Yossef Av-Gay

The full abstract for this thesis is available in the body of the thesis, and will be available when the embargo expires.

View record

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is capable of synthesizing the small sulfur compound ergothioneine (EGT). The physiological role and regulation of EGT biosynthesis in Mtb is unknown. However, mammalian cells deficient in EGT demonstrate augmented oxidative stress and cell death, suggesting that EGT is an antioxidant. Through this work, we identified the Mtb EGT biosynthesis genes and characterized rv3701c (egtD) to encode for a histidine methyltransferase that forms the EGT biosynthetic pathway intermediate, hercynine (N-α-trimethylhistidine). Construction of a knockout (ΔegtD) demonstrated the methyltransferase to be essential for EGT biosynthesis in Mtb.We investigated the role of EGT in protecting Mtb during macrophage infection and observed a small but significant difference in the ex vivo growth and survival of the ΔegtD strain relative to H37Rv wildtype and the complement strains 120 h post-infection of murine macrophages. However, there was no difference in survival of the ΔegtD mutant in human THP-1 cells over this time period suggesting an alternative role for EGT in Mtb physiology and pathogenesis.Upon further analysis we found EgtD to be phosphorylated by and interact with the Mtb Serine/Threonine Protein Kinase PknD. Phosphorylation of EgtD both in vitro and inside Escherichia coli identified phosphorylation at site Thr-213, permitting us to generate an EgtD phosphomimetic and phosphoablative mutant strains with our ΔegtD mutant. In vitro analysis, demonstrated phosphorylated EgtD to produce significantly lower quantities of methylated histidine relative to the non-phosphorylated form. Further quantification of intracellular EGT levels in the Mtb EgtD phosphomutants and Mtb PknD transposon mutant identified PknD to negatively regulate EGT biosynthesis. From these results, we identified that EGT biosynthesis is up-regulated in response to nutrient starvation. Under these conditions, the Mtb ΔegtD mutant was unable to maintain viability compared to its parental wildtype and complement strains. As starvation induces a non-replicative state in Mtb, these findings indicate that EGT plays a role in mediating persistent infection or disease latency. Further metabolic analysis identified Mtb intracellular EGT levels to be directly correlated with carbon source type and availability, suggesting a role for EGT in long-term energy storage.

View record

To survive in the human host macrophage, Mycobacterium tuberculosis, the etiological agent of Tuberculosis, infects human macrophages and inhibits two key cellular events required for the elimination of invading organisms; phagosome acidification and fusion with lysosomes. This is partially dependent on Protein tyrosine phosphatase A (PtpA) secreted into the cytosol of the macrophage. PtpA first disrupts phagosome acidification by binding to subunit H of the proton pump, and second, inhibits phagosome-lysosome fusion by dephosphorylating and inactivating the membrane fusion regulator, hVPS33B. The ability of M. tuberculosis to actively interfere with host trafficking events allows this pathogen to replicate and persist inside the macrophage and prevent antigen presentation required to initiate an adaptive immune response. In this work, we explored the global macrophage’s response to infection emphasizing on PtpA’s role in this process. We analyzed the macrophage global proteomic responses and focused on the activity of signalling pathways by determining the phosphorylation status of host proteins upon infection with M. tuberculosis strains. We found that PtpA affects the macrophage’s response by modulating various proteins involved in RNA metabolism, immunity and defence, and cellular respiration pathways. We further show that PtpA promotes M. tuberculosis survival by dephosphorylating the host kinase GSK3α on amino acid Y²⁷⁹ leading to inhibition of GSK3α and arrest of macrophage apoptosis. GSK3α has pro- and anti-apoptotic activities and dephosphorylation of Y²⁷⁹ inhibits its ability to initiate apoptosis. In this regard, activation of the host apoptosis executioner, caspase-3, is blocked in M. tuberculosis-infected macrophages compared to cells infected with ΔptpA mutant strain. Taken together, these findings reveal one of the long-sought effectors behind the inhibition of apoptosis of the host by virulent M. tuberculosis. Moreover, we are the first to simultaneously determine proteome-wide protein expression levels of human macrophages infected with M. tuberculosis and outline molecular signatures of the global and PtpA-dependent proteomic patterns of macrophages during infection. We have now established that PtpA significantly contributes to successful infection and survival of M. tuberculosis inside the macrophage. Understanding the mechanism of action of PtpA during M. tuberculosis infection may lead to the development of novel anti-mycobacterial drugs.

View record

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.

View record