Crosstalk between cyclic-di-guanosine monophosphate and the sensor kinase MtrB regulates MtrA-dependent genes, bacterial growth, biofilm formation and lysosomal trafficking of Mycobacterium tuberculosis.

Cyclic-di-guanosine monophosphate (c-di-GMP) plays an important role in bacterial signalling networks. C-di-GMP exerts a regulatory function through binding to diverse molecules that include transcription factors, riboswitches and sensor kinases (SKs), thereby regulating diverse processes. Here, we demonstrate the crosstalk between c-di-GMP and the SK MtrB of Mycobacterium tuberculosis. MtrB phosphorylates and regulates its cognate response regulator MtrA. C-di-GMP binds directly to the cytosolic domain of MtrB to inhibit its autophosphorylation. C-di-GMP levels in M. tuberculosis were manipulated by overexpressing a c-di-GMP synthesizing enzyme ydeH and a degrading enzyme rv1357c. We demonstrate that overexpression of ydeH lowers growth of the bacterium both in vitro and in M. tuberculosis grown in macrophages. This is in conformity with lowered expression of mtrA and selected genes of the mtrA regulon involved in cell wall turnover in the ydeH-overexpressing strain compared to the parent strain. We also demonstrate that overexpression of ydeH in M. tuberculosis hinders biofilm formation, whereas overexpression of rv1357c has the opposite effect. Neither of the two genes could rescue the biofilm defective phenotype of the MtrB knock out mutant (ΔmtrB), suggesting that c-di-GMP exerts its role on biofilm formation through MtrB. Finally, we show by fluorescence microscopy that the trafficking of M. tuberculosis overexpressing ydeH is significantly higher than that of the parent strain and that this is linked to reduced expression of the MtrB-dependent genes esxG and esxH, which play a role in subversion of lysosomal trafficking of M. tuberculosis. These results provide important new insight into the crosstalk between c-di-GMP and MtrB in M. tuberculosis.