Mycobacterium tuberculosis (Mtb) is the etiological agent of tuberculosis, an infectious disease which causes 1.5 million deaths annually (1). The pe/ppe gene family comprises approximately 10% of the coding capacity of the Mtb genome and secreted PE and PPE proteins are strongly implicated in virulence (2). Previous work in the West Laboratory has demonstrated that overexpression of ppe19 in mycobacteria, results in increased intracellular infection burdens within immortalised murine macrophages.
This investigation is the first to purify PPE19, both as a fusion protein and in its native tertiary conformation. Moreover, utilisation of a mycobacterial two-hybrid assay has identified a putative PE binding partner of PPE19, with a focus of current study being verification of this interaction. Application of purified PPE19 to primary macrophages resulted in significant upregulation of pro-inflammatory cytokines, whilst increased macrophage uptake of fluorescent microspheres occurred when spheres were coated with this protein.
To investigate PPE19 function at the cellular level, an Mtb strain with constitutive ppe19 expression (Mtb-ppe19) was constructed. Through repeated infections of a macrophage cell line, Mtb-ppe19 demonstrated significantly enhanced internalisation into macrophages, as well as adherence to their outer surface, relative to wild-type Mtb. An Mtb ppe19 knockout mutant was then created (Δppe19), and ppe19 expression restored in two complemented strains, using native (Δppe19-Cnat) or constitutive promoters (Δppe19-Ccon). These three strains were utilised in a murine infection model, to analyse the in vivo effect of abolished ppe19 expression. The lack of a discernible difference in infection burden within the Δppe19-infected group, relative to wild-type-infected mice, suggests functional redundancy by which Mtb compensates for the loss of PPE19. Subsequently, two highly homologous proteins, PPE18 and PPE60, have been identified and the interaction of this trio with host immunity is now a primary focus of ongoing study.