A MyD88-deficient mouse model reveals a role for Nramp1 in Campylobacter jejuni infection

Abstract

Campylobacter jejuni is a major worldwide cause of enteric illnesses. Adult immunocompetent mice are not susceptible to C. jejuni infection. However, we show here that mice deficient in the adaptor protein myeloid differentiation factor 88 (MyD88), which is required for signaling through most Toll-like receptors, can be stably colonized by C. jejuni but not by isogenic derivatives carrying mutations in known virulence genes. We also found that Nramp1 deficiency increases the mouse susceptibility to C. jejuni infection when administered systemically. These results indicate that MyD88-deficient mice could be a useful model to study C. jejuni colonization and reveal a potential role for Nramp1 in the control of this bacterial pathogen.

FIG. 1.

MyD88 is required for C. jejuni-induced Erk activation. BMDMs obtained from myd88^+/+ and myd88^−/− mice were infected with C. jejuni (wild type or ΔflaA as indicated) or S. enterica serovar Typhimurium (as a positive control [35]) for the indicated times, and Erk activation was determined by Western immunoblot analysis of cell extracts using an antibody directed to the phosphorylated (activated) form of Erk (phospho Erk). To ascertain equal loading, blots were reprobed with an antibody directed to Erk (total Erk).

FIG. 2.

MyD88 is required for C. jejuni-induced cytokine production. BMDMs obtained from myd88^+/+ and myd88^−/− mice were infected with wild-type (wt) C. jejuni or an isogenic ΔflaA mutant, and at 7 h after infection, the levels of TNF-α (A) and IL-6 (B) were determined as indicated in Materials and Methods. Values represent the means ± standard deviations of three independent measurements and were standardized by considering the stimulation of myd88^+/+ mice by wild-type C. jejuni to be 100%.

FIG. 3.

MyD88-deficient mice are efficiently colonized by C. jejuni. myd88^+/+ and myd88^−/− mice were inoculated orally or intraperitoneally (IP) with the C. jejuni 81-176 wild-type strain. Colonization was evaluated by determining the number of CFU in the feces at different times after infection. Each circle denotes the CFU of an individual animal.

FIG. 4.

C. jejuni colonizes the systemic tissues of MyD88-deficient mice. myd88^+/+ and myd88^−/− mice were inoculated orally or intraperitoneally (IP) with the C. jejuni 81-176 wild-type strain. At different times after infection, the numbers of C. jejuni CFU in the spleen (SP) (squares), liver (LIV) (circles), mesenteric lymph nodes (MLN) (×), and intestine (INT) (triangles) were determined by plating different dilutions of the tissue lysates. Each symbol represents the CFU of an individual animal.

FIG. 5.

Nramp1 deficiency increases C. jejuni mouse colonization after systemic administration. myd88^−/− nramp1^−/− and myd88^−/− nramp1^+/+ mice were inoculated intraperitoneally (IP) with the C. jejuni 81-176 wild-type strain. Colonization was evaluated by determining the number of CFU in the feces at different times after infection (A). Colonization of systemic tissues was evaluated 4 weeks after infection by determining the number of C. jejuni CFU in the liver and intestine (B). Each square denotes the CFU of an individual animal.

FIG. 6.

Nramp1 deficiency does not alter C. jejuni mouse colonization after oral administration. myd88^−/− nramp1^−/−, and myd88^−/− nramp1^+/+ mice were inoculated orally with the C. jejuni 81-176 wild-type strain. Colonization was evaluated by determining the number of CFU in the feces at different times after infection (A). Colonization of systemic tissues was evaluated 4 weeks after infection by determining the number of C. jejuni CFU in the liver and intestine (B). Each square denotes the CFU of an individual animal.

FIG. 7.

The absence of Nramp1 results in an increased ability of C. jejuni to survive within macrophages. BMDMs obtained from nramp1^+/+ and nramp1^−/− mice were infected with C. jejuni, and at different times after infection, the number of intracellular CFU was determined by the gentamicin protection assay (A). The relative survival index (B) represents the ratio between CFU obtained from nramp1^+/+ and CFU obtained from nramp1^−/− mouse macrophages. Alternatively, viable bacteria were enumerated by flow cytometry (C) as indicated in Materials and Methods. The relative survival index obtained by this method is shown in panel D. Values represent the means ± standard deviations of three independent determinations.

FIG. 8.

Recruitment of Nramp1 to the C. jejuni-containing vacuole. Mouse BMDMs were transduced with a vector encoding Nramp1 fused to GFP. At 24 h after transduction, cells were infected with C. jejuni, and 60 min after infection, cells were fixed, stained with an anti-C. jejuni antibody, and visualized by fluorescence microscopy as indicated in Materials and Methods.

FIG. 9.

C. jejuni virulence mutants are defective for colonization of MyD88-deficient mice. myD88^−/− mice were inoculated orally or intraperitoneally (IP) with equal numbers of the C. jejuni 81-176 wild-type (wt) strain and its isogenic derivatives carrying mutations in pglF or Cj1418c. Colonization was evaluated by determining the number of CFU in the feces at different times after infection (A and B). Each circle or square denotes the CFU obtained from an individual animal. Colonization of systemic tissues was evaluated at 3 weeks after infection by determining the number of C. jejuni CFU in the liver and intestine (C and D). To test the complementation of the pglF mutant, myd88^−/− mice were inoculated orally with equal numbers of the C. jejuni 81-176 pglF::kan mutant strain and its complemented derivative [pglF::kan(+pglF)] (E and F). Each circle or square denotes the CFU obtained from an individual animal.