Protein kinase B controls Mycobacterium tuberculosis growth via phosphorylation of the global transcriptional regulator Lsr2

Abstract

Mycobacterium tuberculosis is able to persist in the body through months of multi-drug therapy. Mycobacteria possess a wide range of regulatory proteins, including the essential protein kinase B (PknB), that control transitions between growth states. Here, we establish that depletion of PknB in replicating M. tuberculosis results in transcriptional adaptations that implicate the DNA-binding protein Lsr2 in coordinating these changes. We show that Lsr2 is phosphorylated by PknB, and that phosphorylation of Lsr2 at threonine 112 is important for M. tuberculosis growth and survival under hypoxic conditions. Fluorescence anisotropy and electrophoretic mobility shift assays demonstrate that phosphorylation reduces Lsr2 binding to DNA, and ChIP-sequencing confirms increased DNA binding of a phosphoablative (T112A) Lsr2 mutant in M. tuberculosis. Altered expression of target genes in T112A Lsr2 compared to wild type Lsr2 M. tuberculosis offers further evidence that phosphorylation mediates expression of the Lsr2 regulon. Structural studies reveal increased dynamics of the Lsr2 DNA binding domain from a T112D phosphomimetic Lsr2 mutant, providing a molecular basis for decreased DNA binding by phosphorylated Lsr2. Our findings suggest that, the essential protein kinase, PknB controls M. tuberculosis growth and adaptations to the changing host environment by phosphorylating the global transcriptional regulator Lsr2. expression (WhiB6) 24 . The transcriptional signature of PknB-depletion resembled features of intracellular growth, which significantly overlapped with M. tuberculosis macrophage-derivedRNA profiles from several studies as reflected by hypergeometric probability values: 6.7x10 -23 25 ; 7.34x10 -18 26 ; 3.57x10 -17 27 (Fig. 1). This profile was exemplified by induction of pathways involved in mycobactin synthesis (mbtB/C/D), complex lipid phthiocerol dimycocerosate (PDIM) biosynthesis (fadD26, ppsA/B/C/D), metabolism of alternative lipid carbon sources, the glyoxylate shunt (icl), the methylcitrate cycle (prpD/C, prpR) and triacylglycerol synthase (tgs1). The isoniazid inducible genes (iniB/A/C), responsive to cell wall stress and cell wall targeting drugs were also upregulated 28 . Four of the nine genes coding for alternative ribosomal proteins, rpmB1, rpmB2, rpmG1, rpsN2 29 were induced by PknB depletion.The 34 genes that were significantly repressed in PknB-depleted bacteria included pknB itself (6 fold down-regulated, as expected); nuoA/B/C, encoding subunits of NADH dehydrogenase I, which is part of the aerobic respiratory chain, and several genes involved in intermediary metabolism (Table S1). Overall these changes in replicating bacteria were comparable, in number of differentially expressed genes, to depletion of other regulators, for example DosR 30 . This is in contrast to the large-scale changes in gene expression after treatment with an inhibitor of PknB and PknA that would likely impact M. tuberculosis viability 31 . In summary, PknB depletion in replicating bacteria resulted in co-ordinated change...