Environmental signals controlling production of hemagglutinin/protease in Vibrio cholerae

Abstract

Vibrio cholerae hemagglutinin/protease (Hap) was induced upon nutrient limitation and strongly repressed by glucose. Hap was not produced in a mutant defective in the cyclic AMP (cAMP) receptor protein, suggesting that intracellular cAMP levels mediate Hap expression. No difference was found in Hap production between an rpoS deletion mutant and its isogenic wild-type precursor, indicating that the alternate sigma(s) factor is not essential for Hap expression. Based on these and previous results, we discuss the role of Hap in the pathogenesis of cholera.

FIG. 1

Time course of Hap production. (A) Strains C7258 (□) and 638 (▪) were grown from a single colony in 5 ml of TSB medium at 30°C. Overnight cultures were diluted 1:1,000 in 100 ml of fresh TBS in a 500-ml flask and incubated at 30°C with shaking (200 rpm). Samples were withdrawn periodically for OD₅₉₅ readings. (B) Samples were taken at 4-h intervals from the cultures described above for determination of azocasein activity. Open bar, strain C7258; shaded bar, strain 638. Enzyme activities are the averages of at least three independent cultures. Standard deviations are indicated with error bars.

FIG. 2

Kinetics of Hap production. Strain C7258 was grown from a single colony in 5 ml of TSB medium at 30°C. An overnight culture was diluted 1:1,000 in fresh TSB and incubated for 8 h with shaking (200 rpm) at 30°C. Aliquots of this culture were centrifuged, the cells were resuspended in 1 volume of each of the different media described below, and incubation continued for 4 h. Samples were taken at 1-h intervals for OD₅₉₅ readings and determination of azocasein activity. (A) Cells were resuspended in original 8-h spent medium (▴), original 8-h spent medium boiled for 20 min (○), or 1× fresh TBS (▵). (B) Cells were resuspended in original 8-h spent medium (▴), 1× fresh TBS (▵), 0.5× fresh TBS (▪), or 0.25× fresh TBS (□). Mutant strain 638 (●) was cultivated as described for C7258, and the 8-h cell pellet was resuspended in 1 volume of 0.25× fresh TBS. (C) Cells were resuspended in original 8-h spent medium (▴) and original 8-h spent medium supplemented with 0.4% (22 mM) d-glucose (⊠). Azocasein activities are the averages of at least three independent cultures. Standard deviations are indicated with error bars.

FIG. 3

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis of Hap production. An 8-h culture of strain C7258 was divided into aliquots, the cells were resuspended in 1 volume of the different media described below, and incubation continued at 30°C with shaking (200 rpm) for 4 h. The cultures were centrifuged, and 2 ml of each supernatant was concentrated 10-fold by centrifuging it through Centricon-10 centrifugal filters. The retentate was adjusted to 0.2 ml, and 0.02-ml aliquots were analyzed in an SDS–12% PAGE gel. (A) SDS-PAGE. Lane 1, cells resuspended in the original 8-h spent medium; lane 2, cells resuspended in 1× fresh TBS; lane 3, cells resuspended in 0.25× fresh TBS; lane 4, cells resuspended in 0.25× fresh TBS supplemented with 0.4% d-glucose; lane 5, concentrated supernatant of mutant 638; lane 6, pure Hap (2.5 μg). (B) Western blot. Identical samples were loaded in a second gel and transferred to a polyvinylidene difluoride membrane. Hap was detected by using a rabbit anti-Hap serum and peroxidase-conjugated goat anti-rabbit immunoglobulin G. The total azocasein units loaded per lane are indicated below the blot. Molecular masses were calculated with reference to SDS molecular mass standards (low range) from Bio-Rad laboratories.