Abstract
The Mycobacterium tuberculosis protein kinase B (PknB) comprises an intracellular kinase domain, connected through a transmembrane domain to an extracellular region that contains four PASTA domains. The present study describes the comprehensive analysis of different domains of PknB in the context of viability in avirulent and virulent mycobacteria. We find stringent regulation of PknB expression necessary for cell survival, with depletion or overexpression of PknB leading to cell death. Although PknB-mediated kinase activity is essential for cell survival, active kinase lacking the transmembrane or extracellular domain fails to complement conditional mutants not expressing PknB. By creating chimeric kinases, we find that the intracellular kinase domain has unique functions in the virulent strain, which cannot be substituted by other kinases. Interestingly, we find that although the presence of the C-terminal PASTA domain is dispensable in the avirulent M. smegmatis, all four PASTA domains are essential in M. tuberculosis. The differential behavior of PknB vis-à-vis the number of essential PASTA domains and the specificity of kinase domain functions suggest that PknB-mediated growth and signaling events differ in virulent compared with avirulent mycobacteria. Mouse infection studies performed to determine the role of PknB in mediating pathogen survival in the host demonstrate that PknB is not only critical for growth of the pathogen in vitro but is also essential for the survival of the pathogen in the host.
Highlights
Mycobacterium tuberculosis protein kinase B (PknB) plays a critical role in modulating cell division and cell wall synthesis
M. tuberculosis H37Rv-pptr-PknB (Rv-pptr-B) mutant strain was created with the help of a suicide delivery plasmid, in which the pknB gene in the genome was replaced with a pristinamycin- inducible copy [19] (Fig. 1F)
Analysis carried out with M. tuberculosis strains revealed a considerable diminution in the levels of PknB when Rv-pptr-B was grown in the absence of pristinamycin and substantial overexpression of PknB in Rv::PknB strain upon induction with IVN (Figs. 1G and 2A)
Summary
Mycobacterium tuberculosis PknB plays a critical role in modulating cell division and cell wall synthesis. The differential behavior of PknB vis-à-vis the number of essential PASTA domains and the specificity of kinase domain functions suggest that PknB-mediated growth and signaling events differ in virulent compared with avirulent mycobacteria. The presence of a eukaryotic-like serine/ threonine protein kinase (STPK) in a prokaryote was first reported in 1991 (pkn in Myxococcus xanthus) [3] These “eukaryotic-like” STPKs play important roles in bacterial cellular processes, including cell division, cell wall synthesis, cell metabolism, and dormancy exit (4 –7). The extracellular domain of PknB is predicted to have four conserved PASTA (penicillin-binding protein and serine/threonine kinase-associated) domains [22, 25] This domain has been suggested to play a role in the recognition of D-alanyl-D-alanine dipeptides used to build up the peptidoglycan layers [25]. Infection studies with a mouse model reveal that depletion of PknB results in clearance of pathogen from the host tissues, indicating definitively that PknB is essential for survival of the pathogen within the host
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