Protein Kinases and Nitric Oxide in the Stealthy Survival of Mycobacterium tuberculosis

Differentially culturable (DC) Mycobacterium tuberculosis (Mtb) are characterised by an inability to grow on solid agar, although their presence can be uncovered by extinction dilution in resuscitation media. Subpopulations of bacilli in this distinct physiological state can be detected in tuberculosis patients, especially following treatment. Several host factors have been implicated in the generation of DC-Mtb, including reactive oxygen species and nitric oxide (NO). Little is known about the characteristics of these cells, further investigation of which could aid in their detection and eradication.

In this study, a previously established model for DC-Mtb generation using the nitric oxide donor NOD was employed for comprehensive characterisation and investigation of the importance of serine/threonine protein kinase signalling for DC-Mtb formation and resuscitation. It was established that NOD rapidly released NO in cells, unlike commercial donors DETA NONOate and sodium nitrite, that breakdown spontaneously and did not induce DC-Mtb.

The project findings suggest that the serine/threonine protein kinase PknG and PknB have distinct roles in DC-Mtb formation and resuscitation. The role of PknG was investigated through energy and redox state assessments and metabolomics, indicating that PknG upregulation during NOD treatment improves the resuscitative capacity of DC-Mtb, presumably by enabling glutamate catabolism. In contrast, dysregulation of PknB expression was detrimental for resuscitation. To understand the basis of this impaired resuscitation two predicted substrates of PknB, MtrA (implicated in regulation of rpf genes expression) and glutamate racemase MurI (dramatically upregulated during transition to DC-Mtb) were investigated.

Results obtained in this project showed that the response regulator MtrA was able to bind rpfA promoter regions, which was reduced by PknB phosphorylation, thus providing a molecular mechanism for PknB-mediated regulation of rpf expression. MurI was confirmed to be phosphorylated by PknB in vitro. Furthermore, the importance of MurI phosphorylation was demonstrated in complementation experiments using a M. smegmatis ∆murI mutant. Experiments with phosphomimetic versions of MurI indicated potential alterations to protein oligomeric state and modulation of MurI activity.

The findings of this thesis provide insights into the nature of NO-induced DC-Mtb generation, as well as the role of kinase signalling, which could be useful for development of tuberculosis diagnostics and therapeutics.