Investigating the role of PPE50 from mycobacterium tuberculosis. Insights into the evolution and pathogenesis of tuberculosis

Abstract

Tuberculosis, caused by M. tuberculosis, remains a significant global health challenge. This thesis explores the highly understudied member of the PE/PPE family of proteins in M. tuberculosis, PPE50, to elucidate its role in TB evolution, pathogenesis, and potential as a therapeutic candidate. Investigating the phylogenetics of PPE50 in a globally representative dataset of MTBC species and strains revealed PPE50 to exist as eight variants, with PPE50-381 as the ancestral variant. These variants also showed lineage specificity, indicating possible involvement in the host-pathogen co-evolution of TB, designating PPE50 as a phylogeographically associated protein. In silico characterisation of the identified PPE50 variants provided insights into their structure, subcellular localisation, and function. Significant structural variation was predicted between the variants, highlighting the importance of considering species, strain, and lineage-specific variation in the design of therapeutics. Recombinant PPE50-381 (rPPE50-381), the likely ancestral and progenitor variant, was expressed and purified for further experimental characterisations. Here, three approaches were explored: a maltose-binding protein (MBP) fusion, an intein fusion, and a histidine tag. The histidine tag was the most effective approach for obtaining soluble, stable, and functional rPPE50-381. Using PMA-differentiated THP-1 cells as a macrophage model, rPPE50-381 was shown to bind to these cells without causing cytotoxicity. RT-qPCR analysis then revealed that rPPE50-381 induces a dual-inflammatory response, defined by an initial upregulated pro-inflammatory phase, followed by a later upregulated pro- and anti-inflammatory phase. This highlights a function of PPE50-381 in modulating the host immune response. Overall, this thesis furthers our understanding of the PE/PPE family protein PPE50 and provides insights into the evolution and pathogenesis of tuberculosis. The optimised methodologies herein provide a framework to recover and characterise not only the other variants, but also other complex PE/PPE family proteins in M. tuberculosis. The insights gained significantly inform the development of therapeutics with global efficacy, as well as highlight PPE50 as a potential therapeutic candidate.