An altered immune response, but not individual cationic antimicrobial peptides, is associated with the oral attenuation of Ara4N-deficient Salmonella enterica serovar Typhimurium in mice

Abstract

Salmonella enterica serovar Typhimurium (S. Typhimurium) uses two-component regulatory systems (TCRS) to respond to stimuli in the local microenvironment. Upon infection, the Salmonella TCRSs PhoP-PhoQ (PhoPQ) and PmrA-PmrB (PmrAB) are activated by environmental signals in the intestinal lumen and within host cells. TCRS-mediated gene expression results in lipopolysaccharide (LPS) modification and cationic antimicrobial peptide resistance. The PmrA-regulated pmrHFIJKLM operon mediates 4-amino-4-deoxy-L-arabinose (Ara4N) production and attachment to the lipid A of LPS. A ΔpmrF S. Typhimurium strain cannot produce Ara4N, exhibits increased sensitivity to cationic antimicrobial peptide (CAMP)-mediated killing, and attenuated virulence in mice upon oral infection. CAMPs are predicted to play a role in elimination of Salmonella, and may activate PhoPQ and PmrAB in vivo, which could increase bacterial resistance to host defenses. Competition experiments between wild type (WT) and ΔpmrF mutant strains of S. Typhimurium indicated that selection against this mutant first occurs within the intestinal lumen early during infection. However, CRAMP and active cryptdins alone are not responsible for elimination of Ara4N-deficient bacteria in vivo. Investigation into the early immune response to ΔpmrF showed that it differed slightly from the early immune response to WT S. Typhimurium. Further investigation into the early immune response to infection of Peyer's patches suggests a role for IL-13 in the attenution of the ΔpmrF mutant strain. Thus, prominent CAMPs present in the mouse intestine are not responsible for the selection against the ΔpmrF strain in this location, but limited alterations in innate immune induction were observed that affect bacterial survival and virulence.

Figure 1. S. Typhimurium PhoP-PhoQ and PmrA-PmrB modified LPS.

(A.) Unmodified LPS of S. typhimurium. Unmodified LPS predominates when S. Typhimurium is grown in vitro in LB broth or on plates. (B.) PhoPQ- and PmrAB-modified S. Typhimurium LPS. This highly modified form of LPS is found when S. Typhimurium in grown in vivo. (C.) LPS modifications capable of being made by the Ara4N-deficient strain of S. Typhimurium.

Figure 2. Survival analysis of BALB/c mice infected with WT or mutant S. Typhimurium.

BALB/c (WT) mice were infected via the oral route with either 10⁴ CFU of WT, ΔpmrF or PmrA^c S. Typhimurium or with 10² CFU of WT or ΔpmrF S. Typhimurium via the IP route. The graph depicts representative results from the virulence assays, which were performed in triplicate.

Figure 3. Survival of WT and ΔpmrF S. Typhimurium in WT and CRAMP KO FvB mice.

FvB (WT) and background-matched CRAMP KO mice were infected via the oral route with a 1∶1 mixture of WT and ΔpmrF S. Typhimurium (or a 1∶1 mixture of two strains with WT virulence) containing 5×10⁵ CFU of each strain. Graphed values represent the competitive index (CI) ratio (output ratio/input ratio) of ΔpmrF/WT (or WT/WT) bacteria recovered from the caecum and ileum of each mouse after 48 hrs of infection. Each value graphed as the same shape represents the CI ratio calculated from the specified organ of one WT or CRAMP KO mouse; (*) indicates p≤0.005.

Figure 4. Survival of WT and ΔpmrF S. Typhimurium in BALB/c and CRAMP KO mice.

BALB/c (WT) and background-matched CRAMP KO mice were infected via the oral route with a 1∶1 mixture of WT S. Typhimurium and ΔpmrF bacteria containing 10⁸ CFU of each strain. Bacteria were recovered from the lumen contents, Peyer’s patches, mesenteric lymph nodes (MLN) and spleen from 24 BALB/c and 22 CRAMP KO mice 96 hrs. p.i. Graphed values represent the competitive index (CI) ratio (output ratio/input ratio) of ΔpmrF/WT bacteria recovered from each mouse organ (Salmonella was not recovered from each organ). Each value graphed as the same shape represents the CI ratio calculated from the specified organ of one WT or CRAMP KO mouse. There are no significant differences between any of the observed average CI ratios (p≥0.05).

Figure 5. In vivo survival of WT and ΔpmrF S. Typhimurium in C57BL/6 and MMP7 KO mice.

C57BL/6 (WT) and background-matched MMP7 (Matrilysin-deficient) mice were infected via the oral route with a 1∶1 mixture of WT and WT S. Typhimurium, or WT and ΔpmrF S. Typhimurium containing 10⁸ CFU of each strain. Graphed values represent the competitive index (CI) ratio output ratio/input ratio) of WT/WT (A.) or ΔpmrF/WT (B.) recovered from the livers and spleens of MMP7 KO mice or C57BL/6 mice at 96 hrs. p.i. Each value graphed as the same shape represents the CI ratio calculated from the specified organ of one WT or MMP7 KO mouse. There are no significant differences between any of the observed average CI ratios (p≥0.05).

Figure 6. Immune response to oral infection with 10⁸ CFU of WT, ΔpmrA, or ΔpmrF S. Typhimurium.

(A.) Significant changes (2-fold or greater relative to uninfected controls) in immune-related gene expressionfrom mouse Peyer’s patches infected with WT, ΔpmrA, or ΔpmrF S. Typhimurium relative to PBS mock-infected Peyer’s patches. (B.) Hematoxylin and Eosin staining, and immunohistochemical stainging of Peyer’s patches infected with WT, pmrA, pmrF S. Typhimurium, or mock-infected with PBS for 48 hrs. Neutrophils were stained using Ly6G antibodies. Macrophages were stained using F4/80 antibodies. GC- germinal center.

Figure 7. Survival of IL-13 KO BALB/c mice infected with WT or ΔpmrF S. Typhimurium.

IL-13 KO mice on a BALB/c background were infected by oral route with 10⁴ CFU of WT or ΔpmrF S. Typhimurium (n = 5 mice), and were monitored for morbidity and mortality.

Figure 8. Histology of BALB/c and IL-13 KO Peyer’s patches infected with WT or ΔpmrF S. Typhimurium.

Hematoxylin and Eosin staining of Peyer’s patches infected orally with 10⁸ CFU of WT or ΔpmrF S. Typhimurium, or mock-infected with PBS for 48 hrs. GC- germinal center.