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PubMed1995

Role of enteric nervous system in Shigella dysenteriae type 1 toxin-induced fluid secretion in rabbit ileum.

Intracellular-free calcium concentrations were measured in the intestinal epithelial cells isolated from the rabbit ileum treated with Shigella dysenteriae type 1 toxin. There is almost a five-fold increase in the [Ca+2]i levels in the epithelial cells isolated from the toxin-treated rabbit ileum compared to control. The [Ca+2]i levels were also measured in the presence or absence of tetrodotoxin, a neurotoxin, and atropine, a muscarinic cholinergic antagonist. These drugs significantly decreased the [Ca+2]i levels of the cells isolated from the toxin-treated rabbit ileum. Furthermore, the unidirectional fluxes of Na+ and Cl- were determined in both the S. dysenteriae type 1 toxin-treated and control rabbit ileum. Dantrolene significantly stimulated net absorption of these ions in the toxin-treated ileum, suggesting the involvement of [Ca+2]i in the regulation of intestinal electrolyte transport. To evaluate the role of neural mechanisms, the transport of these ions was also determined in the presence and absence of tetrodotoxin and atropine. The presence of these drugs caused significant absorption of Na+ and Cl- in the toxin-treated compared to their absence. These findings suggest that the enteric nervous system plays an important role in S. dysenteriae type 1 toxin-induced diarrhoea, and the release of neurotransmitters causes changes in the intracellular calcium levels, ultimately changing electrolyte transport across the rabbit ileum.

Animals↗

PubMed2003

Distribution of the secondary type III secretion system locus found in enterohemorrhagic Escherichia coli O157:H7 isolates among Shiga toxin-producing E. coli strains.

The ability of the complete genome sequence of enterohemorrhagic Escherichia coli O157 led to the identification of a 17-kb chromosomal region which contained a type III secretion system gene cluster at min 64.5. This locus contains open reading frames whose amino acid sequences show high degrees of similarity with those of proteins that make up the type III secretion apparatus, which is encoded by the inv-spa-prg locus on a Salmonella SPI-1 pathogenicity island. This locus was designated ETT2 (E. coli type III secretion 2) and consisted of the epr, epa, and eiv genes. ETT2 was found in enteropathogenic E. coli strains and also in some non-O157 Shiga toxin-producing E. coli (STEC) strains, but most of them contained a truncated portion of ETT2. Most O157 isolates had a complete collection of toxin-encoding genes eae and hlyA and the ETT2 locus, while most O26 strains had toxin-encoding genes eae and hlyA genes but an incomplete ETT2 locus. Thus, an intact copy of ETT2 might mark a pathogenic distinction for particular STEC strains. Therefore, the presence of the ETT2 locus can be used for identification of truly pathogenic STEC strains and for molecular fingerprinting of the epidemic strains in humans and animals.

Animals↗

PubMed2004

[Progress in type III secretion system].

Many Gram-negative bacteria use the type III secretion system (TTSS) to inject toxin proteins into host cells, then cause the plague disease, typhoid fever, diarrhea disease etc. Chaperone proteins is one of four components of MTSS and play an important role in helping the secretion of toxin proteins. Although these proteins had been found for almost ten years, the detailed function of chaperones is enigmatic. In this review, a brief introduction is given about the progress of classification, function of chaperones and characteristics of the interactions between chaperones and substrates.

Gram-Negative Bacteria↗

PubMed2000

Bordetella pertussis adenylate cyclase toxin as a tool to analyze molecular interactions in a bacterial two-hybrid system.

Bordetella pertussis secretes a calmodulin-activated adenylate cyclase toxin (CyaA) that is able to enter into eukaryotic cells. We took advantage of the modular structure of the catalytic domain of CyaA to design a genetic system that can detect protein-protein interactions in Escherichia coli. This bacterial two-hybrid system is based on the functional complementation between two complementary fragments, T25 and T18, of the catalytic domain of CyaA, in an E. coli cya strain. This bacterial two-hybrid system could find applications in the studies of structure/function relationships of proteins, in functional analysis of genomes, in high-throughput screening of interacting ligands and in design of new therapeutic agents.

