Suppression of Virulence of Toxigenic Vibrio cholerae by Anethole through the Cyclic AMP (cAMP)-cAMP Receptor Protein Signaling System

Use of natural compounds as antivirulence drugs could be an alternative therapeutic approach to modify the outcome of bacterial infections, particularly in view of growing resistance to available antimicrobials. Here, we show that sub-bactericidal concentration of anethole, a component of sweet fennel seed, could suppress virulence potential in O1 El Tor biotype strains of toxigenic Vibrio cholerae, the causative agent of the ongoing 7^th cholera pandemic. The expression of cholera toxin (CT) and toxin coregulated pilus (TCP), the major virulence factors of V. cholerae, is controlled through a regulatory cascade involving activation of ToxT with synergistic coupling interaction of ToxR/ToxS with TcpP/TcpH. We present evidence that anethole inhibits in vitro expression of CT and TCP in a toxT-dependent but toxR/toxS-independent manner and through repression of tcpP/tcpH, by using bead-ELISA, western blotting and quantitative real-time RT-PCR assays. The cyclic AMP (cAMP)-cAMP receptor protein (CRP) is a well-studied global signaling system in bacterial pathogens, and this complex is known to suppress expression of tcpP/tcpH in V. cholerae. We find that anethole influences the virulence regulatory cascade by over-expressing cyaA and crp genes. Moreover, suppression of toxigenic V. cholerae-mediated fluid accumulation in ligated ileum of rabbit by anethole demonstrates its potentiality as an antivirulence drug candidate against the diseases caused by toxigenic V. cholerae. Taken altogether, these results revealing a mechanism of virulence inhibition in V. cholerae by the natural compound anethole, may have relevance in designing antivirulence compounds, particularly against multiple antibiotic resistant bacterial pathogens.     

Analysis of gyrB and toxR Gene Sequences of Vibrio hollisae and Development of gyrB- and toxR-Targeted PCR Methods for Isolation of V. hollisae from the Environment and Its Identification

Isolation of Vibrio hollisae strains, particularly from the environment, is rare. This may be due, in part, to the difficulty encountered when using conventional biochemical tests to identify the microorganism. In this study, we evaluated whether two particular genes may be useful for the identification of V. hollisae. The two genes are presumed to be conserved among the bacterial species (gyrB) or among the species of the genus Vibrio (toxR). A portion of the gyrB sequence of V. hollisae was cloned by PCR using a set of degenerate primers. The sequence showed 80% identity with the corresponding Vibrio parahaemolyticus gyrB sequence. The toxR gene of V. hollisae was cloned utilizing a htpG gene probe derived from the V. parahaemolyticus htpG gene, which is known to be linked to the toxR gene in V. hollisae. The coding sequence of the cloned V. hollisae toxR gene had 59% identity with the V. parahaemolyticus toxR coding sequence. The results of DNA colony hybridization tests using the DNA probes derived from the two genes of V. hollisae indicated that these gene sequences could be utilized for differentiation of V. hollisae from other Vibrio species and from microorganisms found in marine fish. PCR methods targeting the two gene sequences were established. Both PCR methods were shown to specifically detect the respective target sequences of V. hollisae but not other organisms. A strain of V. hollisae added at a concentration of 1 to 10² CFU/ml to alkaline peptone water containing a seafood sample could be detected by a 4-h enrichment incubation in alkaline peptone water at 37°C followed by quick DNA extraction with an extraction kit and 35-cycle PCR specific for the V. hollisae toxR gene. We conclude that screening of seafood samples by this 35-cycle, V. hollisae toxR-specific PCR, followed by isolation on a differential medium and identification by the above htpG- and toxR-targeted PCR methods, can be useful for isolation from the environment and identification of V. hollisae.     

Isolation and Characterization of Vibrio tubiashii Outer Membrane Proteins and Determination of a toxR Homolog

Outer membrane proteins (OMPs) expressed by Vibrio tubiashii under different environmental growth conditions were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, N-terminal amino acid sequencing, and PCR analyses. Results showed the presence of a 38- to 40-kDa OmpU-like protein and ompU gene, a maltoporin-like protein, several novel OMPs, and a regulatory toxR homolog.     

