Modulation of inducible nitric oxide synthase expression by the attaching and effacing bacterial pathogen citrobacter rodentium in infected mice

Abstract

Citrobacter rodentium belongs to the attaching and effacing family of enteric bacterial pathogens that includes both enteropathogenic and enterohemorrhagic Escherichia coli. These bacteria infect their hosts by colonizing the intestinal mucosal surface and intimately attaching to underlying epithelial cells. The abilities of these pathogens to exploit the cytoskeleton and signaling pathways of host cells are well documented, but their interactions with the host's antimicrobial defenses, such as inducible nitric oxide synthase (iNOS), are poorly understood. To address this issue, we infected mice with C. rodentium and found that iNOS mRNA expression in the colon significantly increased during infection. Immunostaining identified epithelial cells as the major source for immunoreactive iNOS. Finding that nitric oxide (NO) donors were bacteriostatic for C. rodentium in vitro, we examined whether iNOS expression contributed to host defense by infecting iNOS-deficient mice. Loss of iNOS expression caused a small but significant delay in bacterial clearance without affecting tissue pathology. Finally, immunofluorescence staining was used to determine if iNOS expression was localized to infected cells by staining for the C. rodentium virulence factor, translocated intimin receptor (Tir), as well as iNOS. Interestingly, while more than 85% of uninfected epithelial cells expressed iNOS, fewer than 15% of infected (Tir-positive) cells expressed detectable iNOS. These results demonstrate that both iNOS and intestinal epithelial cells play an active role in host defense during C. rodentium infection. However, the selective expression of iNOS by uninfected but not infected cells suggests that this pathogen has developed mechanisms to locally limit its exposure to host-derived NO.

FIG. 1.

Infection by wild-type (wt) C. rodentium but not the type III secretion mutant ΔescD leads to increased iNOS mRNA expression in colonic tissues. RT-PCR for iNOS demonstrated that infection of immunocompetent C57BL/6 mice with wild-type C. rodentium leads to a progressive and significant elevation in iNOS expression, as seen on days 6 and 10 p.i., compared to that in uninfected (−) mice. In contrast, infection with the ΔescD mutant did not induce iNOS mRNA expression over the same time course. The housekeeping gene GAPDH was used to normalize RNA abundance.

FIG. 2.

C. rodentium infection leads to iNOS expression by colonic epithelial cells. Immunohistochemistry for immunoreactive iNOS was performed on colonic tissues taken from uninfected mice (A), as well as mice at day 10 p.i. (B and C). Little if any immunoreactive iNOS was detectable in the colons of C57BL/6 mice prior to infection (panel A) (original magnification, ×100). By day 10 p.i., iNOS expression was seen along much of the mucosal surface (arrows) of infected C57BL/6 mice, including deep in the crypts (arrowheads) and by some scattered cells in the lamina propria (panel B) (original magnification, ×100). Under higher magnification, iNOS expression could be seen focused at the apical surface of many epithelial cells (arrows). A few lamina propria mononuclear cells were also found to express iNOS (arrowheads) (panel C) (original magnification, ×400).

FIG. 3.

The NO donor SNP and the NO releaser GSNO are bacteriostatic for C. rodentium in vitro. The addition of SNP (A) or GSNO (B) to bacterial cultures led to a dose-dependent suppression of bacterial growth over a period of 4 h. The cultures were grown in LB at pH 7.0 for SNP and pH 5.0 for GSNO. The asterisks denote a significant reduction in bacterial growth compared to that in untreated cultures (∗, P < 0.05).

FIG. 4.

Following oral infection, both wild-type and iNOS-deficient mice were heavily colonized by C. rodentium by day 6 p.i. The numbers of bacteria recovered from the colons of individual infected wild-type C57BL/6 mice (filled circles) and iNOS-deficient mice (open circles) on days 6, 10, 14, 18, 21, and 24 p.i. are presented. Each group contained four or five mice. The asterisks denote the recovery of significantly more bacteria (mean value) recovered from iNOS-deficient mice compared to that recovered from wild-type mice at days 18, 21, and 24 p.i. (∗, P < 0.05.).

FIG. 5.

C. rodentium bacteria at day 10 p.i. penetrate deeper into the colonic crypts of iNOS-deficient mice than in wild-type mice. (A) During the course of C. rodentium infection in wild-type C57BL/6 mice, the majority of the bacteria were found attached to superficial epithelial cells (arrowheads) and within the lumen. There is little evidence of bacteria penetrating deeply into colonic crypts. (C) Under higher magnification, one can see individual bacteria (arrowheads) on the surface of these superficial epithelial cells while the base of the crypts are uninfected. (B) In contrast, bacteria infecting iNOS-deficient mice at day 10 p.i. were found to penetrate deeper into crypts, sometimes reaching even the base of the glands. The asterisks identify crypts that are heavily infected with C. rodentium reaching to the base of the crypt. (D) At higher magnification, one can see several crypts filled down to their base with C. rodentium (arrowheads). This effect was transient with the infection in iNOS-deficient mice, again becoming superficial at later time points in the infection. Original magnification for all, ×400.

FIG. 6.

Both wild-type C57BL/6 mice (solid bars) and iNOS-deficient mice (open bars) undergo significant increases in colonic crypt heights during C. rodentium infection. No significant differences in the mean crypt heights (in micrometers) were found between these two mouse strains over the first 24 days of infection. The data represent the means from three independent experiments in which each group contained five mice, and error bars represent standard errors.

FIG. 7.

Immunofluorescence staining shows that infected cells in colonic tissue sections can be identified by their immunoreactivity for Tir but that Tir generally does not colocalize to cells expressing iNOS. (A) Colonic tissue section from a C. rodentium-infected mouse (day 10 p.i.) showing the lumenal surface of the colonic mucosa. Most host cells in this field of view (using DAPI to stain their nuclei blue) are superficial epithelial cells, while C. rodentium LPS is shown in green and Tir is shown in red. Large clusters of C. rodentium bacteria can be seen attached to these superficial epithelial cells during infection (original magnification, ×1,000). In the inset, at a higher magnification, one can see that Tir staining is not found inside the bacteria (arrow) but rather that it occurs outside of the bacteria (arrowhead), presumably translocated from the bacteria into the underlying epithelial cell and focused at the tip of actin pedestals. (B) This field of view shows the superficial colonic mucosa in both the upper and lower parts of the field, with the lumen in the center. By using the staining of Tir as a marker for infected cells, double immunostaining for Tir (red) and iNOS (green) shows that there is very little colocalization between cells expressing Tir and those expressing iNOS. The majority of the epithelial cells on the mucosal surface on the lower half of the picture are infected (Tir positive) but do not express iNOS, while the cells at the top express iNOS but are not infected and do not express Tir (original magnification, ×630). (Inset) At a higher magnification, one can see individual Tir foci in red (arrowhead) on the apical surface of one or two infected cells. These infected cells are the only iNOS-negative cells in a long stretch of uninfected epithelial cells expressing iNOS (green) at their apical surface.