Host-Derived Sialic Acids Are an Important Nutrient Source Required for Optimal Bacterial Fitness In Vivo

Abstract

A major challenge facing bacterial intestinal pathogens is competition for nutrient sources with the host microbiota.Vibrio cholerae is an intestinal pathogen that causes cholera, which affects millions each year; however, our knowledge of its nutritional requirements in the intestinal milieu is limited. In this study, we demonstrated that V. cholerae can grow efficiently on intestinal mucus and its component sialic acids and that a tripartite ATP-independent periplasmic SiaPQM strain, transporter-deficient mutant NC1777, was attenuated for colonization using a streptomycin-pretreated adult mouse model. In in vivo competition assays, NC1777 was significantly outcompeted for up to 3 days postinfection. NC1777 was also significantly outcompeted in in vitro competition assays in M9 minimal medium supplemented with intestinal mucus, indicating that sialic acid uptake is essential for fitness. Phylogenetic analyses demonstrated that the ability to utilize sialic acid was distributed among 452 bacterial species from eight phyla. The majority of species belonged to four phyla, Actinobacteria (members of Actinobacillus, Corynebacterium, Mycoplasma, and Streptomyces), Bacteroidetes (mainly Bacteroides, Capnocytophaga, and Prevotella), Firmicutes (members of Streptococcus, Staphylococcus, Clostridium, and Lactobacillus), and Proteobacteria (including Escherichia, Shigella, Salmonella, Citrobacter, Haemophilus, Klebsiella, Pasteurella, Photobacterium, Vibrio, and Yersinia species), mostly commensals and/or pathogens. Overall, our data demonstrate that the ability to take up host-derived sugars and sialic acid specifically allows V. cholerae a competitive advantage in intestinal colonization and that this is a trait that is sporadic in its occurrence and phylogenetic distribution and ancestral in some genera but horizontally acquired in others.

Importance: Sialic acids are nine carbon amino sugars that are abundant on all mucous surfaces. The deadly human pathogen Vibrio cholerae contains the genes required for scavenging, transport, and catabolism of sialic acid. We determined that the V. cholerae SiaPQM transporter is essential for sialic acid transport and that this trait allows the bacterium to outcompete noncatabolizers in vivo. We also showed that the ability to take up and catabolize sialic acid is prevalent among both commensals and pathogens that colonize the oral cavity and the respiratory, intestinal, and urogenital tracts. Phylogenetic analysis determined that the sialic acid catabolism phenotype is ancestral in some genera such as Yersinia, Streptococcus, and Staphylococcus and is acquired by horizontal gene transfer in others such as Vibrio, Aeromonas, and Klebsiella. The data demonstrate that this trait has evolved multiple times in different lineages, indicating the importance of specialized metabolism to niche expansion.

FIG 1

Growth analysis of V. cholerae N16961 on mouse intestinal mucus and mucus sugars. (A) V. cholerae growth in M9 minimal medium supplemented with mucus (M9M). (B) Maximum density (OD₅₉₅) of V. cholerae grown in M9 supplemented with glucose, N-acetylglucosamine (GlcNAc), galactose, gluconate, mannose, ribose, or Neu5Ac. (C) V. cholerae growth in LB media. Data are presented as averages of results of two biological replicates with three technical replicates. Error bars represent standard errors of the means (SEM).

FIG 2

Growth analysis of V. cholerae N16961 with sialic acid derivatives as sole carbon sources. V. cholerae N16961, V. cholerae NC1777 (ΔsiaM), E. coli BW25113, and E. coli JW3193 (ΔnanT) in M9 Neu5Ac (A), M9 Neu5Gc (B), or M9 KDN (C) or E. coli JW3193 in M9 Neu5Ac, M9 Neu5Gc, or M9 KDN (D) were complemented with siaPQM and tested for restored growth on sialic acid derivatives. Growth curves represent results of at least two biological replicates performed in triplicate. Error bars represent SEM.

FIG 3

Vibrio cholerae in vivo competition assays. In vivo competition assays were performed using streptomycin-pretreated mice which were orally inoculated with an equal amounts of SAM2338 (ΔlacZ) and NC1777 (ΔsiaM). (A) Competitive index data were determined by homogenizing fecal pellets collected at 6, 12, 24, 48, and 72 h postinoculation followed by plating on LB X-Gal for a blue/white screen. The competitive index is calculated as follows: CI = ratio out_{(NC1777 / SAM2338)}/ratio in_{(NC1777/SAM2338)}. (B) Upon completion of the in vivo persistence assay (72 h), mice were sacrificed, gastrointestinal tracts were removed and sectioned into small intestine (SI), large intestine (LI), and cecum (CE), and CFU values were calculated. In vivo assays were performed with two biological replicates and n = 10 mice. Error bars represent SEM. Statistics were calculated using a one-sample t test with the means compared to the hypothetical CI of 1. Asterisks represent the following: ***, P < 0.01.

FIG 4

In vitro competition assays. Results of assays of in vitro competition between V. cholerae strain SAM2338 (ΔlacZ) and the sialic acid transporter mutant NC1777 (ΔsiaM) are presented. (A) Competition assays were performed in M9G, LB, or M9 supplemented with small-intestine (SI), large-intestine (LI), or cecum mucus. (B) Time course in vitro competition assays were performed at 3, 6, 9, 12, and 24 h postinoculation in either LB or LB supplemented with glucose (0.4% [wt/vol]). In vitro data represent averages of results of two biological replicates, and error bars represent SEM. Statistics were calculated using an unpaired Student’s t test with a 95% confidence interval. All samples were compared to M9G to determine statistical significance. Asterisks represent the following: **, P < 0.05.

FIG 5

NanA phylogeny. A phylogenetic tree of NanA is shown. The tree was constructed using the neighbor-joining tree method and the p-distance model of evolutionary distances as parameters and MEGA6 (75–77). Letters indicate major bacterial phyla present in the corresponding NanA clade. Colors indicate the major phylogenetic groups that contain nanA. Red, Actinobacteria; purple, Bacteroidetes; lime green, Firmicutes; dark green, Fusobacteria; dark red, Planctomycetes; blue, Proteobacteria; yellow, eukaryotes; gray, Spirochaetes.