Vibrio cholerae VexH encodes a multiple drug efflux pump that contributes to the production of cholera toxin and the toxin co-regulated pilus

The resistance-nodulation-division (RND) efflux systems are ubiquitous transporters that function in antimicrobial resistance. Recent studies showed that RND systems were required for virulence factor production in Vibrio cholerae. The V. cholerae genome encodes six RND efflux systems. Three of the RND systems (VexB, VexD, and VexK) were previously shown to be redundant for in vitro resistance to bile acids and detergents. A mutant lacking the VexB, VexD, and VexK RND pumps produced wild-type levels of cholera toxin (CT) and the toxin co-regulated pilus (TCP) and was moderately attenuated for intestinal colonization. In contrast, a RND negative mutant produced significantly reduced amounts of CT and TCP and displayed a severe colonization defect. This suggested that one or more of the three uncharacterized RND efflux systems (i.e. VexF, VexH, and VexM) were required for pathogenesis. In this study, a genetic approach was used to generate a panel of V. cholerae RND efflux pump mutants in order to determine the function of VexH in antimicrobial resistance, virulence factor production, and intestinal colonization. VexH contributed to in vitro antimicrobial resistance and exhibited a broad substrate specificity that was redundant with the VexB, VexD, and VexK RND efflux pumps. These four efflux pumps were responsible for in vitro antimicrobial resistance and were required for virulence factor production and intestinal colonization. Mutation of the VexF and/or VexM efflux pumps did not affect in vitro antimicrobial resistance, but did negatively affect CT and TCP production. Collectively, our results demonstrate that the V. cholerae RND efflux pumps have redundant functions in antimicrobial resistance and virulence factor production. This suggests that the RND efflux systems contribute to V. cholerae pathogenesis by providing the bacterium with protection against antimicrobial compounds that are present in the host and by contributing to the regulated expression of virulence factors.     

Induction,resuscitation and quantitative real-time polymerase chain reaction analyses of viable but nonculturable Vibrio vulnificus in artificial sea water

Vibrio vulnificus, an important food-borne pathogen, is known to enter viable but nonculturable (VBNC) state under low temperature and low nutrition stress conditions. Present study examined the time required for induction of VBNC state and temperature which induces resuscitation of V. vulnificus YJ016. The change in cell morphology and gene expression during VBNC state and in resuscitated cells was also examined. V. vulnificus incubated in artificial sea water at 4 °C entered VBNC state after considerably extended time (70 days). An increase in temperature by 6 °C from the VBNC induction temperature (4 °C) resulted in resuscitation of VBNC cells; however, maximum resuscitation was observed when VBNC cells were held at 23 °C for 24 h. VBNC cells changed their morphology from comma shape to coccoid shape. Two rounds of induction of VBNC and resuscitation were possible with V. vulnificus cells; however, there was progressive reduction in number of resuscitated cells and after 190 days cells failed to resuscitate. Significant up-regulation of genes related to membrane proteins [porinH (10.4-fold), ompU (2.9-fold)], regulatory proteins [envZ (5.6-fold), toxR (4.5-fold), toxS (4.8-fold)], oxidative stress related protein katG (2.3-fold), cell division/maintenance proteins [ftsZ (4.3), mreB (6.5-fold)] and resuscitating promoter factor yeaZ (fourfold) was observed during resuscitation with respect to VBNC state indicating that these genes play a role during resuscitation. Gene expression data presented here would enhance our understanding of resuscitation of V. vulnificus from VBNC state. The results also highlight the importance of maintenance of low temperature during storage of seafood.     

