Experimental Characterization of <italic>Mycobacterium tuberculosis</italic> Adenosine Nucleotide Binding and Ser/Thr/Tyr Phosphosignaling

Abstract

Tuberculosis (TB) is responsible for 1.3 million deaths each year and is a global health burden of overwhelming proportions. The success of <italic>Mycobacterium tuberculosis</italic> (<italic>Mtb</italic>), the causative agent of TB, lies in part with the pathogen's ability to persist under growth-limiting stress to cause disease months to years later. With drug resistant strains on the rise, the need for novel therapeutics is urgent. A fundamental problem for understanding <italic>Mtb</italic> metabolism and pathogenesis and for the development of novel therapeutics is the lack of reliable annotation for many <italic>Mtb</italic> proteins. In addition, our lack of understanding the molecular basis of persistence further hampers the development of effective therapies. Functional characterization of the proteome and the molecular networks that drive <italic>Mtb</italic> persistence is critical to inform novel therapeutic approaches. This dissertation will detail efforts to: 1. Annotate the <italic>Mtb</italic> ATP binding proteins (ATPome), 2. Characterize the role of serine/threonine protein kinases in growth-limiting adaptions that underlie persistence, and 3. Identify protein Tyr phosphorylation, a new <italic>Mtb</italic> phosphosignaling mechanism. The majority of functional annotation is based on in silico predictions; however, these inferences are often unreliable. In addition, ~25% of the <italic>Mtb</italic> protein-coding genes have no predicted function and are annotated as hypothetical proteins. To address the lack of functional annotation, we used a high-throughput quantitative activity-based protein profiling (ABPP) platform to probe, annotate, and validate the large family of ATP-binding proteins. We experimentally confirmed ~240 in silico predictions and identified 72 hypotheticals as ATP-binding proteins, a number of which are only found in mycobacteria and essential for in vitro growth or infection. These data help further define protein function in the <italic>Mtb</italic> proteome and show the diversity among ATP-binding proteins, which may be harnessed for the development of therapeutics. <italic>Mtb</italic> pathogenesis is characterized by adaptation to growth-limiting stress. The molecular mechanisms underlying adaptation are largely unknown; however, phosphosignaling has been suggested as a key mediator of stress response. To test this idea, we investigated a subset of ATP-binding proteins, the 11 eukaryotic-like serine/threonine protein kinases, to determine their role in mediating <italic>Mtb</italic> persistence under hypoxia, a growth-limiting stress induced by the host. <italic>Mtb</italic> adapts to hypoxia by entering reversible bacteriostasis until ample oxygen levels return and growth resumes. We found an essential serine/threonine protein kinase, PknB, to be a critical mediator of oxygen-dependent replication. Inhibition of PknB activity in aerated culture, and overexpression of PknB in hypoxia compromised <italic>Mtb</italic> viability. These data point to a model in which <italic>Mtb</italic> uses PknB to drive cell growth and division, but depletes PknB under hypoxic conditions to promote bacteriostasis and persistence. With serine/threonine protein kinases established as drug targets, these findings suggest PknB as a target in all stages of the <italic>Mtb</italic> life cycle, including in non-replicating bacilli thought to underlie persistence and latent infection. Bacteria drive cellular responses and physiological processes with a variety of ATP-dependent signaling molecules, including serine/threonine, histidine/aspartate, and tyrosine kinases. <italic>Mtb</italic> lacks canonical tyrosine kinases and no tyrosine phosphorylation was detected in previous phosphoproteomic studies, leading to the current notion that <italic>Mtb</italic> does not support protein Tyr phosphorylation. However, protein Tyr phosphorylation is widespread and found in most bacterial phyla, leading us to reevaluate the presence of Tyr phosphorylation in <italic>Mtb</italic>. We show that several serine/threonine protein kinases, including PknB, are in fact dual specificity kinases that are themselves regulated in part by tyrosine phosphorylation, and that <italic>Mtb</italic> supports extensive tyrosine phosphorylation in vivo. These data identify a previously unrecognized arm of <italic>Mtb</italic> phosphosignaling and may present new ways to target <italic>Mtb</italic>.