Impact of pe_pgrs33 Gene Polymorphisms on Mycobacterium tuberculosis Infection and Pathogenesis

Abstract

PE_PGRS33 is a surface-exposed protein of Mycobacterium tuberculosis (Mtb) which exerts its role in macrophages entry and immunomodulation. In this study, we aimed to investigate the polymorphisms in the pe_pgrs33 gene of Mtb clinical isolates and evaluate their impact on protein functions. We sequenced pe_pgrs33 in a collection of 135 clinical strains, genotyped by 15-loci MIRU-VNTR and spoligotyping and belonging to the Mtb complex (MTBC). Overall, an association between pe_pgrs33 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 pe_pgrs33 against deleterious SNPs. Among a total of 19 pe_pgrs33 alleles identified in this study, 5 were cloned and used to complement the pe_pgrs33 knock-out mutant strain of Mtb H37Rv (MtbΔ33) to assess the functional impact of the respective polymorphisms in in vitro infections of primary macrophages. In human monocyte-derived macrophages (MDMs) infection, large in-frame and frameshift mutations were unable to restore the phenotype of Mtb H37Rv, impairing the cell entry capacity of Mtb, but neither its intracellular replication rate nor its immunomodulatory properties. In vivo studies performed in the murine model of tuberculosis (TB) demonstrated that the MtbΔ33 mutant strain was not impaired in the ability to infect and replicate in the lung tissue compared to the parental strain. Interestingly, MtbΔ33 showed an enhanced virulence during the chronic steps of infection compared to Mtb H37Rv. Similarly, the complementation of MtbΔ33 with a frameshift allele also resulted in a Mtb 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 Mtb.

Inhibition of Transglutaminase 2 as a Potential Host-Directed Therapy Against Mycobacterium tuberculosis

“…The M. tuberculosis strain H37Rv, Mtb complex clinical strains (MTBC), and M. bovis BCG were isolated at the Fondazione Policlinico Gemelli IRCCS, Università Cattolica del Sacro Cuore (25,55). The strains were grown in Middlebrook 7H9 (Difco, Sparks, MD) supplemented with 10% (vol/vol) oleic acidalbumin-dextrose-catalase (OADC; Difco), with 0.2% glycerol (Microbiol, Cagliari, Italy) and 0.05% Tween 80 (Sigma-Aldrich, St. Louis, MO) at 37 • C. Mycobacterial cultures were harvested at late log phase, glycerol was added at 20% final concentration, and 1-ml aliquots stored at −80 • C. All experiments with Mtb strains were carried out in biosafety laboratory level 3 (BSL-3), following standard safety procedures.…”

PE_PGRS3 ofMycobacterium tuberculosisis specifically expressed at low phosphate concentration, and its arginine-rich C-terminal domain mediates adhesion and persistence in host tissues when expressed inMycobacterium smegmatis

“…In previous studies, enhanced persistence in host tissues of Ms strains expressing PE_PGRSs correlated with an increased inflammatory response as measured by the enlargement of the spleen and higher levels of cytokines as TNF‐α (Dheenadhayalan, Delogu, & Brennan, ; Espitia et al, ; Singh et al, ), supporting the immunomodulatory role of PE_PGRSs in TB pathogenesis (Camassa et al, ; Palucci et al, ). Here, the enhanced persistence in the spleen tissue of the Ms recombinant strain expressing PE_PGRS3 compared with the parental strain and the Ms expressing PE_PGRS3ΔCT did not correlate with an increase in the inflammatory parameters as indicated by the cytokines secreted and lack of spleen enlargement.…”

“…Inactivation of PE_PGRS30 (Iantomasi et al, ), ESX‐5 (Bottai et al, ; Sayes et al, ), and MMAR_0242 (Singh et al, ) resulted in an attenuation of Mtb virulence. Conversely, inactivation of PE_PGRS33 (Camassa et al, ) and PPE38/PPE71 (Ates et al, ), which abolished PE_PGRSs secretion, resulted in enhanced Mtb virulence in mice. Moreover, ESX‐5 inactivation in M. marinum resulted in a hypervirulent phenotype in adult zebrafish (Weerdenburg et al, ).…”

“…Briefly, an aliquot for each homogenised spleen was collected and centrifuged to remove tissue residues. Supernatant was then filtered to remove mycobacteria using Ultrafree‐CL devices (Millipore) according to manufacturer's instruction (Camassa et al, ). Cytokine profile was carried out by multiplex beads assay using Bio‐Plex ProTM Mouse Cytokine Th17 panel A 6‐plex (Biorad) on Bio‐Plex MagPIX instrument (Biorad) according to manufacturer's instruction.…”

The PE/PPE Multigene Family of Mycobacteria and TB Vaccines

“…Very recently, Camassa et al [2], have shown that Mtb knock-out mutant of PE_PGRS33 (in Mtb strain H37Rv) acts like a persistor mutant, being more virulent over time than the parental/wildtype in the murine model. It is very unusual for an attenuated mutant to be more virulent in an animal model of the disease and the studies indicate that (1) the Mtb is mostly extracellular in the lung and (2) the mutant is associated with chronic TB disease, not early infection.…”