Novel regulation levels in mycolic acids biosynthesis in mycobacteria.

CABRUJA, M.; GRAMAJO, H; GAGO, G.

Rosario

Congreso; L Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2014

SAIB

Elucidation of mechanisms modulating mycolic acid biosynthesis would shed light on the capacity of M. tuberculosis to adapt and survive within the infected host. Reversible protein phosphorylation is a key mechanism by which environmental signals are transmitted to cause changes in protein expression or activity in prokaryotes. Phosphorylation of proteins by Ser/Thr protein kinases (STPKs) has recently emerged as a major physiological mechanism of regulation in mycobacteria. FasR, is a key regulatory protein for maintaining lipid homeostasis in mycobacteria, as it modulates fatty acid availability by regulating the transcription of the fas gene. In this study, we investigated if phosphorylation of FasR might represent a strategy employed by M. tuberculosis to regulate fatty acid biosynthesis. In vitro phosphorylation experiments were performed using purified M. tuberculosis FasR, PknA and PknB. We show that FasR is efficiently phosphorylated in vitro by several mycobacterial STPKs, particularly by PknB. The identification of the main phosphoacceptors using mass spectrometry analyses will allow us to investigate if phosphorylation of FasR by PknB modulates its biochemical activity. On the other hand, the identification of two sRNAs in mycobacterium that may be involved in lipid biosynthesis were recently published. Mcr16 was found inside fabD gene, which encodes a crucial enzyme in the FasII system. Therefore we pretend to determine if fabD expression is regulated by Mcr16. We propose to analyze by Northern blot experiments the expression levels of these sRNAs in different conditions in M. smegmatis and M. bovis BCG and in strains expressing non physiological levels of the transcriptional regulator FasR. Finally we will construct M. smegmatis and M. bovis BCG overexpression strains for the sRNA Mcr16 to analyze it´s effect over mycolic acid synthesis. Our interest resides in the study of M. tuberculosis, the causative agent of human Tuberculosis, a reemerging disease that is affecting people worldwide, especially those co-infected with HIV. We believe that understanding the regulatory network involved in maintaining lipid homeostasis in M. tuberculosis will provide new tools to combat this disease.