Adenylate Cyclase Toxin↗

PubMed2006

The type III pseudomonal exotoxin U activates the c-Jun NH2-terminal kinase pathway and increases human epithelial interleukin-8 production.

Microbial interactions with host cell signaling pathways are key determinants of the host cell response to infection. Many toxins secreted by bacterial type III secretion systems either stimulate or inhibit the host inflammatory response. We investigated the role of type III secreted toxins of the lung pathogen Pseudomonas aeruginosa in the inflammatory response of human respiratory epithelial cells to infection. Using bacteria with specific gene deletions, we found that interleukin-8 production by these cells was almost entirely dependent on bacterial type III secretion of exotoxin U (ExoU), a phospholipase, although other bacterial factors are involved. ExoU activated the c-Jun NH(2)-terminal kinase pathway, stimulating the phosphorylation and activation of mitogen-activated kinase kinase 4, c-Jun NH(2)-terminal kinase, and c-Jun. This in turn increased levels of transcriptionally competent activator protein-1. Although this pathway was dependent on the lipase activity of ExoU, it was independent of cell death. Activation of mitogen-activated kinase signaling by ExoU in this fashion is a novel mechanism by which a bacterial product can initiate a host inflammatory response, and it may result in increased epithelial permeability and bacterial spread.

Epithelial Cells↗

PubMed2001

Targeting exported substrates to the Yersinia TTSS: different functions for different signals?

Many Gram-negative pathogens utilize a type III secretion system (TTSS) to inject toxins into the cytosol of eukaryotic cells. Previous studies have indicated that exported substrates are targeted to the Yersinia TTSS via the coding regions of their 5' mRNA sequences, as well as by their cognate chaperones. However, recent results from our laboratory have challenged the role of mRNA targeting signals, as we have shown that the amino termini of exported substrates are crucial for type III secretion. Here, we discuss the nature of these amino-terminal secretion signals and propose a model for the secretion of exported substrates by amino-terminal and chaperone-mediated signals. In addition, we discuss the roles of chaperones as regulators of virulence gene expression and present models suggesting that such regulation can occur independently of the delivery of their substrates to the secretion apparatus.

Bacterial Outer Membrane Proteins↗

PubMed2003

Oligomerization of type III secretion proteins PopB and PopD precedes pore formation in Pseudomonas.

Pseudomonas aeruginosa is the agent of opportunistic infections in immunocompromised individuals and chronic respiratory illnesses in cystic fibrosis patients. Pseudomonas aeruginosa utilizes a type III secretion system for injection of toxins into the host cell cytoplasm through a channel on the target membrane (the 'translocon'). Here, we have functionally and structurally characterized PopB and PopD, membrane proteins implicated in the formation of the P.aeruginosa translocon. PopB and PopD form soluble complexes with their common chaperone, PcrH, either as stable heterodimers or as metastable heterooligomers. Only oligomeric forms are able to bind to and disrupt cholesterol-rich membranes, which occurs within a pH range of 5-7 in the case of PopB/PcrH, and only at acidic pH for PcrH-free PopD. Electron microscopy reveals that upon membrane association PopB and PopD form 80 A wide rings which encircle 40 A wide cavities. Thus, formation of metastable oligomers precedes membrane association and ring generation in the formation of the Pseudomonas translocon, a mechanism which may be similar for other pathogens that employ type III secretion systems.

Amino Acid Sequence↗

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↗

PubMed1998

Functional conservation of the effector protein translocators PopB/YopB and PopD/YopD of Pseudomonas aeruginosa and Yersinia pseudotuberculosis.

