Flagella and chemotaxis are required for efficient induction of Salmonella enterica serovar Typhimurium colitis in streptomycin-pretreated mice

Abstract

Salmonella enterica subspecies 1 serovar Typhimurium is a common cause of gastrointestinal infections. The host's innate immune system and a complex set of Salmonella virulence factors are thought to contribute to enteric disease. The serovar Typhimurium virulence factors have been studied extensively by using tissue culture assays, and bovine infection models have been used to verify the role of these factors in enterocolitis. Streptomycin-pretreated mice provide an alternative animal model to study enteric salmonellosis. In this model, the Salmonella pathogenicity island 1 type III secretion system has a key virulence function. Nothing is known about the role of other virulence factors. We investigated the role of flagella in murine serovar Typhimurium colitis. A nonflagellated serovar Typhimurium mutant (fliGHI) efficiently colonized the intestine but caused little colitis during the early phase of infection (10 and 24 h postinfection). In competition assays with differentially labeled strains, the fliGHI mutant had a reduced capacity to get near the intestinal epithelium, as determined by fluorescence microscopy. A flagellated but nonchemotactic cheY mutant had the same virulence defects as the fliGHI mutant for causing colitis. In competitive infections, both mutants colonized the intestine of streptomycin-pretreated mice by day 1 postinfection but were outcompeted by the wild-type strain by day 3 postinfection. Together, these data demonstrate that flagella are required for efficient colonization and induction of colitis in streptomycin-pretreated mice. This effect is mostly attributable to chemotaxis. Recognition of flagellar subunits (i.e., flagellin) by innate immune receptors (i.e., Toll-like receptor 5) may be less important.

FIG. 1.

Characterization of flagellum mutants. (A) Supernatant proteins from overnight Salmonella cultures were prepared as described in Material and Methods. They were separated by 10% polyacrylamide gel electrophoresis and stained with Coomassie brilliant blue. Major bands resulting from secreted proteins via the flagellum apparatus or the SPI-1 TTSS are indicated. M913, fliGHI::Tn10; M935, cheY::Tn10; SB225, sipA::aphT; SB220, sipC::aphT; SB169, sipB::aphT; SB302, invJ::aphT; SB161, ΔinvG. (B) Southern blot analysis of wild-type Salmonella, M913, and M935. Chromosomal DNA was digested with EcoRV (lanes E) or HindIII (lanes H), and the resulting fragments were separated on a 1% agarose gel prior to Southern blotting. Hybridization was performed with fluorescein-labeled probes corresponding to the fliGHI (right panel) and cheY (left panel) regions (see Materials and Methods). Asterisks indicate identities tentatively assigned based on the findings of Komoriya et al. (32). wt, wild type.

FIG. 2.

Analysis of Salmonella wild-type and fliGHI mutant at 48 h p.i. Two groups of five streptomycin-pretreated mice were infected for 2 days with 5 × 10⁷ CFU of serovar Typhimurium strain SL1344 (wild type) or M913 (fliGHI). (A to D) Bacterial loads in the cecal contents (A), the mLN (B), the spleen (C), and the liver (D). The dotted lines indicate the limits of detection, and the solid horizontal lines indicate the medians. (E) Histopathological analyses. HE-stained sections of cecal tissue were scored for edema in the submucosa (black bars), PMN infiltration (inf.) (medium gray bars), reductions in the numbers of goblet cells (dark grey bars), and desquamation, erosion, and ulceration of the epithelial layer (light gray bars) (see Materials and Methods). The scores are expressed as stacked vertical bars. The total pathological score (sum of the separate scores) was statistically analyzed by using the exact Mann-Whitney U test (in comparison to SL1344). S. Tm, Salmonella serovar Typhimurium; wt, wild type; stat. anal., statistical analysis; NS, not statistically significant (P ≥ 0.05).

FIG. 3.

Time course of Salmonella wild-type and fliGHI mutant infection. Two groups of 15 streptomycin-pretreated mice were treated with 10% bicarbonate p.o. prior to infection with 5 × 10⁷ CFU of serovar Typhimurium strains SL1344 (wild type) and M913 (fliGHI). After 10, 24, and 48 h five mice from each group were sacrificed. (A to D) Bacterial loads in the cecal contents (A), the mLN, (B), the spleen, (C), and the liver (D). The dotted lines indicate the limits of detection, and the solid horizontal lines indicate the medians. (E) Histopathological analysis. HE-stained sections of cecal tissue were scored for edema in the submucosa (black bars), PMN infiltration (medium gray bars), reductions in the numbers of goblet cells (dark grey bars), and desquamation, erosion, and ulceration of the epithelial layer (light gray bars) (see Materials and Methods). The scores are expressed as stacked vertical bars. The total pathological score (sum of the separate scores) and the bacterial load were statistically analyzed by using the exact Mann-Whitney U test (in comparison to wild-type strain SL1344). S. Tm, Salmonella serovar Typhimurium; wt, wild type; stat. anal., statistical analysis; NS, not statistically significant (P ≥ 0.05).

