Allelic-Specific Regulation of xCT Expression Increases Susceptibility to Tuberculosis by Modulating microRNA-mRNA Interactions

Abstract

xCT forms part of the x_c^- cysteine-glutamate antiporter which inhibits antimicrobial inflammatory immune functions and thus increases susceptibility to tuberculosis (TB). However, the associations between xCT gene polymorphisms and susceptibility to TB, as well as whether these modulate xCT expression or affect treatment with the xCT inhibitor sulfasalazine (SASP), are unclear. In the present study, we genotyped xCT polymorphisms in a large Chinese cohort and found that the single-nucleotide polymorphism (SNP) rs13120371 was associated with susceptibility to TB. The rs13120371 AA genotype was strongly associated with an increased risk of TB and increased xCT mRNA expression levels compared to those with the GG or AG genotype. rs13120371 is located on the 3' untranslated (UTR) region of the xCT gene, in the putative binding site for miR-142-3p, and the results of luciferase reporter assays indicated that the rs13120371 AA genotype inhibited the binding of miR-42-3p to xCT. Bacterial burden was also significantly higher in cells with the AA genotype than in those with the GG genotype. Furthermore, pretreatment with SASP alleviated this burden in cells with the AA genotype but conferred no benefit in cells with the GG phenotype. In summary, we identified a functional SNP (rs13120371) in the xCT 3' UTR region that increases susceptibility to TB through interacting with miR-142-3p.IMPORTANCE Tuberculosis (TB) is the leading cause of death from a single infectious agent globally, and the development of multidrug resistance represents a serious health concern, particularly in the developing world. Novel effective treatments are urgently required. xCT expression is known to increase susceptibility to TB, and certain polymorphisms in the gene encoding this protein interrupt the binding of microRNA and prevent its suppression. Taking advantage of the FDA approval for the use of sulfasalazine (SASP), which inhibits xCT-mediated cystine transport in humans, we demonstrate how host genotype-specific therapies tailored to the xCT genotype can improve TB outcomes.

Keywords: genotype; polymorphism; therapy; tuberculosis; xCT.

FIG 1

SNP rs13120371 A is associated with increased severity of TB. (A and B) M. tuberculosis antigen (ESAT-6 protein, or ESAT-6/CFP-10 peptide pool)-specific IFN-γ production by PBMCs from pulmonary TB patients with different rs13120371 genotypes was quantified using an ELISPOT assay. Data are expressed as the number of IFN-γ SFCs per 2 × 10⁵ PBMCs from each subject. The ESR (C) and HRCT (D) scores of in pulmonary TB patients with different rs13120371 genotypes, prior to anti-TB chemotherapy. (E) HRCT scores were also determined in 48 pulmonary TB patients, both before and 2 years after completion of anti-TB chemotherapy. Differences between groups were compared with the ANOVA/Newman-Keuls multiple-comparison test. *, P < 0.05, with comparisons indicated by lines. TB, tuberculosis; PBMC, peripheral blood mononuclear cell; SFC, sphere-forming cells; ESR, erythrocyte sedimentation rate; HRCT, high-resolution computed tomography.

FIG 2

Influence of rs13120371 polymorphisms on xCT mRNA and protein expression levels. PBMCs isolated from healthy controls with different rs13120371 genotypes were cultured in the absence of M. tuberculosis lysate (20 μg/ml) for 24 h. Quantification of xCT mRNA (A) and protein (B) expression. Quantification of CXCL1 (C), CXCL2 (D), and IL1B (E) mRNA expression levels. Differences between groups were compared with the ANOVA/Newman-Keuls multiple-comparison test. *, P < 0.05; **, P < 0.01; with comparisons indicated by lines. PBMC, peripheral blood mononuclear cell; MTB, Mycobacterium tuberculosis.

FIG 3

miR-142-3p suppresses xCT mRNA expression. hsp-miR-142-3p (A) and xCT mRNA (B) expression levels following transfection of Thp-1 cells with control plasmids, miRNA mimics, or miRNA inhibitors. (C) hsp-miR-142-3p and xCT mRNA expression levels after infection of Thp-1 cells with H37Ra M. tuberculosis at various time points. hsp-miR-142-3p (D) and xCT mRNA (E) expression levels in PBMCs isolated from clinical samples. HD (n = 10), TB (n = 10), cured (n = 10). *, P < 0.05; **, P < 0.01; ***, P < 0.001, with comparisons indicated by lines. PBMC, peripheral blood mononuclear cell, HD, healthy donor; TB, tuberculosis; cured, cured tuberculosis.

FIG 4

rs13120371 polymorphisms affect miR-142-3P binding activity. (A) The target region of hsp-miR-142-3p in xCT 3′ UTR was determined, and the gain effect of rs13120371 G was predicted with miRNASNP 2.0 software. (B) HEK 293T cells were transiently cotransfected with pMIR-3′-UTR-A or pMIR-3′-UTR-G constructs, hsa-mir-142-3p-mimic, and a negative scramble control for 24 h. Firefly luciferase activity was normalized to Renilla luciferase activity and quantified. **, P < 0.01, with the comparisons indicated by the line.

FIG 5

rs13120371 polymorphisms impact bacterial clearance and the effect of SASP treatment. (A) PBMCs from healthy controls carrying different rs13120371 genotypes were induced with 20 ng/ml GM-CSF for 7 days. MDMs were infected with H37Ra M. tuberculosis at an MOI of 10 for 6 h, and the bacterial burden was assessed after 72 h. (B) MDMs were pretreated with SASP (200 μM) or dimethyl sulfoxide (DMSO) for 1 h and infected with H37Ra M. tuberculosis at an MOI of 10 for 6 h, and the bacterial burden was assessed after 72 h. ANOVA/Newman-Keuls tests were used for multiple comparisons. **, P < 0.01; ***, P < 0.001, with comparisons indicated by lines. PBMC, peripheral blood mononuclear cell; SASP, sulfasalazine.