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Vibrio cholerae hemolysin is required for lethality, developmental delay, and intestinal vacuolation in Caenorhabditis elegans.

Cinar HN, Kothary M, Datta AR, Tall BD, Sprando R, Bilecen K, Yildiz F, McCardell B - PLoS ONE (2010)

Bottom Line: To determine the role of other virulence factors in V. cholerae pathogenesis, we used a CT and TCP independent infection model in the nematode Caenorhabditis elegans and identified the hemolysin A (hlyA) gene as a factor responsible for animal death and developmental delay.We demonstrated a correlation between the severity of infection in the nematode and the level of hemolytic activity in the V. cholerae biotypes.Our data strongly suggest that HlyA is a virulence factor in C. elegans infection leading to lethality and developmental delay presumably through intestinal cytopathic changes.

View Article: PubMed Central - PubMed

Affiliation: Division of Virulence Assessment, Food and Drug Administration, Laurel, Maryland, USA. hediye.cinar@fda.hhs.gov

ABSTRACT

Background: Cholera toxin (CT) and toxin-co-regulated pili (TCP) are the major virulence factors of Vibrio cholerae O1 and O139 strains that contribute to the pathogenesis of disease during devastating cholera pandemics. However, CT and TCP negative V. cholerae strains are still able to cause severe diarrheal disease in humans through mechanisms that are not well understood.

Methodology/principal findings: To determine the role of other virulence factors in V. cholerae pathogenesis, we used a CT and TCP independent infection model in the nematode Caenorhabditis elegans and identified the hemolysin A (hlyA) gene as a factor responsible for animal death and developmental delay. We demonstrated a correlation between the severity of infection in the nematode and the level of hemolytic activity in the V. cholerae biotypes. At the cellular level, V. cholerae infection induces formation of vacuoles in the intestinal cells in a hlyA dependent manner, consistent with the previous in vitro observations.

Conclusions/significance: Our data strongly suggest that HlyA is a virulence factor in C. elegans infection leading to lethality and developmental delay presumably through intestinal cytopathic changes.

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Related in: MedlinePlus

hlyA is required for killing during V. cholerae infection in C. elegans.Lethality analysis was performed in glp-4(bn2) worms that were fed with indicated bacterial strains (Table 1). Agar plates were kept at 25°C and scored for survivors at 24–48 hour intervals. Data were plotted according to a Kaplan-Meier method and survival curves were compared using the logrank test. p<0.005. CVD110 and CVD109 represent the hlyA deficient and hlyA containing vaccine strains respectively, and they are isogenic with E7946. strR, streptomycin resistance; ΔhlyA, hlyA deletion; CVD110 strR/pHNC44:hlyA is the complementation strain. A) Comparison of lethality caused by vaccine strains CVD110 (hlyA-), CVD109 (hlyA+) and V. cholerae WT strain E7946. CVD110 exposed worms: median survival–10 days, CVD109 exposed worms: median survival–5 days, E7946 exposed worms: median survival-5 days. p<0.0001 for CVD110 versus CVD109 and for CVD110 versus E7946. p = 0.3455 for E7946 versus CVD109. B) Comparison of lethality caused by hlyA deletion mutant, WT strain E7946 and CVD110. p<0.0001 for E7946 strR versus E7946 strR ΔhlyA and for E7946 strR versus CVD110 strR. p = 0.1383 for E7946 strR ΔhlyA versus CVD110 strR. C) Comparison of lethality caused by CVD110, CVD110 with hlyA expressing plasmid and CVD109. p<0.0001 for each curve comparison in this graph.
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pone-0011558-g001: hlyA is required for killing during V. cholerae infection in C. elegans.Lethality analysis was performed in glp-4(bn2) worms that were fed with indicated bacterial strains (Table 1). Agar plates were kept at 25°C and scored for survivors at 24–48 hour intervals. Data were plotted according to a Kaplan-Meier method and survival curves were compared using the logrank test. p<0.005. CVD110 and CVD109 represent the hlyA deficient and hlyA containing vaccine strains respectively, and they are isogenic with E7946. strR, streptomycin resistance; ΔhlyA, hlyA deletion; CVD110 strR/pHNC44:hlyA is the complementation strain. A) Comparison of lethality caused by vaccine strains CVD110 (hlyA-), CVD109 (hlyA+) and V. cholerae WT strain E7946. CVD110 exposed worms: median survival–10 days, CVD109 exposed worms: median survival–5 days, E7946 exposed worms: median survival-5 days. p<0.0001 for CVD110 versus CVD109 and for CVD110 versus E7946. p = 0.3455 for E7946 versus CVD109. B) Comparison of lethality caused by hlyA deletion mutant, WT strain E7946 and CVD110. p<0.0001 for E7946 strR versus E7946 strR ΔhlyA and for E7946 strR versus CVD110 strR. p = 0.1383 for E7946 strR ΔhlyA versus CVD110 strR. C) Comparison of lethality caused by CVD110, CVD110 with hlyA expressing plasmid and CVD109. p<0.0001 for each curve comparison in this graph.

