The outer core lipopolysaccharide of Salmonella enterica serovar Typhi is required for bacterial entry into epithelial cells

Abstract

Salmonella enterica serovar Typhi causes typhoid fever in humans. Central to the pathogenicity of serovar Typhi is its capacity to invade intestinal epithelial cells. The role of lipopolysaccharide (LPS) in the invasion process of serovar Typhi is unclear. In this work, we constructed a series of mutants with defined deletions in genes for the synthesis and polymerization of the O antigen (wbaP, wzy, and wzz) and the assembly of the outer core (waaK, waaJ, waaI, waaB, and waaG). The abilities of each mutant to associate with and enter HEp-2 cells and the importance of the O antigen in serum resistance of serovar Typhi were investigated. We demonstrate here that the presence and proper chain length distribution of the O-antigen polysaccharide are essential for serum resistance but not for invasion of epithelial cells. In contrast, the outer core oligosaccharide structure is required for serovar Typhi internalization in HEp-2 cells. We also show that the outer core terminal glucose residue (Glc II) is necessary for efficient entry of serovar Typhi into epithelial cells. The Glc I residue, when it becomes terminal due to a polar insertion in the waaB gene affecting the assembly of the remaining outer core residues, can partially substitute for Glc II to mediate bacterial entry into epithelial cells. Therefore, we conclude that a terminal glucose in the LPS core is a critical residue for bacterial recognition and internalization by epithelial cells.

FIG. 1.

Analysis of LPS from serovar Typhi strain Ty2 and O-antigen mutants. LPS samples from equal numbers of bacterial cells (1 × 10⁷ CFU) were loaded in each lane and were analyzed by Tricine/SDS-polyacrylamide gel electrophoresis on a 14% acrylamide gel followed by silver staining. The strains are Ty2 (wild type [wt]), M985 (Ty2 Δwzz), M1224 (Ty2 Δwzy), MSS1 (Ty2 ΔwbaP::cat), and M8 (Ty2 ΔrfaH::cat).

FIG. 2.

(A) Genetic organization of genes for core biosynthesis in serovar Typhi. The targets of the mutations obtained in this study are shown in gray. (B) Proposed structures of the inner core, outer core, and O antigen of serovar Typhi. Kdo, 3-deoxy-d-manno-octulosonic acid. (C) Analysis of LPS from serovar Typhi strain Ty2 and core mutants. LPS samples from equal numbers of bacterial cells (2.5 × 10⁷ CFU) were loaded in each lane and were analyzed by Tricine/SDS-polyacrylamide gel electrophoresis on a 14% acrylamide gel followed by silver staining. The strains are Ty2 (wild type [wt]), MSS1 (Ty2 ΔwbaP::cat), M115 (Ty2 ΔwaaK), M102 (Ty2 ΔwaaJ), M1015 (Ty2 ΔwaaI), M109 (Ty2 ΔwaaB), M108 (Ty2 ΔwaaB::aph), and M113 (Ty2 ΔwaaG).

FIG. 3.

Invasion of HEp-2 epithelial cells by serovar Typhi Ty2 and O-antigen mutants. The strains are Ty2 (wild type [wt]), M985 (Ty2 Δwzz), M1224 (Ty2 Δwzy), MSS1 (Ty2 ΔwbaP::cat), and M8 (Ty2 ΔrfaH::cat). Invasion assays were performed in triplicate on at least three independent occasions. Averages ± standard errors (error bars) are shown. The value that was significantly different from that of the wild type (P < 0.05) by the one-way ANOVA test and Tukey posttest is indicated by an asterisk.

FIG. 4.

Invasion of HEp-2 epithelial cells by serovar Typhi Ty2 and core mutants. The strains are Ty2 (wild type [wt]), M115 (Ty2 ΔwaaK), M102 (Ty2 ΔwaaJ), M1015 (Ty2 ΔwaaI), M109 (Ty2 ΔwaaB), M108 (Ty2 ΔwaaB::aph), and M113 (Ty2 ΔwaaG). Invasion assays were performed in triplicate on at least three independent occasions. Averages ± standard errors (error bars) are shown. Values that were significantly different from that of the wild-type Ty2 strain (P < 0.05) by the one-way ANOVA test and Tukey posttest are indicated by asterisks.

FIG. 5.

(A) Analysis of LPS from serovar Typhi Ty2 and complemented core mutants. LPS samples from equal numbers of bacterial cells (2.5 × 10⁷ CFU) were loaded in each lane and were analyzed by Tricine/SDS-polyacrylamide gel electrophoresis on a 14% acrylamide gel followed by silver staining. (B) Invasion of HEp-2 epithelial cells by serovar Typhi Ty2 and complemented core mutants. Invasion assays were performed in triplicate on at least three independent occasions. Values represent invasion of each strain relative to invasion of Ty2/pGEM-T. The strains are Ty2 (wild type [wt])/pGEM-T, M102 (Ty2 ΔwaaJ)/pGEM-T, M1015 (Ty2 ΔwaaI)/pGEM-T, M109 (Ty2 ΔwaaB)/pGEM-T, M108 (Ty2 ΔwaaB::aph)/pGEM-T, M102 (Ty2 ΔwaaJ)/pJC102, M1015 (Ty2 ΔwaaI)/pJC1015, M109 (Ty2 ΔwaaB)/pJC109, and M108 (Ty2 ΔwaaB::aph)/pJC109.

FIG. 6.

Adherence of serovar Typhi Ty2 and mutants on HEp-2 epithelial cells. The strains are Ty2 (wild type [wt]), M985 (Ty2 Δwzz), M1224 (Ty2 Δwzy), MSS1 (Ty2 ΔwaaP::cat), M8 (Ty2 ΔrfaH::cat), M115 (Ty2 ΔwaaK), M102 (Ty2 ΔwaaJ), M1015 (Ty2 ΔwaaI), M109 (Ty2 ΔwaaB), M108 (Ty2 ΔwaaB::aph), and M113 (Ty2 ΔwaaG). Adhesion assays were performed in triplicate on at least three independent occasions. Averages ± standard errors (error bars) are shown. Values that are significantly different from that of the wild-type Ty2 (P < 0.05) by the one-way ANOVA test and Tukey posttest are indicated by asterisks.

FIG. 7.

Motility phenotype of serovar Typhi Ty2 and mutants. Swimming motility on soft agar was performed as described in Materials and Methods. Bars represent the migration of each mutant relative to the migration of the wild-type strain. The strains are Ty2 (wild type [wt]), M985 (Ty2 Δwzz), M1224 (Ty2 Δwzy), MSS1 (Ty2 ΔwaaP::cat), M8 (Ty2 ΔrfaH::cat), M115 (Ty2 ΔwaaK), M102 (Ty2 ΔwaaJ), M1015 (Ty2 ΔwaaI), M109 (Ty2 ΔwaaB), M108 (Ty2 ΔwaaB::aph), and M113 (Ty2 ΔwaaG). Averages ± standard errors (error bars) are shown. Experiments were performed in duplicate on three independent occasions. The value that is significantly different from that of the wild-type Ty2 (P < 0.05) by the one-way ANOVA test and Tukey posttest is indicated by an asterisk.