Conformational instability of the cholera toxin A1 polypeptide

Cholera toxin (CT) moves from the cell surface to the endoplasmic reticulum (ER) by vesicular transport. In the ER, the catalytic CTA1 subunit dissociates from the holotoxin and enters the cytosol by exploiting the quality control system of ER-associated degradation (ERAD). It is hypothesized that CTA1 triggers its ERAD-mediated translocation into the cytosol by masquerading as a misfolded protein, but the process by which CTA1 activates the ERAD system remains unknown. Here, we directly assess the thermal stability of the isolated CTA1 polypeptide by biophysical and biochemical methods and correlate its temperature-dependent conformational state with susceptibility to degradation by the 20S proteasome. Measurements with circular dichroism and fluorescence spectroscopy demonstrated that CTA1 is a thermally unstable protein with a disordered tertiary structure and a disturbed secondary structure at 37 °C. A protease sensitivity assay likewise detected the temperature-induced loss of native CTA1 structure. This protease-sensitive conformation was not apparent when CTA1 remained covalently associated with the CTA2 subunit. Thermal instability in the dissociated CTA1 polypeptide could thus allow it to appear as a misfolded protein for ERAD-mediated export to the cytosol. In vitro, the disturbed conformation of CTA1 at 37 °C rendered it susceptible to ubiquitin-independent degradation by the core 20S proteasome. In vivo, CTA1 was also susceptible to degradation by a ubiquitin-independent proteasomal mechanism. ADP-ribosylation factor 6, a cytosolic eukaryotic protein that enhances the enzymatic activity of CTA1, stabilized the heat-labile conformation of CTA1 and protected it from in vitro degradation by the 20S proteasome. Thermal instability in the reduced CTA1 polypeptide has not been reported before, yet both the translocation and degradation of CTA1 may depend upon this physical property.     

Immunological properties and ganglioside recognitions by Campylobacter jejuni-enterotoxin and cholera toxin

Abstract The immunological properties of Campylobacter jejuni enterotoxin (CJT) and cholera toxin (CT) were compared by enzyme-linked immunosorbent assay (ELISA) and Western blotting analysis with antiserum against each toxin. Antibody against CJT recognized the 68, 54 and 43 kDa polypeptides of CJT and the 11 kDa subunit of CT, whereas antibody against CT recognized the 68 and 54 kDa polypeptides of CJT and 11 kDa subunit of CT. The immunological reactions between the heterogenous combinations of toxins and the antibodies were weaker than those between the homogenous systems. Thus, different antigenicity was found in CJT and CT at the subunit level, although they possessed cross-reactive epitope(s). The binding of CJT and CT to gangliosides was also examined. CJT and CT bound to GM₁ ganglioside preferentially than to other ganglioside species. However, CJT did not bind to GD₁b in spite of the fact that CT preferred GD₁b. This suggests that both toxins recognize different receptors on the surface of the target cell. This study is the first demonstration of the different properties between CJT and CT in immunological character and ganglioside recognition.     

A Bistable Switch and Anatomical Site Control Vibrio cholerae Virulence Gene Expression in the Intestine

A fundamental, but unanswered question in host-pathogen interactions is the timing, localization and population distribution of virulence gene expression during infection. Here, microarray and in situ single cell expression methods were used to study Vibrio cholerae growth and virulence gene expression during infection of the rabbit ligated ileal loop model of cholera. Genes encoding the toxin-coregulated pilus (TCP) and cholera toxin (CT) were powerfully expressed early in the infectious process in bacteria adjacent to epithelial surfaces. Increased growth was found to co-localize with virulence gene expression. Significant heterogeneity in the expression of tcpA, the repeating subunit of TCP, was observed late in the infectious process. The expression of tcpA, studied in single cells in a homogeneous medium, demonstrated unimodal induction of tcpA after addition of bicarbonate, a chemical inducer of virulence gene expression. Striking bifurcation of the population occurred during entry into stationary phase: one subpopulation continued to express tcpA, whereas the expression declined in the other subpopulation. ctxA, encoding the A subunit of CT, and toxT, encoding the proximal master regulator of virulence gene expression also exhibited the bifurcation phenotype. The bifurcation phenotype was found to be reversible, epigenetic and to persist after removal of bicarbonate, features consistent with bistable switches. The bistable switch requires the positive-feedback circuit controlling ToxT expression and formation of the CRP-cAMP complex during entry into stationary phase. Key features of this bistable switch also were demonstrated in vivo, where striking heterogeneity in tcpA expression was observed in luminal fluid in later stages of the infection. When this fluid was diluted into artificial seawater, bacterial aggregates continued to express tcpA for prolonged periods of time. The bistable control of virulence gene expression points to a mechanism that could generate a subpopulation of V. cholerae that continues to produce TCP and CT in the rice water stools of cholera patients.     

