UBC Theses and Dissertations
Role of Mycobacterium tuberculosis protein tyrosine phosphatase A in the pathogenesis of tuberculosis Wong, Dennis Dick-Hang
One of the main mechanisms by which the etiological agent of tuberculosis (TB), Mycobacterium tuberculosis (Mtb), survives in the host macrophage is by its capacity to arrest phagosome acidification and fusion with lysosomes. This Mtb feature is associated with phagosomal exclusion of the Vacuolar H⁺-ATPase (V-ATPase) proton pump, which normally drives luminal acidification of membranous organelles. Although this phenomenon has been known for 20 years, the mechanism by which Mtb blocks phagosome acidification remains obscure. Research on Mtb pathophysiology shows that a wide array of Mtb lipid and protein molecules contribute to maintaining the mycobacterial phagosome in an immature state. We have previously found that Mtb protein-tyrosine Phosphatase A (PtpA) is required for Mtb infection of human macrophages. PtpA is secreted into the macrophage cytosol to inactivate the human VPS33B, a component of the Class C VPS Complex that regulates endosomal membrane fusion. VPS33B dephosphorylation by PtpA results in the inhibition of phagosome-lysosome fusion. In this work, we demonstrated that, in addition to its phosphatase activity, PtpA is also capable of binding to subunit H of the macrophage V-ATPase complex, indicating that PtpA can directly disrupt phagosome acidification. Indeed, we found that a Mtb strain expressing a V-ATPase-binding defective mutant PtpA protein failed to inhibit phagosome acidification, and expression of wild-type PtpA protein in E. coli-infected macrophages is sufficient to block acidification. Furthermore, we showed that the Class C VPS complex associates with V-ATPase during phagosome maturation, identifying a novel role for V-ATPase in coordinating endocytic membrane fusion. PtpA interaction with host V-ATPase is required for the previously reported dephosphorylation of VPS33B and subsequent exclusion of V-ATPase from the phagosome during Mtb infection. Taken together, these findings reveal, for the first time, the long-sought mechanism behind the lack of acidification in the mycobacterial phagosome. Interestingly, we found that PtpA is also a substrate for the newly identified Mtb protein tyrosine kinase PtkA, which is encoded within a shared operon with PtpA, indicating a regulatory control of PtpA during Mtb infection. Understanding the pathophysiological importance of PtpA in Mtb infection might contribute to the development of novel antitubercular therapeutics.
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