Abstract
Protein phosphorylation plays a key role in Mycobacterium tuberculosis (Mtb) physiology and pathogenesis. We have previously shown that a secreted protein tyrosine phosphatase, PtpA, is essential for Mtb inhibition of host macrophage acidification and maturation, and is a substrate of the protein tyrosine kinase, PtkA, encoded in the same operon. In this study, we constructed a ∆ptkA deletion mutant in Mtb and found that the mutant exhibited impaired intracellular survival in the THP-1 macrophage infection model, correlated with the strain’s inability to inhibit macrophage phagosome acidification. By contrast, the mutant displayed increased resistance to oxidative stress in vitro. Proteomic and transcriptional analyses revealed upregulation of ptpA, and increased secretion of TrxB2, in the ΔptkA mutant. Kinase and protein-protein interaction studies demonstrated that TrxB2 is a substrate of PtkA phosphorylation. Taken together these studies establish a central role for the ptkA-ptpA operon in Mtb pathogenesis.
Highlights
The etiological agent of tuberculosis (TB), Mycobacterium tuberculosis (Mtb), remains one of the most devastating infectious agents in the world
Genomic DNA isolated from the parental wild type (WT) strain and the ∆ptkA mutant strain were subjected to Southern hybridization analysis to confirm that the gene encoding PtkA was replaced with the hygromycin resistance cassette as outlined in Fig. 1a and b
Since ptkA and ptpA are present in the same polycistronic operon, and PtpA is known to be essential for Mtb pathogenesis[15,20,26] as well as being the phosphorylation substrate of PtkA19,23, we hypothesized that PtkA might be required for Mtb intracellular growth within human macrophages
Summary
The etiological agent of tuberculosis (TB), Mycobacterium tuberculosis (Mtb), remains one of the most devastating infectious agents in the world. The Mtb cell wall glycolipid ManLAM co-operates with the secreted acid phosphatase SapM to block phagosome fusion with late endosomes Both prevent phagosomal accumulation of phosphatidylinositol 3-phosphate (PI3P), which mediates the recruitment of membrane trafficking proteins such as the early endosomal antigen, EEA19,10. Mtb possesses a wide repertoire of signal transduction systems, including eleven “two-component” systems, eleven eukaryotic-like serine/threonine protein kinases (PknA-PknL), two protein tyrosine phosphatases (PtpA and PtpB), and the newly identified protein tyrosine kinase, PtkA8,18–22. These signaling proteins play key roles in bacterial adaptation and response to host defense mechanisms. This suggests that PtkA and PtpA may act cooperatively to provide a function essential for Mtb pathogenesis
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