Impact of pe_pgrs33 Gene Polymorphisms on Mycobacterium tuberculosis Infection and Pathogenesis

Abstract

PE_PGRS33 is a surface-exposed protein of <i>Mycobacterium tuberculosis</i> (<i>Mtb</i>) which exerts its role in macrophages entry and immunomodulation. In this study, we aimed to investigate the polymorphisms in the <i>pe_pgrs33</i> gene of <i>Mtb</i> clinical isolates and evaluate their impact on protein functions. We sequenced <i>pe_pgrs33</i> in a collection of 135 clinical strains, genotyped by 15-loci MIRU-VNTR and spoligotyping and belonging to the <i>Mtb</i> complex (MTBC). Overall, an association between <i>pe_pgrs33</i> alleles and MTBC genotypes was observed and a dN/dS ratio of 0.64 was obtained, suggesting that a purifying selective pressure is acting on <i>pe_pgrs33</i> against deleterious SNPs. Among a total of 19 <i>pe_pgrs33</i> alleles identified in this study, 5 were cloned and used to complement the <i>pe_pgrs33</i> knock-out mutant strain of <i>Mtb</i> H37Rv (<i>Mtb</i>Δ33) to assess the functional impact of the respective polymorphisms in <i>in vitro</i> infections of primary macrophages. In human monocyte-derived macrophages (MDMs) infection, large in-frame and frameshift mutations were unable to restore the phenotype of <i>Mtb</i> H37Rv, impairing the cell entry capacity of <i>Mtb</i>, but neither its intracellular replication rate nor its immunomodulatory properties. <i>In vivo</i> studies performed in the murine model of tuberculosis (TB) demonstrated that the <i>Mtb</i>Δ33 mutant strain was not impaired in the ability to infect and replicate in the lung tissue compared to the parental strain. Interestingly, <i>Mtb</i>Δ33 showed an enhanced virulence during the chronic steps of infection compared to <i>Mtb</i> H37Rv. Similarly, the complementation of <i>Mtb</i>Δ33 with a frameshift allele also resulted in a <i>Mtb</i> strain capable of causing a surprisingly enhanced tissue damage in murine lungs, during the chronic steps of infection. Together, these results further support the role of PE_PGRS33 in the pathogenesis and virulence of <i>Mtb</i>.