Experimental Characterization of Mycobacterium tuberculosis Adenosine Nucleotide Binding and Ser/Thr/Tyr Phosphosignaling
Year: 2014
Type: dissertation
Abstract: Tuberculosis (TB) is responsible for 1.3 million deaths each year and is a global health burden of overwhelming proportions. The success of Mycobacterium tuberculosis (Mtb), 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 Mtb metabolism and pathogenesis and for the development of novel therapeutics is the lack of reliable annotation for many Mtb 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 Mtb persistence is critical to inform novel therapeutic approaches. This dissertation will detail efforts to: 1. Annotate the Mtb 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 Mtb phosphosignaling mechanism. The majority of functional annotation is based on in silico predictions; however, these inferences are often unreliable. In addition, ~25% of the Mtb 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 Mtb proteome and show the diversity among ATP-binding proteins, which may be harnessed for the development of therapeutics. Mtb 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 Mtb persistence under hypoxia, a growth-limiting stress induced by the host. Mtb 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 Mtb viability. These data point to a model in which Mtb 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 Mtb 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. Mtb lacks canonical tyrosine kinases and no tyrosine phosphorylation was detected in previous phosphoproteomic studies, leading to the current notion that Mtb 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 Mtb. 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 Mtb supports extensive tyrosine phosphorylation in vivo. These data identify a previously unrecognized arm of Mtb phosphosignaling and may present new ways to target Mtb.
Author: Corrie Ortega
Language: English
Subfield: Infectious Diseases
Field: Medicine
Domain: Health Sciences
Open Access status: green
OpenAlex ID: W2271963659