Proteome remodeling in theMycobacterium tuberculosisPknG knockout: molecular evidence for the role of this kinase in cell envelope biogenesis and hypoxia response

Abstract

Mycobacterium tuberculosis, the ethiological agent of tuberculosis, is among the deadliest human pathogens. One of M. tuberculosis pathogenic hallmarks is its ability to persist in a dormant state in the host for long periods, reinitiating the infectious cycle when favorable environmental conditions are found. Thus, it is not surprising that this pathogen has developed different mechanisms to withstand the stressful conditions found in the host. In particular, the Ser/Thr protein kinase PknG has gained special relevance since it regulates nitrogen metabolism and facilitates bacterial survival inside macrophages. Nevertheless, the molecular mechanisms underlying these effects are far from being elucidated. To further investigate these issues, we performed quantitative proteomics analyses of protein extracts from M. tuberculosis H37Rv and a mutant derivative lacking pknG. Our results showed that in the absence of PknG the mycobacterial proteome was remodeled since 5.7% of the proteins encoded by M. tuberculosis presented significant changes in its relative abundance when compared to the wild-type strain. The main biological processes affected by pknG deletion were the biosynthesis of cell envelope components and the response to hypoxic conditions. As many as 13 DosR-regulated proteins were underrepresented in the pknG deletion mutant, including the distinctive Hrp-1, which was found to be 12-fold decreased according to Parallel Reaction Monitoring experiments. Altogether, the results presented here allow us to postulate that PknG regulation of bacterial adaptation to stress conditions might be an important mechanism underlying its reported effect on intracellular bacterial survival.

Bacterial effector kinases and strategies to identify their target host substrates

“…The kinase activity of PknG is essential to M. tuberculosis pathogenicity (Walburger et al, 2004) and has therefore been a potential antibiotic target for tuberculosis treatments (Gil et al, 2013;Swain et al, 2021). Studies in PknG's role in pathogenesis show that this effector contains versatile activities, such as E3-ubiquitin ligase activity for host protein ubiquitination Shariq et al, 2022) and regulatory activity for metabolism, virulence, and stress response within M. tuberculosis (Khan et al, 2017;Rieck et al, 2017;Lima et al, 2021). In this section, we focus on a newly identified host protein that is phosphorylated by PknG and approaches used to identify this host substrate.…”