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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

V. cholerae causes developmental delay in C. elegans via hlyA gene.A) Illustrative pictures of gonad and vulva in each developmental stage. Black arrowhead indicates vulva. B) Synchronized L1 stage N2 worms (n = 50 per condition) were fed the indicated bacterial strains on agar plates for 48 hours at 20°C. The developmental stage of growing worms was determined under Nomarski optics using gonad development as milestones.
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pone-0011558-g002: V. cholerae causes developmental delay in C. elegans via hlyA gene.A) Illustrative pictures of gonad and vulva in each developmental stage. Black arrowhead indicates vulva. B) Synchronized L1 stage N2 worms (n = 50 per condition) were fed the indicated bacterial strains on agar plates for 48 hours at 20°C. The developmental stage of growing worms was determined under Nomarski optics using gonad development as milestones.

Mentions: Because the worm lethality assay measures the longevity of non-reproducing worm populations and allows observations only for adult animals, we wanted to evaluate other possible outcomes in relation to C. elegans infection that could be attributed to hlyA. To characterize how the exposure to V. cholerae affects the life cycle of C. elegans, we fed strain E7946 to wild type growing worms and examined their development using two approaches. In the first assay, synchronized L1 stage worms were initiated on bacterial feeding, and were examined under Nomarski optics at 48 hours for subsequent attainment of larval stages. To identify developmental stages, we used the size and shape of the somatic gonad and vulva as developmental landmarks (Methods, and Fig. 2A). While all the worms that were fed with the control bacterium OP50 reached L4 stage and beyond at 48 hours, only 45% of the E7946 fed worms attained L4 stage during this time (Fig. 2B), suggesting that a developmental delay was induced by V. cholerae exposure. Using this assay, we evaluated the effects of hlyA on larval development and found that all L1 worms that were fed on the hlyA-deficient V. cholerae strain HNC45 reached L4 stage and beyond at 48 hours (Fig. 2B).


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)

V. cholerae causes developmental delay in C. elegans via hlyA gene.A) Illustrative pictures of gonad and vulva in each developmental stage. Black arrowhead indicates vulva. B) Synchronized L1 stage N2 worms (n = 50 per condition) were fed the indicated bacterial strains on agar plates for 48 hours at 20°C. The developmental stage of growing worms was determined under Nomarski optics using gonad development as milestones.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2903476&req=5

pone-0011558-g002: V. cholerae causes developmental delay in C. elegans via hlyA gene.A) Illustrative pictures of gonad and vulva in each developmental stage. Black arrowhead indicates vulva. B) Synchronized L1 stage N2 worms (n = 50 per condition) were fed the indicated bacterial strains on agar plates for 48 hours at 20°C. The developmental stage of growing worms was determined under Nomarski optics using gonad development as milestones.
Mentions: Because the worm lethality assay measures the longevity of non-reproducing worm populations and allows observations only for adult animals, we wanted to evaluate other possible outcomes in relation to C. elegans infection that could be attributed to hlyA. To characterize how the exposure to V. cholerae affects the life cycle of C. elegans, we fed strain E7946 to wild type growing worms and examined their development using two approaches. In the first assay, synchronized L1 stage worms were initiated on bacterial feeding, and were examined under Nomarski optics at 48 hours for subsequent attainment of larval stages. To identify developmental stages, we used the size and shape of the somatic gonad and vulva as developmental landmarks (Methods, and Fig. 2A). While all the worms that were fed with the control bacterium OP50 reached L4 stage and beyond at 48 hours, only 45% of the E7946 fed worms attained L4 stage during this time (Fig. 2B), suggesting that a developmental delay was induced by V. cholerae exposure. Using this assay, we evaluated the effects of hlyA on larval development and found that all L1 worms that were fed on the hlyA-deficient V. cholerae strain HNC45 reached L4 stage and beyond at 48 hours (Fig. 2B).

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