Rv0805, a novel regulator of central carbon metabolism and cell envelope properties in mycobacteria

Abstract

Metabolic flexibility is one of the key factors that underpin mycobacterial physiology and pathogenesis. During infection, mycobacteria shift towards utilising host-derived fatty acids, lipids, cholesterol and cholesterol-esters. This, coupled with elevation in virulence polyketide lipid synthesis, induces critical changes in host-pathogen interaction that affect key signalling events inside macrophages that favour intracellular survival of mycobacteria. However, assimilation of cholesterol leads to elevated levels of propionyl-CoA, which can result in extreme metabolic toxicity unless utilised efficiently. In a high throughout analysis, the gene encoding Rv0805, present in slow-growing, pathogenic mycobacterial species and characterized as a cAMP phosphodiesterase, was identified as one of the genes essential for cholesterol utilisation in mycobacteria. Previous reports from our laboratory suggested that cAMP hydrolysis is not the main function of Rv0805. Therefore, to elucidate the biological role of Rv0805 in mycobacteria, M. bovis BCG was chosen as a model system, and the effects of deletion of the orthologue of rv0805 (bcg_0857) were analysed. Analysis of growth and measurement of metabolites and their turnover using 13C flux approaches showed that BCG_0857 is crucial for growth in media containing cholesterol and propionate. BCGΔ0857 showed impaired propionate assimilation and depletion of CCM metabolites, when propionate was provided as the sole carbon source. Based on gene expression analysis of enzymes involved in the metabolism of propionate, it appeared that the BCGΔ0857 strain experienced a depletion in its carbon reservoir because of increased lipid synthesis. Supporting this, analysis of polar and apolar lipids from the cell envelope of BCGΔ0857 showed anomalies in the levels of PDIMs, PGL and AC2PIM2. Changes in cell envelope composition rendered BCGΔ0857 susceptible to cell wall perturbants, lipophilic antibiotics and led to perturbation of intrabacterial pH homeostasis. Induction of sigE and clgR further supported the presence of a stressed cell envelope in BCGΔ0857. Induction of sigE was attributed to the abberrant accumulation of an unkown phosphodiester substrate of BCG_0857, as evidenced by the fact that the catalytic activity of BCG_0857 was critical for rescuing growth and cell wall-related defects. To conclude, the findings show how a cell envelope localised metallophosphoesterase plays an important regulatory function in central carbon metabolism and generation of an optimum cell envelope in slow growing mycobacteria. Based on this knowledge, we suggest that MtbΔrv0805 would be compromised in intracellular survival during infection and future anti-mycobacterial strategies could be designed that target Rv0805.