Granisetron, a 5-HT3 receptor blocker, partly inhibited the fluid secretion caused by bile salt. 4. The effects of nifedipine, felodipine, R-felodipine, H186/86 (t-butyl analogue of felodipine) on the fluid secretion caused by cholera toxin or sodium deoxycholate were studied. Both secretory states were markedly attenuated in a dose dependent manner by all calcium channel blockers tested regardless of their effects on arterial pressure. 5. It is concluded that both cholera toxin and bile salt activate ENS, at least in part, via a release of 5-HT from the enterochromaffin cells. The antisecretory effect calcium channel blockers is partly explained by an inhibition of this release of 5-HT.

Animals↗

PubMed2002

Evidence for a type III secretion system in Aeromonas salmonicida subsp. salmonicida.

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.

ADP Ribose Transferases↗

PubMed2006

Bordetella bronchiseptica modulates macrophage phenotype leading to the inhibition of CD4+ T cell proliferation and the initiation of a Th17 immune response.

Bordetella bronchiseptica is a Gram-negative bacterium equipped with several colonization factors that allow it to establish a persistent infection of the murine respiratory tract. Previous studies indicate that B. bronchiseptica adenylate cyclase toxin (ACT) and the type III secretion system (TTSS) synergize to drive dendritic cells into an altered phenotype to down-regulate the host immune response. In this study, we examined the effects of B. bronchiseptica ACT and TTSS on murine bone marrow-derived macrophages. We demonstrate that ACT and TTSS are required for the inhibition of Ag-driven CD4+ T cell proliferation by bacteria-infected macrophages. We identify PGE2 as the mediator of this inhibition, and we show that ACT and the TTSS synergize to increase macrophage production of PGE2. We further demonstrate that B. bronchiseptica can modulate normal macrophage function and drive the immune response toward a Th17 phenotype classified by the significant production of IL-17. In this study, we show that B. bronchiseptica-infected macrophages can induce IL-17 production from naive CD4+ splenocytes, and that lung tissues from B. bronchiseptica-infected mice exhibit a strong Th17 immune response. ACT inhibited surface expression of CD40 and CD86, suppressed TNF-alpha production, and up-regulated IL-6 production. TTSS also synergized with ACT to up-regulate IL-10 and PGE2 secretion. These findings indicate that persistent colonization by B. bronchiseptica may rely on the ability of the bacteria to differentially modulate both macrophage and dendritic cell function leading to an altered adaptive immune response and subsequent bacterial colonization.

Adenylate Cyclase Toxin↗

PubMed2026

Genome sequences of pirAB+ and pirAB- Vibrio campbellii strains isolated from shrimp ponds with mortality outbreaks carry type VI secretion systems.

Vibrio campbellii strains PH1401 and PH1409 were isolated from shrimp ponds with documented mortality outbreaks in the Philippines. PH1401 and PH1409 share identity with V. campbellii strain BoB-53. Whole-genome analysis reveals full-length pirAB binary toxin genes in PH1401. Both strains carry three type VI secretion systems.

Vibrio campbellii↗

PubMed2005

The Pseudomonas aeruginosa PA14 type III secretion system is expressed but not essential to virulence in the Caenorhabditis elegans-P. aeruginosa pathogenicity model.

The Pseudomonas aeruginosa type III secretion system (TTSS), enabling direct injection of toxins into host cells, has been shown to be crucial to virulence in several models of P. aeruginosa pathogenesis. Using the strain PA14 and its isogenic mutant, PA14exsA, we investigated the role of the TTSS during infection of the nematode Caenorhabditis elegans. Although C. elegans N2 was killed by PA14 in an infection like process over 48 to 72 h the same effect was observed following infection with PA14exsA, implying that a functional TTSS was not essential for virulence. This was despite the TTSS being actively expressed during C. elegans infection as demonstrated by the use of green fluorescent reporter constructs and RT-PCR. However, compared to the wild type PA14, PA14exsA did display a reduced rate of killing of C. elegans strain AU1 which harbours a mutation in the sek-1 gene encoding a MAP kinase involved in nematode innate immunity. A fuller understanding of the mechanism of resistance to type III attack in C. elegans may lead to the identification and development of novel therapeutic targets affording protection to TTSS products in man.

Animals↗