Virulent Yersinia species cause systemic infections in rodents, and Y. pestis is highly pathogenic for humans. Pseudomonas aeruginosa, on the other hand, is an opportunistic pathogen, which normally infects only compromised individuals. Surprisingly, these pathogens both encode highly related contact-dependent secretion systems for the targeting of toxins into eukaryotic cells. In Yersinia, YopB and YopD direct the translocation of the secreted Yop effectors across the target cell membrane. In this study, we have analysed the function of the YopB and YopD homologues, PopB and PopD, encoded by P. aeruginosa. Expression of the pcrGVHpopBD operon in defined translocation-deficient mutants (yopB/yopD) of Yersinia resulted in complete complementation of the cell contact-dependent, YopE-induced cytotoxicity of Y. pseudotuberculosis on HeLa cells. We demonstrated that the complementation fully restored the ability of Y. pseudotuberculosis to translocate the effector molecules YopE and YopH into the HeLa cells. Similar to YopB, PopB induced a lytic effect on infected erythrocytes. The lytic activity induced by PopB could be prevented if the erythrocytes were infected in the presence of sugars larger than 3 nm in diameter, indicating that PopB induced a pore of similar size compared with that induced by YopB. Our findings show that the contact-dependent toxin-targeting mechanisms of Y. pseudotuberculosis and P. aeruginosa are conserved at the molecular level and that the translocator proteins are functionally interchangeable. Based on these similarities, we suggest that the translocation of toxins such as ExoS, ExoT and ExoU by P. aeruginosa across the eukaryotic cell membrane occurs via a pore induced by PopB.

Amino Acid Sequence↗

PubMed2006

The unusual extended signal peptide region of the type V secretion system is phylogenetically restricted.

The plasmid encoded toxin, Pet, is a prototypical member of the serine protease autotransporters of the Enterobacteriaceae. In addition to the passenger and beta-domains typical of autotransporters, in silico predictions indicate that Pet possesses an unusually long N-terminal signal sequence. The signal sequence can be divided into five regions termed N1 (charged), H1 (hydrophobic), N2, H2 and C (cleavage site) domains. The N1 and H1 regions, which we have termed the extended signal peptide region, demonstrate remarkable conservation. In contrast, the N2, H2 and C regions demonstrate significant variability and are reminiscent of typical Sec-dependent signal sequences. Despite several investigations, the function of the extended signal peptide region remains obscure and surprisingly it has not been proven that the extended signal peptide region is actually synthesized as part of the signal sequence. Here, we demonstrate that the extended signal peptide region is present only in Gram-negative bacterial proteins originating from the classes Beta- and Gammaproteobacteria, and more particularly only in proteins secreted via the Type V secretion pathway: autotransporters, TpsA exoproteins of the two-partner system and trimeric autotransporters. In vitro approaches demonstrate that the DNA region encoding the extended signal peptide region is transcribed and translated.

Amino Acid Sequence↗

PubMed2004

Pathogenesis of Helicobacter pylori infection.

Research in the last year has provided new insights into the function of the the cag-associated type IV secretion system and the vacuolating toxin VacA. A quite new aspect was disclosed by the finding that Helicobacter pylori in Mongolian gerbils colonizes a very distinct topology in the gastric mucous layer, obviously providing optimal conditions for long-term survival. Further research activities focused on H. pylori ammonia and metal metabolism as well as on bacterial stress defence mechanisms. Differential expression of approximately 7% of the bacterial genome was found at low pH suggesting that H. pylori has evolved a multitude of acid-adaptive mechanisms. VacA was shown to interrupt phagosome maturation in macrophage cell lines as well as to modulate and interfere with T lymphocyte immunological functions. Gastric mucosa as well as the H. pylori-infected epithelial cell line AGS strongly express IL-8 receptor A and B, which might contribute to the augmentation of the inflammatory response. Accumulating evidence implicates genetic variation in the inflammatory response to H. pylori in the etiology of the increased risk of gastric cancer after H. pylori infection. The chronic imbalance between apoptosis and cell proliferation is the first step of gastric carcinogenesis. In this regard, it was demonstrated that coexpression of two H. pylori proteins, CagA and HspB, in AGS cells, caused an increase in E2F transcription factor, cyclin D3, and phosphorylated retinoblastoma protein. Taken together, we now have a better understanding of the role of different virulence factors of H. pylori. There is still a lot to be learned, but the promising discoveries summarized here, demonstrate that the investigation of the bacterial survival strategies will give novel insights into pathogenesis and disease development.

