A rapid change in virulence gene expression during the transition from the intestinal lumen into tissue promotes systemic dissemination of Salmonella

Abstract

Bacterial pathogens causing systemic disease commonly evolve from organisms associated with localized infections but differ from their close relatives in their ability to overcome mucosal barriers by mechanisms that remain incompletely understood. Here we investigated whether acquisition of a regulatory gene, tviA, contributed to the ability of Salmonella enterica serotype Typhi to disseminate from the intestine to systemic sites of infection during typhoid fever. To study the consequences of acquiring a new regulator by horizontal gene transfer, tviA was introduced into the chromosome of S. enterica serotype Typhimurium, a closely related pathogen causing a localized gastrointestinal infection in immunocompetent individuals. TviA repressed expression of flagellin, a pathogen associated molecular pattern (PAMP), when bacteria were grown at osmotic conditions encountered in tissue, but not at higher osmolarity present in the intestinal lumen. TviA-mediated flagellin repression enabled bacteria to evade sentinel functions of human model epithelia and resulted in increased bacterial dissemination to the spleen in a chicken model. Collectively, our data point to PAMP repression as a novel pathogenic mechanism to overcome the mucosal barrier through innate immune evasion.

Figure 1. TviA represses flagellar and invasion gene expression in both S. Typhi and S. Typhimurium.

S. Typhi (A, C, E, and G) and S. Typhimurium (B, D, F, and H) strains were grown under tviA-inducing conditions (SOB broth). Relative levels of flhD (A and B), flgB (C and D), fliC (E and F), and hilA (G and H) mRNA were measured by real-time qRT-PCR. The serotype and the genotype of the bacterial strains are indicated below each graph. Bars represent the geometric mean of three independent experiments ± standard error. Asterisks indicate the statistical significance of differences between data sets: * (P<0.05) or ** (P<0.01).

Figure 2. TviA represses flagellar gene expression under conditions mimicking tissue osmolarity.

(A) A S. Typhi flhC::lacZYA mutant (SW197, dark gray bars), a S. Typhi ΔviaB flhC::lacZYA mutant (SW186, open bars), a S. Typhimurium flhC::MudJ mutant (SW335, light gray bars) and a S. Typhimurium ΔphoN::tviA flhC::MudJ mutant (SW316, black bars) were grown in tryptone yeast extract medium and β-galactosidase activity was measured. NaCl was added at the concentrations to increase osmolarity. Bars represent the geometric mean of three independent experiments ± standard error. Asterisks indicate statistical significance between data sets: * (P<0.05) or ** (P<0.01). (B) The S. Typhi wild-type strain (Ty2), a ΔviaB mutant (SW347), a ΔtviB-vexE mutant (SW74), and a ΔviaB ΔfliC mutant (SW483) were grown in tissue culture medium (DMEM) and FliC expression was detected by Western blot using H antiserum d. (C) The S. Typhimurium wild-type strain (IR715), a ΔphoN mutant (AJB715), a ΔphoN::tviA mutant (SW474) and a ΔphoN ΔfliC fljB mutant (SW681) were cultured in tissue culture medium (DMEM) and expression of FliC was detected by Western blot using Salmonella H antiserum i. Expression of GroEL was determined to ensure equal loading of samples, (αGroEL). Approximate position of standard proteins with known molecular mass is indicated.

Figure 3. TviA is involved in a rapid decrease in flagella expression in serum.

(A – B) A plasmid (pDW5) encoding GFP was introduced into S. Typhimurium strains. FliC expression of a S. Typhimurium ΔphoN mutant (AJB715[pDW5]), a ΔphoN::tviA mutant (SW474[pDW5]), and a ΔphoN ΔfliC fljB mutant (SW681[pDW5]) was detected by flow cytometry. Strains were grown for two hours in tryptone yeast extract broth (TYE) containing 0.3 M NaCl (A) or in murine serum (B). (C) Time dependent repression of FliC expression exerted by TviA. A S. Typhimurium ΔphoN mutant and a ΔphoN::tviA mutant were grown in tryptone yeast extract broth containing 0.3 M NaCl and subsequently transferred into tissue culture medium (MEM). FliC expression was detected by Western blot using Salmonella H antiserum i. Expression of GroEL was determined to ensure equal loading of samples (αGroEL). Approximate position of standard proteins with known molecular mass is indicated.

Figure 4. TviA-mediated flagellin repression in S. Typhi reduces the ability of model epithelia to serve as sentinels by detecting flagellin on their basolateral surface.

(A and B) The S. Typhi wild-type strain (Ty2), a ΔtviB-vexE mutant (SW74), a ΔviaB mutant (SW347), and a ΔviaB ΔfliC mutant (SW483) were grown in tissue culture medium (MEM) and then added to the basolateral compartment of polarized T84 epithelial cells. Alternatively, purified flagellin was added to the basolateral or apical compartment as indicated. 3 hours later, relative expression of the chemokines CXCL1 (A) and CCL20 (B) was measured by real time qRT-PCR. (C) The S. Typhimurium wild-type strain (IR715), a ΔphoN mutant (AJ715), a ΔphoN::tviA mutant (SW474), and a ΔphoN ΔfliC fljB mutant (SW681) were grown in tissue culture medium (MEM) and then added to the basolateral compartment of polarized T84 epithelial cells. CXCL1 transcript levels were quantified by real time qRT-PCR. Bars represent the geometric mean of three independent experiments ± standard error. Asterisks indicate the statistical significance of differences between data sets: * (P<0.05); ns: not statistically significant.

Figure 5. Introduction of the tviA regulatory gene into the S. Typhimurium chromosome promotes increased systemic dissemination in chickens.

Groups of five 4-day-old chicks were inoculated orally with a S. Typhimurium ΔphoN mutant, a ΔphoN::tviA mutant or a ΔphoN ΔfliC fljB mutant. At 8 hours after inoculation, the bacterial load in the cecal contents (A) and the spleen (B) was determined. Bars represent the geometric mean ± standard error. Asterisks indicate the statistical significance of differences between data sets: ** (P<0.01); ns: not statistically significant.

Figure 6. Proposed model of TviA-mediated changes in gene expression, which occur during the transition of bacteria from the intestinal lumen into tissue.

Due to elevated osmolarity, tviA is not expressed in the intestinal lumen, allowing expression of invasion genes (T3SS-1) and flagella. In the lamina propria, a relative decrease in osmolarity leads to the expression of tviA. Under these conditions, TviA is available as a co-repressor for RcsB, resulting in reduced invasion gene and flagellar gene expression and activation of genes in the viaB locus, resulting in capsule (Vi antigen) production.