Impact of pe_pgrs33 Gene Polymorphisms on Mycobacterium tuberculosis Infection and Pathogenesis.

Serena Camassa,Riccardo Manganelli,Samuel Jouny,Michela Sali,Stefano Rocca,Mariachiara Minerva,Francesco Ria,Giovanni Delogu,Ivana Palucci,Raffaella Iantomasi,Tiziana Cubeddu,Priscille Brodin,Elisa Petruccioli,Maurizio Sanguinetti,Flavio De Maio,Delia Goletti

doi:10.3389/fcimb.2017.00137

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.

Highlights

Mycobacterium tuberculosis (Mtb), the main etiological agent of tuberculosis (TB) in humans, still represents one of the most feared pathogens at global level, with 10.4 million new cases of TB and 1.4 million deaths in 2015 (World Health Organization, 2016)
According to the phylogeographical distribution described for the Mtb complex (MTBC) by Gagneux and colleagues (Gagneux, 2012), our results showed how all MTBC superlineages are represented in the metropolitan area of Rome, except for the recently described ancient superlineage denominated 7 or Ethiopian (Firdessa et al, 2013), and that the Euro-American superlineage is the most prevalent
We investigated the genetic variability of pe_pgrs33 in a collection of 135 randomly selected MTBC clinical isolates and assessed in in vitro and in vivo models the impact of large sequence deletions on PE_PGRS33 function at early and late phases of Mtb infection

Summary

Introduction

Mycobacterium tuberculosis (Mtb), the main etiological agent of tuberculosis (TB) in humans, still represents one of the most feared pathogens at global level, with 10.4 million new cases of TB and 1.4 million deaths in 2015 (World Health Organization, 2016). According to recent comparative genomic studies, Mtb originated in Africa between 70,000 and 35,000 years ago from a genetic bottleneck of its progenitor (Gutierrez et al, 2005; Hershberg et al, 2008; Wirth et al, 2008), followed by the clonal expansion of 7 genetically homogeneous Mtb complex (MTBC) superlineages that ravaged human communities for centuries (Portevin et al, 2011; Gagneux, 2012; Firdessa et al, 2013). Despite the genetic homogeneity shared by MTBC superlineages compared to other bacterial pathogens, the presence of small sequence variations in MTBC genome is responsible for differential pathogenetic properties (Gagneux and Small, 2007; Portevin et al, 2011). The PE_PGRS and PPE_MPTR protein subfamilies, which are the most recently evolved within the respective families, are characterized by the presence of polymorphic regions at their C-terminus that vary in sequence and size (Gey van Pittius et al, 2006; McEvoy et al, 2012). pe_pgrs genes show GCrich repetitive sequences (PGRS) which encode Gly-Gly-Ala/X repeats and ppe_mptr genes present polymorphic tandem repeats (MPTR) encoding Asn-(X-Gly)2-X-Asn-X-Gly repeats (Poulet and Cole, 1995; Sampson, 2011; Soldini et al, 2011; McEvoy et al, 2012)

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