Effects of polyamines on Vibrio cholerae virulence properties

Abstract

Vibrio cholerae is the causative agent of the severe enteric disease cholera. To cause cholera the bacterium must be able to synthesize both cholera toxin (CT) and toxin-coregulated pilus (TCP) which mediates autoagglutination and is required for colonization of the small intestine. Only a few environmental signals have been shown to regulate V. cholerae virulence gene expression. Polyamines, which are ubiquitous in nature, and have been implicated in regulating virulence gene expression in other bacteria, have not been extensively studied for their effect on V. cholerae virulence properties. The objective of this study was to test the effect of several polyamines that are abundant in the human intestine on V. cholerae virulence properties. All of the polyamines tested inhibited autoagglutination of V. cholerae O1 classical strain in a concentration dependent manner. Putrescine and cadaverine decreased the synthesis of the major pilin subunit, TcpA, spermidine increased its production, and spermine had no effect. Putrescine and spermidine led to a decrease and increase, respectively, on the relative abundance of TCP found on the cell surface. Spermine led to a small reduction in cholera toxin synthesis whereas none of the other polyamines had an effect. The polyamines did not affect pili bundling morphology, but caused a small reduction in CTXφ transduction, indicating that the TCP present on the cell surface may not be fully functional. We hypothesize the inhibition of autoagglutination is likely to be caused by the positively charged amine groups on the polyamines electrostatically disrupting the pili-pili interactions which mediate autoagglutination. Our results implicate that polyamines may have a protective function against colonization of the small intestine by V. cholerae.

Figure 1. Polyamines used in this study.

Figure 2. Effect of polyamines on V. cholerae autoagglutination.

V. cholerae was cultured in the presence of increasing concentrations of putrescine, cadaverine, spermidine and spermine under virulence factor inducing conditions. Negative controls were grown in media with a pH of 8.5 at 37°C. For the experimental cultures, the first row of values is mmoles of polyamines added to the media as a result of polyamine supplementation; the second row of values is the molarity of the polyamines in the culture. Sedimentation index was calculated as 1– (Final A₅₉₅/Initial A₅₉₅). Each sample is the average of three biological replicates with error bars representing the standard deviation. Pair-wise Student’s t-tests were performed to compare SI’s of cultures grown in the absence of polyamines to cultures grown with each different polyamine concentration. Stars indicate p<0.01).

Figure 3. Effect of polyamines on TcpA levels.

A. Western blots. Extracts of cells grown in the absence or presence of polyamines were separated by SDS-PAGE, blotted on PVDF membrane, and TcpA was detected using an anti-TcpA antibody. Negative controls were grown in media with a pH of 8.5 at 37°C. The loading control was probed using an antibody against the α-subunit of the E. coli RNA polymerase. Although the α-subunit of the E. coli RNA polymerase has a higher molecular weight (36 kD) than TcpA (20.5 kD), the images were inverted vertically to aid ease of comparison. Blots shown are representatives of three different experiments performed with three biological replicates. The vertical breaks in the gel images indicate that the lanes in which the cell extracts from cultures grown with cadaverine were not immediately next to the negative control lane. B. Quantification of TcpA levels. TcpA band densities were normalized to the loading control and then compared to those obtained from cells grown without polyamines. TcpA levels are calculated as: normalized band densities in the presence of polyamines/normalized band densities in the absence of polyamines. Each sample is the average of three biological replicates with error bars representing the standard deviation. Pair-wise Student’s t-tests were performed to compare SI’s of cultures grown in the absence of polyamines to cultures grown with each different polyamine concentration. Stars indicate p<0.01. Put: putrescine, Cad: cadaverine, Spd: spermidine, Spm: spermine.

Figure 4. Effect of polyamines on cell-associated TCP levels.

Cell-associated TCP was quantified using a whole-cell ELISA as described in Materials and Methods. Values are reported as TCP Levels and were obtained using the following formula: A₄₂₀ with polyamines/A₄₂₀ without polyamines. Each sample is the average of three biological replicates with error bars representing the standard deviation. Pair-wise Student’s t-tests were performed to compare SI’s of cultures grown in the absence of polyamines to cultures grown with each different polyamine Stars indicate p<0.01. Put: putrescine, Cad: cadaverine, Spd: spermidine, Spm: spermine.

Figure 5. Effect of polyamines on TCP bundling.

Representative TEM images showing rope-like bundles of TCP from cultures grown in the absence of polyamines (A,B) and in the presence of putrescine (C,D), cadaverine (E,F), spermidine (G,H) and spermine (I,J).