Human genetic variation in GLS2 is associated with development of complicated Staphylococcus aureus bacteremia

Abstract

The role of host genetic variation in the development of complicated Staphylococcus aureus bacteremia (SAB) is poorly understood. We used whole exome sequencing (WES) to examine the cumulative effect of coding variants in each gene on risk of complicated SAB in a discovery sample of 168 SAB cases (84 complicated and 84 uncomplicated, frequency matched by age, sex, and bacterial clonal complex [CC]), and then evaluated the most significantly associated genes in a replication sample of 240 SAB cases (122 complicated and 118 uncomplicated, frequency matched for age, sex, and CC) using targeted sequence capture. In the discovery sample, gene-based analysis using the SKAT-O program identified 334 genes associated with complicated SAB at p<3.5 x 10-3. These, along with eight biologically relevant candidate genes were examined in the replication sample. Gene-based analysis of the 342 genes in the replication sample using SKAT-O identified one gene, GLS2, significantly associated with complicated SAB (p = 1.2 x 10-4) after Bonferroni correction. In Firth-bias corrected logistic regression analysis of individual variants, the strongest association across all 10,931 variants in the replication sample was with rs2657878 in GLS2 (p = 5 x 10-4). This variant is strongly correlated with a missense variant (rs2657879, p = 4.4 x 10-3) in which the minor allele (associated here with complicated SAB) has been previously associated with lower plasma concentration of glutamine. In a microarray-based gene-expression analysis, individuals with SAB exhibited significantly lower expression levels of GLS2 than healthy controls. Similarly, Gls2 expression is lower in response to S. aureus exposure in mouse RAW 264.7 macrophage cells. Compared to wild-type cells, RAW 264.7 cells with Gls2 silenced by CRISPR-Cas9 genome editing have decreased IL1-β transcription and increased nitric oxide production after S. aureus exposure. GLS2 is an interesting candidate gene for complicated SAB due to its role in regulating glutamine metabolism, a key factor in leukocyte activation.

Fig 1. Regional association plot surrounding GLS2 in the overall replication sample.

The–log₁₀ p-values for individual SNV association tests are plotted against chromosomal position. The strongest association is at intronic variant rs2657878 (purple diamond). The next strongest result is at rs937115 (blue circle), an intronic variant in modest linkage disequilibrium (r²<0.2) with rs2657878 in the 1000 Genomes November 2014 European sample. Three additional variants (red circles), including missense variant rs2657879, are in strong linkage disequilibrium (r²>0.8) with rs2657878.

Fig 2

GLS2 transcript is suppressed in (A) patients with S. aureus or E. coli blood stream infection and in (B) RAW 264.7 macrophages challenged with S. aureus. Data represent two independent experiments each with six biological replicates.

Fig 3

(A) Knockdown of Gls2 decreased IL-1β mRNA and (B) enhanced NO production in RAW 264.7 macrophages challenged with S. aureus. Data represent two independent experiments, with six biological replicates for IL-1β (A) and three biological replicates for NO production (B).