Two genes on A/J chromosome 18 are associated with susceptibility to Staphylococcus aureus infection by combined microarray and QTL analyses

Abstract

Although it has recently been shown that A/J mice are highly susceptible to Staphylococcus aureus sepsis as compared to C57BL/6J, the specific genes responsible for this differential phenotype are unknown. Using chromosome substitution strains (CSS), we found that loci on chromosomes 8, 11, and 18 influence susceptibility to S. aureus sepsis in A/J mice. We then used two candidate gene selection strategies to identify genes on these three chromosomes associated with S. aureus susceptibility, and targeted genes identified by both gene selection strategies. First, we used whole genome transcription profiling to identify 191 (56 on chr. 8, 100 on chr. 11, and 35 on chr. 18) genes on our three chromosomes of interest that are differentially expressed between S. aureus-infected A/J and C57BL/6J. Second, we identified two significant quantitative trait loci (QTL) for survival post-infection on chr. 18 using N(2) backcross mice (F(1) [C18A]xC57BL/6J). Ten genes on chr. 18 (March3, Cep120, Chmp1b, Dcp2, Dtwd2, Isoc1, Lman1, Spire1, Tnfaip8, and Seh1l) mapped to the two significant QTL regions and were also identified by the expression array selection strategy. Using real-time PCR, 6 of these 10 genes (Chmp1b, Dtwd2, Isoc1, Lman1, Tnfaip8, and Seh1l) showed significantly different expression levels between S. aureus-infected A/J and C57BL/6J. For two (Tnfaip8 and Seh1l) of these 6 genes, siRNA-mediated knockdown of gene expression in S. aureus-challenged RAW264.7 macrophages induced significant changes in the cytokine response (IL-1 beta and GM-CSF) compared to negative controls. These cytokine response changes were consistent with those seen in S. aureus-challenged peritoneal macrophages from CSS 18 mice (which contain A/J chromosome 18 but are otherwise C57BL/6J), but not C57BL/6J mice. These findings suggest that two genes, Tnfaip8 and Seh1l, may contribute to susceptibility to S. aureus in A/J mice, and represent promising candidates for human genetic susceptibility studies.

Figure 1. Overall strategy for identifying genes associated with susceptibility to S. aureus in A/J mice.

Figure 2. A/J and C57BL/6J mice exhibit different susceptibility to S. aureus that is not primarily due to deficiency in Complement C5.

C57BL/6J and A/J were injected via intraperitoneal (i.p.) route (10⁷ CFU/g) with S. aureus strains Sanger 476 (Figure 2A) and MW2 (Figure 2B) (n = 15 mice for each strain). (Figure 2C) C57BL/6J and A/J mice were injected via tail vein route (10⁷ CFU/g) with Sanger 476 (n = 10 mice for each strain). Comparison of survival curves was performed by use of log rank test. Values of P<0.05 were considered significant. (Figure 2D) Deficiency of Complement C5 is not the primary determinant of susceptibility to S. aureus in A/J mice. Three C5-deficient mouse strains (C57BL/10SnJ, B10.D2/oSnJ, B10.D2-Hc0 H2d H2-T18c/o2SnJ, and NOD/LtJ) were injected i.p. (10⁷ CFU/g) with Sanger 476 (n = 10 mice for each strain).

Figure 3. Bacterial load in peritoneal fluid and kidneys is higher in chromosome substituted strain (CSS) 8, CSS11 and CSS18 mice after S. aureus injection.

(A) Bacterial load in peritoneal fluid. C57BL/6J, A/J, and CSS mice were injected (i.p.) with Sanger 476 (10⁷ CFU/g) and euthanized 24 h later (n = 5 mice for each murine strain). (B) Bacterial load in kidneys. C57BL/6J, A/J, and CSS mice were injected (i.p.) with Sanger 476 (10⁷ CFU/g) and euthanized 24 h later (n = 5 mice for each murine strain). P-values were calculated from the F-test. Values of P<0.05 were considered significant.

Figure 4. Ten genes on chromosome 18 were identified by both QTL mapping and expression array for susceptibility to S. aureus.

(A) Chromosome 18 LOD score plot for susceptibility to S. aureus in N₂ backcross mice. Six to eight-week-old backcross mice were injected i.p. with 1×10⁷ CFU/g S. aureus and observed every 8 h for 5 days. The horizontal dashed lines indicate the thresholds for significant (P = 0.05) and suggestive (P = 0.63) LOD score determined by the J/qtl permutation test using 1,000 permuted data sets. The microsatellite markers used for determining genotypes of N₂ backcross mice are presented along the X-axis. The differentially expressed genes are indicated on chromosome 18. Genes identified within significant or suggestive QTL were indicated with * or /†, respectively. (B) Quantitative real-time PCR validation of 10 genes (March3, Cep120, Chmp1b, Dcp2, Dtwd2, Isoc1, Lman1, Spire1, Tnfaip8, and Seh1l) mapping within the two identified QTLs on chr.18 that were also identified by the expression-array selection strategy. Expression values of genes were normalized to 18S rRNA. Six genes (Dtwd2, Tnfaip8, Isoc1, Lman1, Chmp1b, and Seh1l) were significantly differentially expressed between A/J and C57BL/6J infected with S. aureus in the significant QTLs on chromosome 18. Values of P<0.05 were considered significant (n = 5, paired two-tailed t-test).

Figure 5. RAW264.7 cells transfected with Tnfaip8 and Seh1l siRNA and peritoneal macrophages from C57BL/6J and CSS18 exhibited significant and consistent patterns of change in the cytokine production (IL-1β and GM-CSF).

(A) RAW264.7 cells were treated with negative control, Tnfaip8 or Seh1l siRNA and incubated with S. aureus for 5 h. (B) Peritoneal macrophages from CSS18 and C57BL/6J were incubated with S. aureus for 48 h. Macrophage culture supernatants were collected at two time points (pre- and post- S. aureus stimulation) for cytokine/chemokine analysis. IL-1β and GM-CSF were determined by Luminex-based multiplex cytokine assay. Means and standard deviations of three independent experiments are shown. Values of P<0.05 were considered significant (unpaired two-tailed t-test).