Adaptation, Physiological↗

PubMed1993

Involvement of the myenteric plexus in the cholera toxin-induced net fluid secretion in the rat small intestine.

BACKGROUND: The enteric nervous system is responsible in vivo for most of the change in fluid transport induced by cholera toxin. The aim of the present study was to investigate the importance of the myenteric plexus in the Intramural reflex responsible for this secretion. METHODS: Long-term ablation of the myenteric plexus was achieved by serosal application of benzalkonium chloride on jejunal segments in rats. RESULTS: The treated segments without functioning myenteric plexus showed a normal net fluid absorption. Cholera toxin in this segment only induced a reduction of fluid absorption, whereas in a nontreated ileal segment it concomitantly induced a conspicuous net fluid secretion. Intravenous hexamethonium did not change the cholera toxin response in the treated jejunal segments, whereas vasoactive intestinal polypeptide elicited a marked secretion. CONCLUSIONS: Benzalkonium chloride treatment eliminated the ability of cholera toxin to induce intestinal secretion. Thus, all afferent fibers in the intramural secretory reflex activated by cholera toxin are probably conveyed via the myenteric plexus, which functions as the integrating center in the enteric nervous system. The Ussing chamber technique using stripped intestinal preparations cannot be used when studying effects of luminal secretagogues.

Animals↗

PubMed2025

Type VI secretion system activity at lethal antibiotic concentrations leads to overestimation of weapon potency.

Competition assays are a mainstay of modern microbiology, offering a simple and cost-effective means to quantify microbe-microbe interactions in vitro. Here, we demonstrate a key weakness of this method that arises when competing microbes interact via toxins, such as those secreted via the type VI secretion system (T6SS). Time-lapse microscopy reveals that T6SS-armed Acinetobacter baylyi bacteria can maintain lethal T6SS activity against E. coli target cells, even under selective conditions intended to eliminate A. baylyi. Further, this residual killing creates a density- and T6SS-dependent bias in the apparent recovery of E. coli, leading to a misreporting of competition outcomes where target survival is low. We also show that incubating A. baylyi/E. coli co-cultures in liquid antibiotic prior to selective plating can substantially correct this bias. Our findings demonstrate the need for caution when using selective plating as part of T6SS competition assays, or assays involving other toxin-producing bacteria.

Type VI Secretion Systems↗

PubMed2026

Identification of Specific Virulence Factors of Pseudomonas Strains in the Biocontrol of the Potato Pest Tecia solanivora.

Tecia solanivora (The Guatemalan potato tuber moth) is a major potato pest, responsible for up to 20% of crop losses and a significant economic impact. Certain Pseudomonas exhibit insecticidal activity and produce virulence factors with cytotoxic and antimicrobial properties, positioning them as promising candidates for biological control. This study evaluated seven Pseudomonas strains with insecticidal activity and identified key virulence factors involved. The strains demonstrated varying degrees of insecticidal activity, with Pseudomonas protegens strains CHA0 and 59C being the most lethal, causing over 75% mortality and triggering a systemic melanization response in the insects. Genomic analysis revealed 175 virulence-related genes shared across all strains and 16 genes specific to the highly insecticidal ones, including genes for antimicrobial compounds and insect toxins. Mutational analysis confirmed the roles of hydrogen cyanide, 2,4-diacetylphloroglucinol, pyoluteorin, Fit toxin, and two-partner secretion systems in P. protegens CHA0 insecticidal activity. This strain also exhibited insecticidal effects on adult T. solanivora and delayed egg hatching and pupal emergence. In microcosm assays, P. protegens CHA0 reduced tuber damage caused by T. solanivora larvae by up to 38%. These results suggest that P. protegens CHA0 is a promising biocontrol agent, providing a sustainable alternative to chemical pesticides to control T. solanivora.

Animals↗

PubMed1990

Roles of the disulfide bond and the carboxy-terminal region of the S1 subunit in the assembly and biosynthesis of pertussis toxin.

