Novel Role of VisP and the Wzz System during O-Antigen Assembly in Salmonella enterica Serovar Typhimurium Pathogenesis

Abstract

Salmonella enterica serovars are associated with diarrhea and gastroenteritis and are a helpful model for understanding host-pathogen mechanisms. Salmonella enterica serovar Typhimurium regulates the distribution of O antigen (OAg) and presents a trimodal distribution based on Wzy polymerase and the Wzz_ST (long-chain-length OAg [L-OAg]) and Wzz_fepE (very-long-chain-length OAg [VL-OAg]) copolymerases; however, several mechanisms regulating this process remain unclear. Here, we report that LPS modifications modulate the infectious process and that OAg chain length determination plays an essential role during infection. An increase in VL-OAg is dependent on Wzy polymerase, which is promoted by a growth condition resembling the environment of Salmonella-containing vacuoles (SCVs). The virulence- and stress-related periplasmic protein (VisP) participates in OAg synthesis, as a ΔvisP mutant presents a semirough OAg phenotype. The ΔvisP mutant has greatly decreased motility and J774 macrophage survival in a colitis model of infection. Interestingly, the phenotype is restored after mutation of the wzz_ST or wzz_fepE gene in a ΔvisP background. Loss of both the visP and wzz_ST genes promotes an imbalance in flagellin secretion. L-OAg may function as a shield against host immune systems in the beginning of an infectious process, and VL-OAg protects bacteria during SCV maturation and facilitates intramacrophage replication. Taken together, these data highlight the roles of OAg length in generating phenotypes during S Typhimurium pathogenesis and show the periplasmic protein VisP as a novel protein in the OAg biosynthesis pathway.

Keywords: LPS; O antigen; Salmonella; VisP; lipopolysaccharide.

FIG 1

OAg changes are driven by intracellular conditions. (A) Electrophoresis profile of LPS in LB and N-minimal media. E. coli strain O55:B5 (lane 1) and the S. Typhimurium WT (lanes 2 and 3), ΔvisP (lanes 4 and 5), and complemented visP⁺ (lanes 6 and 7) strains were used. (B) qRT-PCR analysis of wzy polymerase in LB and N-minimal media. Relative fold expression in the WT, ΔvisP, Δwzz_fepE, Δwzz_ST, ΔvisP wzz_fepE, and ΔvisP wzz_ST strains is shown. **, P < 0.01; ****, P < 0.0001.

FIG 2

Roles of OAg and VisP in S. Typhimurium pathogenesis. (A) qRT-PCR analysis of genes involved in the OAg biosynthesis pathway. relative fold expression of the pmrB, wzz_fepE, wzz_ST, and phoP genes in the WT, ΔvisP, and complemented visP⁺ strains grown in LB is shown. (B to D) J774 phagocytosis (B), internalization (C), and long-period replication (D) of the WT, ΔvisP, and complemented visP⁺ strains. (E and F) qRT-PCR analysis of the sifA (N-minimal growth condition) (E) and mgtB (LB growth condition) (F) genes. Relative fold expression in the WT, ΔvisP, and complemented visP⁺ strains is shown. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

FIG 3

The interplay of VisP and PCP determines the final length of OAg. (A) qRT-PCR analysis of wzz expression in LB and N-minimal media. Relative fold expression in the WT, ΔvisP wzz_fepE, and ΔvisP wzz_ST strains is shown. (B) High-resolution electrophoresis (15% SDS-PAGE) and staining with a ProQ Emerald 300 LPS gel staining kit (Thermo Fisher, Waltham, MA, USA) of the LPS profiles of the ΔvisP wzz_fepE (lanes 1 and 2) and ΔvisP wzz_ST (lanes 3 and 4) strains grown in LB and N-minimal media. (C) Gradient SDS-PAGE (4 to 12%) of the LPS profile stained with a ProQ Emerald 300 LPS gel staining kit (Thermo Fisher, Waltham, MA, USA), showing the core lipid A and different OAg chain lengths in the WT, Δwzz_fepE, and Δwzz_ST strains, all grown in LB. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

FIG 4

Longer OAg facilitates intestinal colonization. A colitis model of infection was used in C57BL/6 mice. (A) Day 1 p.i. The log₁₀ CFU of the WT, ΔvisP, Δwzz_fepE, Δwzz_ST, ΔvisP wzz_fepE, and ΔvisP wzz_ST strains recovered from each feces pellet are shown. (B) Day 2 p.i. The log₁₀ CFU of the WT, ΔvisP, Δwzz_fepE, Δwzz_ST, ΔvisP wzz_fepE, and ΔvisP wzz_ST strains recovered from each feces pellet are shown. (C and D) qRT-PCR analysis of Il17a (C) and Il22 (D) performed with RNA extracted from the mouse colon. Relative fold expression in the WT, Δwzz_fepE, Δwzz_ST, ΔvisP wzz_fepE, and ΔvisP wzz_ST strains causing colitis is shown. *, P < 0.05; **, P < 0.01; ****, P < 0.0001.

FIG 5

Changes in flagellar gene expression with different OAgs. (A) qRT-PCR analysis of genes involved in flagellar assembly and motility. relative fold expression of the fliA, flhDC, and motA genes in the WT, ΔvisP, and complemented visP⁺ strains grown in LB is shown. (B) qRT-PCR of the flagellin gene fliC, swimming motility assay, and immunoblotting performed with anti-FliC and anti-RpoA (control) monoclonal antibodies. ***, P < 0.001; ****, P < 0.0001.

FIG 6

Proposed models of OAg outer-leaf remodeling and flagellum-OAg interaction. (A) Model of OAg final chain length in the intestinal lumen (left) and intracellular environment (right). (B) S. Typhimurium flagellum interaction with VL-OAg chains.