Sialic acid catabolism confers a competitive advantage to pathogenic vibrio cholerae in the mouse intestine

Abstract

Sialic acids comprise a family of nine-carbon ketosugars that are ubiquitous on mammalian mucous membranes. However, sialic acids have a limited distribution among Bacteria and are confined mainly to pathogenic and commensal species. Vibrio pathogenicity island 2 (VPI-2), a 57-kb region found exclusively among pathogenic strains of Vibrio cholerae, contains a cluster of genes (nan-nag) putatively involved in the scavenging (nanH), transport (dctPQM), and catabolism (nanA, nanE, nanK, and nagA) of sialic acid. The capacity to utilize sialic acid as a carbon and energy source might confer an advantage to V. cholerae in the mucus-rich environment of the gut, where sialic acid availability is extensive. In this study, we show that V. cholerae can utilize sialic acid as a sole carbon source. We demonstrate that the genes involved in the utilization of sialic acid are located within the nan-nag region of VPI-2 by complementation of Escherichia coli mutants and gene knockouts in V. cholerae N16961. We show that nanH, dctP, nanA, and nanK are highly expressed in V. cholerae grown on sialic acid. By using the infant mouse model of infection, we show that V. cholerae DeltananA strain SAM1776 is defective in early intestinal colonization stages. In addition, SAM1776 shows a decrease in the competitive index in colonization-competition assays comparing the mutant strain with both O1 El Tor and classical strains. Our data indicate an important relationship between the catabolism of sialic acid and bacterial pathogenesis, stressing the relevance of the utilization of the resources found in the host's environment.

FIG. 1.

Schematic diagram of VPI-2 and the nan-nag region in V. cholerae N16961. (a) VPI-2. Black arrows, core genes; bent black arrow, the tRNA^Ser insertion site of VPI-2; thin vertically striped arrow, integrase; checkerboard arrow, restriction-modification system; bold vertically striped arrows, nan-nag region; horizontally striped arrows, phage-like region. (b) nan-nag region. Stippled arrows, mutarotases; light gray arrow, putative regulator; hatched arrows, putative genes involved in the catabolism of sialic acid; black arrows, putative genes involved in the transport of sialic acid; grid pattern arrow, neuraminidase. Above each arrow, the name of the putative gene is indicated. Below each arrow, the locus tag of the gene is indicated. (c) Structure of VPI-2 in V. cholerae MO10 (O139 strain). Black arrows, core genes; bent black arrow, the tRNA^Ser insertion site of VPI-2; thin vertically striped arrow, integrase; horizontally striped arrows, phage-like region.

FIG. 2.

Growth of different V. cholerae strains on minimal medium supplemented with glucose or sialic acid. The strains were incubated at 37°C under aerobic conditions on M9 minimal medium supplemented with glucose (a) or sialic acid (b). N16961 and O395 are VPI-2 positive, whereas MO10 is VPI-2 negative. Plots are represented on natural log scale. O.D., optical density. Error bars indicate standard deviations.

FIG. 3.

Transcriptional analysis of the nan-nag genes. The bars represent expression ratio of the nan-nag genes relative to the expression of rpoB as determined using real-time PCR. V. cholerae N16961 was inoculated on M9 plus sialic acid (S) and M9 plus glucose (G). The RNA was extracted at 1 h and 3 h postinoculation. The genes under study were VC1775 (rpiR), VC1784 (nanH), VC1779 (dctP), VC1776 (nanA), VC1782 (nanK), and VC1781 (nanE). The expression of rpoB was used to normalize our test genes. The paired t test was used to infer statistical significance of the differences in expression of the genes between the two substrates. *, 0.01 < P ≤ 0.1; **, 0.001 < P ≤ 0.01; ***, P ≤ 0.001. Error bars indicate standard deviations.

FIG. 4.

Growth of V. cholerae sialic acid catabolism mutant strains. The constructed mutants and complements for the genes involved in the catabolism of sialic acid were incubated at 37°C under aerobic conditions. Growth was on M9 minimal medium supplemented with sialic acid. O.D., optical density. Error bars indicate standard deviations.

FIG. 5.

Infection dynamics of V. cholerae N16961 and a sialic acid catabolism-deficient strain in the suckling mouse intestine. Three- to 5-day-old CD-1 mice were inoculated with V. cholerae N16961 or V. cholerae SAM1776 (ΔnanA). Six mice were used per strain and time point. The intestines were removed after 1, 3, 6, 9, 12, and 24 h. Serial dilutions of the homogenates were plated on LB containing Sm and the CFU counted. We used the paired t test to compare the numbers of CFU of N16961 and SΑΜ1776 at the same time point. ***, P < 0.0005. Error bars indicate standard deviations.