A Bordetella pertussis expression system was developed to analyze the structure-function relationship, in vivo assembly, and biosynthesis of pertussis toxin. The toxin structural gene was first deleted from the B. pertussis chromosome; into the resulting B. pertussis strain the toxin gene was introduced on a low-copy-number, broad-host-range plasmid. The amount of pertussis toxin produced and secreted with this expression system was in the same order of magnitude as that produced by B. pertussis Tohama I, indicating that although the plasmid may be present in more than one copy per cell, overproduction of the toxin was not achieved in B. pertussis. Expression of mutant pertussis toxin genes in which the codon for Cys-41 was deleted or altered or in which the carboxy-terminal region was deleted showed that both the single intrachain disulfide bond and the carboxy-terminal region of S1 are essential for the stable expression, assembly, and secretion of S1. On the other hand, the B oligomer was efficiently secreted in the culture medium in the absence of the S1 subunit. The secreted B oligomer contained S2, S3, and S4 subunits as evidenced by enzyme-linked immunosorbent assay and was fully functional with respect to haptoglobin binding. Furthermore, the deletion of the hydrophobic carboxy-terminal region has a drastic effect on S1 subunit solubility; however, inclusion of the hydrophobic region was not sufficient for assembly and secretion, indicating that other interactions involving amino acids beyond residue 207 of the S1 subunit are also required.

Amino Acid Sequence↗

PubMed2002

The E. coli alpha-hemolysin secretion system and its use in vaccine development.

Many Gram-negative bacteria use a type I secretion system to translocate proteins, including pore-forming toxins, proteases, lipases and S-layer proteins, across both the inner and outer membranes into the extracellular surroundings. The Escherichia coli alpha-hemolysin (HlyA) secretion system is the prototypical and best characterized type I secretion system. The structure and function of the components of the HlyA secretion apparatus, HlyB, HlyD and TolC, have been studied in great detail. The functional characteristics of this secretion system enable it to be used in a variety of different applications, including the presentation of heterologous antigens in live-attenuated bacterial vaccines. Such vaccines can be an effective delivery system for heterologous antigens, and the use of a type I secretion system allows the antigens to be actively exported from the cytoplasm of the bacterial carrier rather than only becoming accessible to the host immune system after bacterial disintegration.

Bacterial Proteins↗

PubMed1997

Protective immunity against Clostridium difficile toxin A induced by oral immunization with a live, attenuated Vibrio cholerae vector strain.

Clostridium difficile causes pseudomembranous colitis through the action of Rho-modifying proteins, toxins A and B. Antibodies directed against C. difficile toxin A prevent or limit C. difficile-induced colitis. We engineered plasmid pETR14, containing the hlyB and hlyD genes of the Escherichia coli hemolysin operon, to express a fusion protein containing 720 amino acid residues from the nontoxic, receptor-binding, carboxy terminus of C. difficile toxin A and the secretion signal of E. coli hemolysin A. We introduced pETR14 into Vibrio cholerae and found that the toxin A-HlyA fusion protein was secreted by a number of V. cholerae strains and recognized by both monoclonal and polyclonal anti-C. difficile toxin A antibodies. We introduced pETR14 into an attenuated V. cholerae strain, O395-NT, and inoculated rabbits orally with this construct. Colonization studies disclosed that the V. cholerae vector containing pETR14 was recoverable from rabbit ilea up to 5 days after oral inoculation. Vaccination produced significant systemic anti-C. difficile toxin A immunoglobulin G and anti-V. cholerae vibriocidal antibody responses. Vaccination also produced significant protection against toxin A in an ileal loop challenge assay, as assessed by determination of both fluid secretion and histological changes. These results suggest that the hemolysin system of E. coli can be used successfully in V. cholerae vector strains to effect secretion of large heterologous antigens and that a V. cholerae vector strain secreting a nontoxic, immunogenic portion of C. difficile toxin A fused to the secretion signal of E. coli HlyA induces protective systemic and mucosal immunity against this toxin.

Administration, Oral↗

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↗