Host susceptibility to the attaching and effacing bacterial pathogen Citrobacter rodentium

Abstract

Many studies have shown that genetic susceptibility plays a key role in determining whether bacterial pathogens successfully infect and cause disease in potential hosts. Surprisingly, whether host genetics influence the pathogenesis of attaching and effacing (A/E) bacteria such as enteropathogenic and enterohemorrhagic Escherichia coli has not been examined. To address this issue, we infected various mouse strains with Citrobacter rodentium, a member of the A/E pathogen family. Of the strains tested, the lipopolysaccharide (LPS) nonresponder C3H/HeJ mouse strain experienced more rapid and extensive bacterial colonization than did other strains. Moreover, the high bacterial load in these mice was associated with accelerated crypt hyperplasia, mucosal ulceration, and bleeding, together with very high mortality rates. Interestingly, the basis for the increased susceptibility was not due to LPS hyporesponsiveness, as the genetically related but LPS-responsive C3H/HeOuJ and C3H/HeN mouse strains were also susceptible to infection. Analysis of the intestinal pathology in these susceptible strains revealed significant crypt epithelial cell apoptosis (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end label staining) as well as bacterial translocation to the mesenteric lymph nodes. Further studies with infection of SCID (T- and B-lymphocyte-deficient) C3H/HeJ mice demonstrated that loss of lymphocytes had no effect on bacterial numbers but did reduce crypt cell apoptosis and delayed mortality. These studies thus identify the adaptive immune system, crypt cell apoptosis, and bacterial translocation but not LPS responsiveness as contributing to the tissue pathology and mortality seen during C. rodentium infection of highly susceptible mouse strains. Determining the basis for these strains' susceptibility to intestinal colonization by an A/E pathogen will be the focus of future studies.

FIG. 1.

Comparison of mortality among different strains of mice orally infected with 2.5 × 10⁸ CFU of C. rodentium. Infection caused only limited mortality in most mouse strains tested, including C57BL/6 (open squares), BALB/c (filled triangles), 129S1/SvImJ (filled squares), and NIH Swiss (open triangles) mice. In contrast, C3H/HeJ mice (filled circles) suffered 100% mortality during the course of infection. Survival data from one representative experiment out of three are shown, with all three experiments showing similar results. Each data point represents the percentage of mice still surviving from an initial population of 10 mice.

FIG. 2.

C. rodentium infection is heavier and leads to mucosal ulceration and friability in susceptible mice. C. rodentium infection at day 6 p.i. was predominantly superficial in resistant strains, as seen in colonic tissue taken from an infected 129S1/SvImJ mouse (A). Note the superficial location of C. rodentium in the colon (arrows). In contrast, the bacterial load was much heavier in the C3H/HeJ mice, with larger numbers of bacteria (arrows) that penetrated more deeply into colonic crypts (B). The inflammatory response was fairly mild in resistant strains at day 6 p.i., shown here with colonic tissue from an infected C57BL/6 mouse that shows well-preserved tissue morphology (C). In contrast, the inflammatory response was much stronger in the susceptible C3H/HeJ mice (D), resulting in heavy inflammatory cell infiltration and ulceration (arrowheads), as well as the loss of crypt morphology. Various inflammatory cells including neutrophils (arrows) can be seen. Shown at higher magnification, the histology from panel D shows the loss of epithelial cells (arrowhead) and the denuding of the mucosal surface. The mucosal ulceration is accompanied by hyperemia and hemorrhage with numerous red blood cells (arrow) observed in the ulcerated region (E). Aside from overt ulceration, the tissue damage seen in C3H/HeJ mice often led to extensive bleeding into the colonic lumen (F) (see arrow), with the blood in the lumen being indicated by the arrowhead.

FIG. 3.

C. rodentium infection caused rapid mortality in C3H/HeJ (filled circles) and C3H/HeOuJ (open circles) mice, with these two strains suffering 100% mortality by day 12 of infection. In comparison, severe illness was slower to develop in the C3H/HeN mice (crosses), but between days 10 and 21 p.i., all mice of this strain succumbed to the infection. Survival data from one representative experiment out of three are shown, with all three experiments showing similar results. Each data point represents the percentage of mice still surviving from an initial population of 10 mice.

FIG. 4.

Following oral infection, all three C3H strains were heavily colonized by C. rodentium. The mean numbers of bacteria or CFU recovered from the colons of infected C3H/HeJ (black bars), C3H/HeOuJ (shaded bars), and C3H/HeN (open bars) mice are shown in this figure. While C3H/HeJ and C3H/HeOuJ mice showed similar colonization levels at days 4 and 6 p.i., C3H/HeN mice carried significantly smaller bacterial loads (asterisks; P < 0.05) at these time points. By day 10 p.i., colony counts were performed only on C3H/HeN mice, as the majority of the C3H/HeJ and C3H/HeOuJ mice required euthanization (crosses) by this time. Values are the mean colonic C. rodentium CFU ± 1 SEM from three independent experiments, each with groups of four to five mice.

FIG. 5.

TUNEL staining identified significant numbers of apoptotic cells at the base of the colonic crypts in susceptible, but not resistant, mice. As shown in panel A, very few colonic cells were TUNEL positive in tissue sections from C. rodentium-infected resistant mouse strains (shown here with an infected C57BL/6 mouse at day 6 p.i.). In contrast, TUNEL staining (brown) identified numerous cells as TUNEL positive at the base of the crypts in C3H/HeJ mice at day 6 p.i. (B). While not as numerous, TUNEL-positive cells were also seen in the same location in the C3H/HeN mice, also shown at day 6 p.i. (C). At higher magnification, with colonic tissue from a C3H/HeOuJ mouse at day 6 p.i., the location and size of the TUNEL-positive cells identify them as being crypt epithelial cells (D).