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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 growth retardation of worm cultures via hlyA gene.Synchronized L1 stage N2 worms were fed on indicated bacterial strains on agar plates for 72 hours at 22°C. Worms washed into M9 buffer were sorted using COPAS (n = 1000). Images in A to E indicate the composition of worm cultures prior to sorting. Black arrows indicate eggs, white arrows indicate adult nematodes. (F) Curve representing the optical density (EXT) distribution of sorted worms was plotted. E. coli OP50 fed worm populations showed two separable peak domains (indicated with arrows), V. cholerae A1552 fed worms showed a single peak (indicated with an arrowhead) falling between E. coli OP50 induced peaks that is consistent with a population of worms mostly larger than eggs but fail to reach adult sizes EXT, extinction; strR, streptomycin resistance. (G) Population growth of C. elegans fed with hlyA deleted, hlyA intact V. cholerae strains and OP50. Student's t test was used to compare growth. ** denotes a statistical significance of P<0.001 according to Student's t test; *** P<0.0001. Total EXT represents the sum of EXT values for sorted worms per condition.
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pone-0011558-g003: V. cholerae causes growth retardation of worm cultures via hlyA gene.Synchronized L1 stage N2 worms were fed on indicated bacterial strains on agar plates for 72 hours at 22°C. Worms washed into M9 buffer were sorted using COPAS (n = 1000). Images in A to E indicate the composition of worm cultures prior to sorting. Black arrows indicate eggs, white arrows indicate adult nematodes. (F) Curve representing the optical density (EXT) distribution of sorted worms was plotted. E. coli OP50 fed worm populations showed two separable peak domains (indicated with arrows), V. cholerae A1552 fed worms showed a single peak (indicated with an arrowhead) falling between E. coli OP50 induced peaks that is consistent with a population of worms mostly larger than eggs but fail to reach adult sizes EXT, extinction; strR, streptomycin resistance. (G) Population growth of C. elegans fed with hlyA deleted, hlyA intact V. cholerae strains and OP50. Student's t test was used to compare growth. ** denotes a statistical significance of P<0.001 according to Student's t test; *** P<0.0001. Total EXT represents the sum of EXT values for sorted worms per condition.

Mentions: In an independent approach, the COPAS biosorter [30] was used to quantify the optical density distributions of growing worm populations to evaluate the composition of life stages in a given feeding condition. For this assay, synchronized L1 stage worms were fed on hlyA intact (E7946 and CVD109) and hlyA deficient (CVD110 and HNC45) strains of V. cholerae for 72 hours, and the optical density of the worms were evaluated by EXT measurements (Methods). A qualitative assessment of images taken on samples of worm cultures prior to sorting showed that the worms fed with hlyA deficient strains and with E. coli OP50 contained mostly adults and eggs. In contrast, worms fed with the hlyA intact strains contained smaller animals and no eggs (Fig. 3A–E). We observed distinct density distribution curves for E. coli OP50 and V. cholerae A1552 fed worms, which is consistent with the life stage composition of their respective worm cultures (Figure 3F). We compared the total optical density values that reflects the population growth (methods), and found that the worms exposed to hlyA(+) strains showed developmental delay while the worms exposed to hlyA(-) strains grew similar to the worms fed with the OP50 bacteria (Fig. 3G). Altogether, these results confirmed our findings in lethality assays, and further implicated a role for hlyA as a virulence factor that impairs nematode development.


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 growth retardation of worm cultures via hlyA gene.Synchronized L1 stage N2 worms were fed on indicated bacterial strains on agar plates for 72 hours at 22°C. Worms washed into M9 buffer were sorted using COPAS (n = 1000). Images in A to E indicate the composition of worm cultures prior to sorting. Black arrows indicate eggs, white arrows indicate adult nematodes. (F) Curve representing the optical density (EXT) distribution of sorted worms was plotted. E. coli OP50 fed worm populations showed two separable peak domains (indicated with arrows), V. cholerae A1552 fed worms showed a single peak (indicated with an arrowhead) falling between E. coli OP50 induced peaks that is consistent with a population of worms mostly larger than eggs but fail to reach adult sizes EXT, extinction; strR, streptomycin resistance. (G) Population growth of C. elegans fed with hlyA deleted, hlyA intact V. cholerae strains and OP50. Student's t test was used to compare growth. ** denotes a statistical significance of P<0.001 according to Student's t test; *** P<0.0001. Total EXT represents the sum of EXT values for sorted worms per condition.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2903476&req=5

pone-0011558-g003: V. cholerae causes growth retardation of worm cultures via hlyA gene.Synchronized L1 stage N2 worms were fed on indicated bacterial strains on agar plates for 72 hours at 22°C. Worms washed into M9 buffer were sorted using COPAS (n = 1000). Images in A to E indicate the composition of worm cultures prior to sorting. Black arrows indicate eggs, white arrows indicate adult nematodes. (F) Curve representing the optical density (EXT) distribution of sorted worms was plotted. E. coli OP50 fed worm populations showed two separable peak domains (indicated with arrows), V. cholerae A1552 fed worms showed a single peak (indicated with an arrowhead) falling between E. coli OP50 induced peaks that is consistent with a population of worms mostly larger than eggs but fail to reach adult sizes EXT, extinction; strR, streptomycin resistance. (G) Population growth of C. elegans fed with hlyA deleted, hlyA intact V. cholerae strains and OP50. Student's t test was used to compare growth. ** denotes a statistical significance of P<0.001 according to Student's t test; *** P<0.0001. Total EXT represents the sum of EXT values for sorted worms per condition.
Mentions: In an independent approach, the COPAS biosorter [30] was used to quantify the optical density distributions of growing worm populations to evaluate the composition of life stages in a given feeding condition. For this assay, synchronized L1 stage worms were fed on hlyA intact (E7946 and CVD109) and hlyA deficient (CVD110 and HNC45) strains of V. cholerae for 72 hours, and the optical density of the worms were evaluated by EXT measurements (Methods). A qualitative assessment of images taken on samples of worm cultures prior to sorting showed that the worms fed with hlyA deficient strains and with E. coli OP50 contained mostly adults and eggs. In contrast, worms fed with the hlyA intact strains contained smaller animals and no eggs (Fig. 3A–E). We observed distinct density distribution curves for E. coli OP50 and V. cholerae A1552 fed worms, which is consistent with the life stage composition of their respective worm cultures (Figure 3F). We compared the total optical density values that reflects the population growth (methods), and found that the worms exposed to hlyA(+) strains showed developmental delay while the worms exposed to hlyA(-) strains grew similar to the worms fed with the OP50 bacteria (Fig. 3G). Altogether, these results confirmed our findings in lethality assays, and further implicated a role for hlyA as a virulence factor that impairs nematode development.

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