Protein tyrosine phosphatase B (PtpB) is a mycobacterial phosphatase secreted during early infection of macrophages1. It has been demonstrated that PtpB is imperative to the intracellular survival of Mycobacterium tuberculosis, and infections with PtpB-deficient M. bovis BCG results in a reduced capacity to maintain infection2,3,4. Previous studies do not necessarily reflect the true primary site of infection, as it has been demonstrated that alveolar macrophages are the site in animal models5. Although PtpB inhibitors exist they don’t effectively inhibit PtpB activity. Better inhibitors can be generated through a better understanding of the role PtpB6. We, therefore, hypothesise that PtpB assists in the maintenance of infection.
Confocal microscopy analysis of infected macrophages was used to determine when to sample early infection the images were analysed using ImageJ. Label-free shotgun LC-MS/MS analysis was conducted on alveolar-like macrophage cell-line infected with M. bovis BCG wild-type for 0, 3, 11 and 24 hours. The mass spectrometry-based analyses were conducted using MaxQuant, R and Cytoscape.
Analysis of the confocal data indicates that an optimal infection time was 1 hour. Preliminary proteomic results showed 57 differentially expressed proteins between all time points. Functional analysis of the infection shows enrichment of proteins related to signal transduction.
Preliminary evidence suggests that knocking out PtpB alters host signalling, preventing maintenance of infection by the bacterium. These results would be complemented by phosphoproteomic data to identify significant pathways in the infection. Future analysis of the difference in the phosphoproteome of the cell-line infected with M. bovis BCG wild-type and ΔPtpB will be done.