Design and Study of Properties of Avirulent Genetically Modified <Emphasis Type="Italic">Vibrio cholerae</Emphasis> Biovar El Tor Strain with Inactivated Genes of Thermolabile Hemolysin and Effective Expression of Cloned Cholera Toxin B-subunit Gene

The results of the construction of an avirulent strain of V. cholerae genovariant of the O1 serogroup of the El Tor biovar with efficient production of the cholera toxin (CT) B-subunit, which causes the formation of antitoxic immunity during cholera infection, are presented. In the beginning of the study, a modified P18899ΔCTXφchr:TnphoA (Km^RHly^–) strain was obtained via nondirectional transposon mutagenesis on the basis of a nontoxigenic strain of a P18899ΔCTXφHly⁺ genovariant. It lost the ability to produce thermolabile hemolysin, which is an additional toxin. The ctxB gene, which encodes biosynthesis of the CT B-subunit, was further introduced into cells of this strain. To accomplish this, the cointegrative recombinant plasmid pIEM3 was used. It was generated in the process of the fusion of a conjugative plasmid pIEM1 and a nonconjugative one, pCTΔ27, which is a derivative of pBR322 (which carries the cloned gene ctxB). Via restriction analysis, it was established that disjunction of a cointegrate occurred in vibrio cholera cells, followed by the preservation of a multicopy plasmid (pCTΔ27) only. As a result, avirulent clones of Km^RTc^R with a high production level (5–6 μg/mL) of secreted CT B-subunit were obtained, one of which (E99) was selected for further studies. Via real-time PCR, it was discovered that the expression of the ctxB plasmid gene in the cells of a designed strain of V. cholerae, E99, doesn’t depend on the activity of the key regulatory gene toxT, which located on the chromosome. Efficient expression of the ctxB gene in E99 cells allows their use to obtain CT B-subunit in order to produce cholera immunobiological drugs.     

Increased Iron Stores Correlate with Worse Disease Outcomes in a Mouse Model of Schistosomiasis Infection

Schistosomiasis is a significant parasitic infection creating disease burden throughout many of the world''s developing nations. Iron deficiency anemia is also a significant health burden resulting from both nutritional deficit as well as parasitic infection in these countries. In this study we investigated the relationships between the disease outcomes of Schistosoma japonicum infection and iron homeostasis. We aimed to determine if host iron status has an effect on schistosome maturation or egg production, and to investigate the response of iron regulatory genes to chronic schistosomiasis infection. Wild-type C57BL/6 and Transferrin Receptor 2 null mice were infected with S. japonicum, and sacrificed at the onset of chronic disease. Transferrin Receptor 2 null mice are a model of type 3 hereditary hemochromatosis and develop significant iron overload providing increased iron stores at the onset of infection. The infectivity of schistosomes and egg production was assessed along with the subsequent development of granulomas and fibrosis. The response of the iron regulatory gene Hepcidin to infection and the changes in iron status were assessed by real-time PCR and Western blotting. Our results show that Hepcidin levels responded to the changing iron status of the animals, but were not significantly influenced by the inflammatory response. We also show that with increased iron availability at the time of infection there was greater development of fibrosis around granulomas. In conclusion, our studies indicate that chronic inflammation may not be the primary cause of the anemia seen in schistosomiasis, and suggest that increased availability of iron, such as through iron supplementation, may actually lead to increased disease severity.     

Role of Temperate Bacteriophage 139 in Changing Cholera Toxin Production in Vibrio cholerae Classical Biovar

New data were obtained concerning cell sensitivity of pathogenic strains of cholera vibrions, which belong to the serogroup O1 of classical biovar, to the temperate bacteriophage 139, the native host of which is Vibrio cholerae O139. Molecular–genetic and biochemical studies showed that phage 139 integrated into the chromosome of strains V. cholerae O1 can change their toxigenic properties. A change in the production of cholera toxin (CT) in lysogens is associated both with an increase in the activity of the toxR regulatory gene and with an alteration of the structure of a chromosomal DNA region that contains a copy of the operon ctxAB encoding CT biosynthesis.