Identification of a genetic region linked to tolerance to MRSA infection using Collaborative Cross mice

Abstract

Staphylococcus aureus (S. aureus) colonizes humans asymptomatically but can also cause opportunistic infections, ranging from mild skin infections to severe life-threatening conditions. Resistance and tolerance are two ways a host can survive an infection. Resistance is limiting the pathogen burden, while tolerance is limiting the health impact of a given pathogen burden. In previous work, we established that collaborative cross (CC) mouse line CC061 is highly susceptible to Methicillin-resistant S. aureus infection (MRSA, USA300), while CC024 is tolerant. To identify host genes involved in tolerance after S. aureus infection, we crossed CC061 mice and CC024 mice to generate F1 and F2 populations. Survival after MRSA infection in the F1 and F2 generations was 65% and 55% and followed a complex dominant inheritance pattern for the CC024 increased survival phenotype. Colonization in F2 animals was more extreme than in their parents, suggesting successful segregation of genetic factors. We identified a Quantitative Trait Locus (QTL) peak on chromosome 7 for survival and weight change after infection. In this QTL, the WSB/EiJ (WSB) allele was present in CC024 mice and contributed to their MRSA tolerant phenotype. Two genes, C5ar1 and C5ar2, have high-impact variants in this region. C5ar1 and C5ar2 are receptors for the complement factor C5a, an anaphylatoxin that can trigger a massive immune response by binding to these receptors. We hypothesize that C5a may have altered binding to variant receptors in CC024 mice, reducing damage caused by the cytokine storm and resulting in the ability to tolerate a higher pathogen burden and longer survival.

Fig 1. CC061 and CC024 differ in survival after infection with MRSA USA300.

After intravenous infection with MRSA USA300 (underlying data from our previous work [31]) we show: A. Survival time, B. Colonization, C. Percent weight change after infection, and D. Tissue damage in the spleen, liver, heart, lung, and kidney. Dots represent individual mice; red dots represent CC061; blue dots represent CC024. The median and interquartile range are shown for each strain. Mann-Whitney test was performed to determine statistical significance (* = P < 0.05, ** = P < 0.01).

Fig 2. Immune differences between CC061 and CC024.

A. Circulating levels of white blood cells (WBC) and lymphocytes (LYM) in uninfected (U) and infected (I) mice. B. Change in WBC and LYM levels post-infection. C-E. Change in chemokine levels post-infection. Dots represent individual mice; red dots represent CC061; blue dots represent CC024. The median and interquartile range are shown for each strain. Mann-Whitney test was performed to determine statistical significance (ns–no significance, * = P < 0.05, ** = P < 0.01).

Fig 3. The F2 population is similar to F1 in survival and weight change after infection.

A. Survival time B. Percent weight change after infection C. Kidney colonization. Dots represent individual mice. Significance tests were performed between the two parents and F1 and between F1 and F2. The median and interquartile range are shown. Kruskal-Wallis test was performed to test for significance (ns–no significance, * = P < 0.05, ** = P < 0.01, *** = P < 0.001, **** = P < 0.0001). Data for CC061 and CC024 parents is reproduced from our previous work for direct comparison to F1 and F2 generations.

Fig 4. Survival after infection has a significant peak on chromosome 7.

A. LOD plot for rank-transformed survival after infection (number of days). The dotted (Red– 95%, Blue– 90%, Green– 85%) lines represent the significant LOD scores for 999 permutations. B. Founder allele plot for chromosome 7. C. Founder allele contribution at the highest marker on chromosome 7, mean and standard error of the mean are shown. Het–Heterozygous allele for CC061 and CC024. Kruskal-Wallis test was performed to determine statistical significance (ns–no significance, *** = P < 0.001).

Fig 5. Prioritizing genes on chromosome 7 for survival after infection.

A. Genes with high-impact variants shortlisted using the founder effect pattern. B. Kidney mRNA expression values after infection (log-transformed). Mean and standard error are shown. The number of animals is given in brackets. Mann-Whitney test was performed (Mean with standard error and P values are shown).

Fig 6. Gene expression differences between CC061 and CC024.

Enriched pathways in CC024 compared to CC061 A. Before infection B. After infection. Fold enrichment was calculated by using all expressed genes as background.