Mentions: Worms fed with a wild type V. cholerae strain die faster than the ones fed with E. coli OP50, the standard nematode food, or UV-killed V. cholerae, and this lethality seems to be the result of a lethal infection that is independent of the major virulence factors, CT and TCP ([23] and our unpublished results). To determine the role of other virulence factors in nematode killing, we fed worms with V. cholerae vaccine strains that are deficient in several known virulence factors, and assayed lethality under these conditions. The vaccine strain CVD110 lacks the virulence genes zot, ace, ctxA, and hlyA [24], Table 1. The ctxB gene locus was deleted in CVD110 genome, but it was reinserted into the hlyA locus to inactivate hlyA gene and keep the immunogenicity elicited by CtxB protein. For full toxicity, CtxB requires the presence of CtxA, and since CVD110 does not have ctxA gene, the virulence mediated by cholera toxin is lacking in this strain [24]. We observed an attenuated killing response in CVD110 fed worms in comparison to the worms fed with the isogenic wild type V. cholerae strain E7946 (Fig. 1A), suggesting that one or more of these deficient factors might be responsible for increased lethality in C. elegans. When we fed worms with CVD110's immediate parental strain CVD109, which has an intact hlyA, we found that the presence of hlyA gene was sufficient to kill the worms at a rate comparable to that of observed for V. cholerae E7946 (Fig. 1A). To further evaluate the role of hlyA gene, we created the E7946-derived strain HNC45 that has a single locus deletion of hlyA gene. HNC45 fed worms showed decreased lethality similar to that of observed for CVD110 (Fig. 1B). When we reintroduced a functional copy of hlyA into CVD110 via conjugation of a plasmid expressing hlyA, the resulting strain showed lethality when fed to C. elegans (Fig. 1C) similar to that of the wild type strain E7946. Together, these findings indicated that V. cholerae hlyA is responsible for lethality during infection in C. elegans.


Vibrio cholerae hemolysin is required for lethality, developmental delay, and intestinal vacuolation in Caenorhabditis elegans.

Cinar HN, Kothary M, Datta AR, Tall BD, Sprando R, Bilecen K, Yildiz F, McCardell B - PLoS ONE (2010)

hlyA is required for killing during V. cholerae infection in C. elegans.Lethality analysis was performed in glp-4(bn2) worms that were fed with indicated bacterial strains (Table 1). Agar plates were kept at 25°C and scored for survivors at 24–48 hour intervals. Data were plotted according to a Kaplan-Meier method and survival curves were compared using the logrank test. p<0.005. CVD110 and CVD109 represent the hlyA deficient and hlyA containing vaccine strains respectively, and they are isogenic with E7946. strR, streptomycin resistance; ΔhlyA, hlyA deletion; CVD110 strR/pHNC44:hlyA is the complementation strain. A) Comparison of lethality caused by vaccine strains CVD110 (hlyA-), CVD109 (hlyA+) and V. cholerae WT strain E7946. CVD110 exposed worms: median survival–10 days, CVD109 exposed worms: median survival–5 days, E7946 exposed worms: median survival-5 days. p<0.0001 for CVD110 versus CVD109 and for CVD110 versus E7946. p = 0.3455 for E7946 versus CVD109. B) Comparison of lethality caused by hlyA deletion mutant, WT strain E7946 and CVD110. p<0.0001 for E7946 strR versus E7946 strR ΔhlyA and for E7946 strR versus CVD110 strR. p = 0.1383 for E7946 strR ΔhlyA versus CVD110 strR. C) Comparison of lethality caused by CVD110, CVD110 with hlyA expressing plasmid and CVD109. p<0.0001 for each curve comparison in this graph.
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Related In: Results  -  Collection

