Mycobacterium tuberculosis (Mtb) pathogenicity depends on its ability to inhibit phagosome acidification and maturation processes after engulfment by macrophages. Here, we show that the secreted Mtb protein tyrosine phosphatase (PtpA) binds to subunit H of the macrophage vacuolar-H + -ATPase (V-ATPase) machinery, a multisubunit protein complex in the phagosome membrane that drives luminal acidification. Furthermore, we show that the macrophage class C vacuolar protein sorting complex, a key regulator of endosomal membrane fusion, associates with V-ATPase in phagosome maturation, suggesting a unique role for V-ATPase in coordinating phagosome-lysosome 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. These findings show that inhibition of phagosome acidification in the mycobacterial phagosome is directly attributed to PtpA, a key protein needed for Mtb survival and pathogenicity within host macrophages.phagocytosis | vesicular trafficking

Entry into host macrophages and evasion of intracellular destruction mechanisms, including phagosome-lysosome fusion, are critical elements of Mycobacterium tuberculosis (Mtb) pathogenesis. To achieve this, the Mtb genome encodes several proteins that modify host signaling pathways. PtpA, a low-molecular weight tyrosine phosphatase, is a secreted Mtb protein of unknown function. The lack of tyrosine kinases in the Mtb genome suggests that PtpA may modulate host tyrosine phosphorylated protein(s). We report that a genetic deletion of ptpA attenuates Mtb growth in human macrophages, and expression of PtpA-neutralizing antibodies simulated this effect. We identify VPS33B, a regulator of membrane fusion, as a PtpA substrate. VPS33B and PtpA colocalize in Mtb-infected human macrophages. PtpA secretion combined with active-phosphorylated VPS33B inhibited phagosome-lysosome fusion, a process arrested in Mtb infections. These results demonstrate that PtpA is essential for Mtb intracellular persistence and identify a key host regulatory pathway that is inactivated by Mtb.

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In Mycobacterium tuberculosis, signal transduction is mediated by 11 serine/threonine kinases, but no tyrosine kinases have been identified thus far. The protein encoded by the ORF (open reading frame) Rv2232 has been annotated as a member of the HAD (haloacid dehydrogenase-like hydrolase) superfamily, which includes phosphatases, phosphomanno- and phosphogluco-mutases, and haloacid dehydrogenases. In the present paper, we report, on the basis of biochemical and mutational analyses, that the Rv2232-encoded protein, named protein tyrosine kinase A (PtkA) is a bona fide protein tyrosine kinase. The cognate substrate of PtkA is the secreted protein tyrosine phosphatase A (PtpA).

Defining the mechanisms of Mycobacterium tuberculosis (Mtb) persistence in the host macrophage and identifying mycobacterial factors responsible for it are keys to better understand tuberculosis pathogenesis. The emerging picture from ongoing studies of macrophage deactivation by Mtb suggests that ingested bacilli secrete various virulence determinants that alter phagosome biogenesis, leading to arrest of Mtb vacuole interaction with late endosomes and lysosomes. While most studies focused on Mtb interference with various regulators of the endosomal compartment, little attention was paid to mechanisms by which Mtb neutralizes early macrophage responses such as the NADPH oxidase (NOX2) dependent oxidative burst. Here we applied an antisense strategy to knock down Mtb nucleoside diphosphate kinase (Ndk) and obtained a stable mutant (Mtb Ndk-AS) that displayed attenuated intracellular survival along with reduced persistence in the lungs of infected mice. At the molecular level, pull-down experiments showed that Ndk binds to and inactivates the small GTPase Rac1 in the macrophage. This resulted in the exclusion of the Rac1 binding partner p67phox from phagosomes containing Mtb or Ndk-coated latex beads. Exclusion of p67phox was associated with a defect of both NOX2 assembly and production of reactive oxygen species (ROS) in response to wild type Mtb. In contrast, Mtb Ndk-AS, which lost the capacity to disrupt Rac1-p67phox interaction, induced a strong ROS production. Given the established link between NOX2 activation and apoptosis, the proportion of Annexin V positive cells and levels of intracellular active caspase 3 were significantly higher in cells infected with Mtb Ndk-AS compared to wild type Mtb. Thus, knock down of Ndk converted Mtb into a pro-apoptotic mutant strain that has a phenotype of increased susceptibility to intracellular killing and reduced virulence in vivo. Taken together, our in vitro and in vivo data revealed that Ndk contributes significantly to Mtb virulence via attenuation of NADPH oxidase-mediated host innate immunity.