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Abstract
Vibrio vulnificus produces Hemolysin/cytolysin (VvhA), which is one of the most potent exotoxins capable of killing mice at submicrogram levels. However, V. vulnificus growth and vvhA expression are severely repressed and extracellular VvhA produced at low levels is easily inactivated in human body fluids. This study was conducted to obtain additional unequivocal evidence of the enigmatic characteristic of VvhA. V. vulnificus growth was stimulated, vvhA expression was de-repressed, and extracellular VvhA production was increased in cirrhotic ascites, a human ex vivo experimental system, by a mutation of fur encoding ferric uptake regulator, which acts as a transcriptional repressor. However, regardless of the presence or absence of the fur mutation, extracellular VvhA activity was not detected in cirrhotic ascites. These results indicate that VvhA is easily inactivated even when vvhA expression and extracellular VvhA production are maintained at high levels in cirrhotic ascites.
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Figure 1.
Effect of a fur mutation on the production of catechol-siderophore vulnibactin. The two PvvhA::lacZ transcription reporter strains, CMM2103 with wild-type fur and CMM2305 with deleted fur, were cultured in iron-deficient Heart Infusion broths at 37°C for 12 h. (A) Bacterial growth was measured by the optical density of culture aliquots at 600 nm (OD600). (B) Culture supernatants were obtained by centrifugation and catechol concentration in the culture supernatants was determined by the Arnow test. Numeric values are expressed as the means ± standard deviation, from triplicate measurements. The # symbol indicates a statistically significant difference between the two means (p < 0.05 in Student's t-test).
Figure 2.
Effect of a fur mutation on vvhA expression and extracellular VvhA production and activity. The two PvvhA::lacZ transcription reporter strains, CMM2103 with wild-type fur and CMM2305 with deleted fur, were cultured in iron-deficient Heart Infusion broths at 37°C for 12 h. Bacterial growth was measured by the optical density of culture aliquots at 600 nm (OD600), and culture supernatants were obtained by centrifugation. (A) The vvhA expression levels were measured by the Miller method and β-galactosidase activity was expressed as Miller units. (B) Extracellular VvhA activity was measured by the tube hemolytic assay using a 1% human red blood cell suspension. (C) Extracellular VvhA amount was determined by the dot blot method. For comparison on a per cell basis, signal intensities were quantified by densitometry. Numeric values are expressed as the means ± standard deviation, from triplicate measurements. The # symbol indicates a statistically significant difference between the two means (p < 0.05 in Student's t-test).
Figure 3.
Effect of a fur mutation on pilD expression. The two PpilD::lacZ transcription reporter strains, RC176 with wild-type fur and RC180 with deleted fur, were cultured in iron-deficient Heart Infusion broths at 37°C for 12 h. Bacterial growth was measured by the optical density of culture aliquots at 600 nm (OD600), and culture supernatants were obtained by centrifugation. (A) The pilD expression levels were measured by the Miller method and β-galactosidase activity was expressed as Miller units. Numeric values are expressed as the means ± standard deviation, from triplicate measurements. The # symbol indicates a statistically significant difference between the two means (p < 0.05 in Student's t-test).
Figure 4.
Effect of a fur mutation on vvhA expression and extracellular VvhA production and activity in cirrhotic ascites. The two PvvhA::lacZ transcription reporter strains, CMM2103 with wild-type fur and CMM-2305 with deleted fur, were cultured in cirrhotic ascites at 37°C for 12 h. (A) Bacterial growth was measured by the optical density of culture aliquots at 600 nm (OD600). (B) The vvhA expression levels in culture aliquots were measured by the Miller method and β-galactosidase activity was expressed as Miller units. (C) Extracellular VvhA activity in culture supernatants was measured by the tube hemolytic assay using a 1% human red blood cell suspension. (D) Extracellular VvhA amount in culture supernatants was determined by the dot blot method. For comparison on a per cell basis, signal intensities were digitalized by densitometry. Numeric values are expressed as the means ± standard deviation, from triplicate measurements. The # symbol indicates a statistically significant difference between the two means (p < 0.05 in Student's t-test).
Table 1.
Bacterial strains used in this study
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