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pone-0011558-g001: hlyA is required for killing during V. cholerae infection in C. elegans.Lethality analysis was performed in glp-4(bn2) worms that were fed with indicated bacterial strains (Table 1). Agar plates were kept at 25°C and scored for survivors at 24–48 hour intervals. Data were plotted according to a Kaplan-Meier method and survival curves were compared using the logrank test. p<0.005. CVD110 and CVD109 represent the hlyA deficient and hlyA containing vaccine strains respectively, and they are isogenic with E7946. strR, streptomycin resistance; ΔhlyA, hlyA deletion; CVD110 strR/pHNC44:hlyA is the complementation strain. A) Comparison of lethality caused by vaccine strains CVD110 (hlyA-), CVD109 (hlyA+) and V. cholerae WT strain E7946. CVD110 exposed worms: median survival–10 days, CVD109 exposed worms: median survival–5 days, E7946 exposed worms: median survival-5 days. p<0.0001 for CVD110 versus CVD109 and for CVD110 versus E7946. p = 0.3455 for E7946 versus CVD109. B) Comparison of lethality caused by hlyA deletion mutant, WT strain E7946 and CVD110. p<0.0001 for E7946 strR versus E7946 strR ΔhlyA and for E7946 strR versus CVD110 strR. p = 0.1383 for E7946 strR ΔhlyA versus CVD110 strR. C) Comparison of lethality caused by CVD110, CVD110 with hlyA expressing plasmid and CVD109. p<0.0001 for each curve comparison in this graph.
Mentions: Worms fed with a wild type V. cholerae strain die faster than the ones fed with E. coli OP50, the standard nematode food, or UV-killed V. cholerae, and this lethality seems to be the result of a lethal infection that is independent of the major virulence factors, CT and TCP ([23] and our unpublished results). To determine the role of other virulence factors in nematode killing, we fed worms with V. cholerae vaccine strains that are deficient in several known virulence factors, and assayed lethality under these conditions. The vaccine strain CVD110 lacks the virulence genes zot, ace, ctxA, and hlyA [24], Table 1. The ctxB gene locus was deleted in CVD110 genome, but it was reinserted into the hlyA locus to inactivate hlyA gene and keep the immunogenicity elicited by CtxB protein. For full toxicity, CtxB requires the presence of CtxA, and since CVD110 does not have ctxA gene, the virulence mediated by cholera toxin is lacking in this strain [24]. We observed an attenuated killing response in CVD110 fed worms in comparison to the worms fed with the isogenic wild type V. cholerae strain E7946 (Fig. 1A), suggesting that one or more of these deficient factors might be responsible for increased lethality in C. elegans. When we fed worms with CVD110's immediate parental strain CVD109, which has an intact hlyA, we found that the presence of hlyA gene was sufficient to kill the worms at a rate comparable to that of observed for V. cholerae E7946 (Fig. 1A). To further evaluate the role of hlyA gene, we created the E7946-derived strain HNC45 that has a single locus deletion of hlyA gene. HNC45 fed worms showed decreased lethality similar to that of observed for CVD110 (Fig. 1B). When we reintroduced a functional copy of hlyA into CVD110 via conjugation of a plasmid expressing hlyA, the resulting strain showed lethality when fed to C. elegans (Fig. 1C) similar to that of the wild type strain E7946. Together, these findings indicated that V. cholerae hlyA is responsible for lethality during infection in C. elegans.

Bottom Line: To determine the role of other virulence factors in V. cholerae pathogenesis, we used a CT and TCP independent infection model in the nematode Caenorhabditis elegans and identified the hemolysin A (hlyA) gene as a factor responsible for animal death and developmental delay.We demonstrated a correlation between the severity of infection in the nematode and the level of hemolytic activity in the V. cholerae biotypes.Our data strongly suggest that HlyA is a virulence factor in C. elegans infection leading to lethality and developmental delay presumably through intestinal cytopathic changes.

View Article: PubMed Central - PubMed

Affiliation: Division of Virulence Assessment, Food and Drug Administration, Laurel, Maryland, USA. hediye.cinar@fda.hhs.gov

ABSTRACT

Background: Cholera toxin (CT) and toxin-co-regulated pili (TCP) are the major virulence factors of Vibrio cholerae O1 and O139 strains that contribute to the pathogenesis of disease during devastating cholera pandemics. However, CT and TCP negative V. cholerae strains are still able to cause severe diarrheal disease in humans through mechanisms that are not well understood.

Methodology/principal findings: To determine the role of other virulence factors in V. cholerae pathogenesis, we used a CT and TCP independent infection model in the nematode Caenorhabditis elegans and identified the hemolysin A (hlyA) gene as a factor responsible for animal death and developmental delay. We demonstrated a correlation between the severity of infection in the nematode and the level of hemolytic activity in the V. cholerae biotypes. At the cellular level, V. cholerae infection induces formation of vacuoles in the intestinal cells in a hlyA dependent manner, consistent with the previous in vitro observations.

Conclusions/significance: Our data strongly suggest that HlyA is a virulence factor in C. elegans infection leading to lethality and developmental delay presumably through intestinal cytopathic changes.

Show MeSH
Related in: MedlinePlus