FIG. 4.

fliGHI mutant cannot efficiently reach the cecal epithelium. Three mice were inoculated with 10% bicarbonate prior to infection for 24 h with 5 × 10⁷ CFU of M913(pM979) plus 5 × 10⁷ CFU of SL1344(pDsRed), 5 × 10⁷ CFU of SL1344(pM979) plus 5 × 10⁷ CFU of M913(pDsRed), 5 × 10⁷ CFU of SL1344(pM979) plus 5 × 10⁷ CFU of SL1344(pDsRed), 5 × 10⁷ CFU of SL1344(pM979), or 5 × 10⁷ CFU of SL1344(pM968). Cecal tissue sections were stained with DAPI (grey), Alexa-647-phalloidin (blue), a rabbit anti-LPS antibody, and a goat anti-rabbit serum-rhodamine conjugate (red) (see Materials and Methods). Samples of cecal contents from two mice per group were plated to determine the fraction of pM979 or pM968 carrying bacteria (see Materials and Methods) (panel D, grey columns). (A) Cecum section from a mouse infected with M913(pM979) plus SL1344(pDsRed). (B) Cecum section from a mouse infected with SL1344(pM979) plus M913(pDsRed). (C) Strategy for quantification of GFP-positive red Salmonella cells (green circles) and red Salmonella cells (red circles) within 20 μm (dotted line) of the cecal epithelium (blue). (D) Presence of GFP-positive red Salmonella cells within 20 μm of the cecal epithelium (dotted line) as determined by fluorescence microscopy (black bars; average) (scale on the left side). The fraction of serovar Typhiumurium cells carrying pM979 or pM968 in the cecal contents was determined by replica plating (grey bars; average) (scale on the right side). The error bars indicate standard deviations. The plus signs indicate results for the two individual measurements. wt, wild type.

FIG. 5.

Colitis caused by the Salmonella wild-type strain, an fliGHI mutant, and a cheY mutant. Three groups of 10 streptomycin-pretreated mice were treated with 10% bicarbonate p.o. prior to infection for 24 h with 5 × 10⁷ CFU of serovar Typhimurium strains SL1344 (wild type), M913 (fliGHI), and M935 (cheY). The data were obtained from two independent experiments performed with groups of five mice. (A to D) Bacterial loads in the cecal contents (A), the mLN, (B), the spleen, (C), and the liver (D). The dotted lines indicate the limits of detection, and the solid horizontal lines indicate the medians. (E) Histopathological analysis. HE-stained sections of cecal tissue were scored for edema in the submucosa (black bars), PMN infiltration (PMN inf.; medium gray bars), reductions in the numbers of goblet cells (dark grey bars), and desquamation, erosion, and ulceration of the epithelial layer (light gray bars) (see Materials and Methods). The scores are expressed as stacked vertical bars. The total pathological score (sum of the separate scores) and the bacterial load were statistically analyzed by using the exact Mann-Whitney U test (in comparison to wild-type strain SL1344). S. Tm, Salmonella serovar Typhimurium; wt, wild type; stat. anal., statistical analysis; NS, not statistically significant (P ≥ 0.05).

FIG. 6.

CI for intestinal colonization of fliGHI and cheY mutants. Two groups of five streptomycin-pretreated mice were infected for 3 days with 5 × 10⁷ CFU of a 1:1 mixture of serovar Typhimurium strains SL1344 (wild type) and M913 (fliGHI) or strains SL1344 and M935 (cheY). The exact ratio of the wild-type strain to the mutant strain in each inoculum was determined by replica plating (ratio_input). The ratios of the wild-type strain to the mutant strain in fecal pellets on days 1 and 2 p.i. (two or three pellets) and in cecal contents on day 3 p.i. (A), in mLN (B), in the spleen (C), and in the liver (D) were determined by replica plating (ratio_output). CI were calculated with the formula CI = ratio_output/ratio_input. The solid squares, open circles, solid circles, open triangles, and solid triangles indicate the individual mice in each group. The CI for fecal pellets on days 1 and 2 p.i. are indicated by solid circles because data could not be assigned to individual mice. d, day; wt, wild type.