CONSTRUCTION OF A BIOLUMINESCENT REPORTER STRAIN FOR SCREENING AND IDENTIFICATION OF NOVEL INHIBITORS OF FASR, A TRANSCRIPTIONAL REGULATOR ESSENTIAL FOR VIRULENCE OF Mycobacterium tuberculosis.

DESCHUTTER, S.; COLACCINI, F; GRAMAJO, H; GAGO, G.

Congreso; XVII SAMIGE Meeting; 2022

SAMIGE

Mycobacterium tuberculosis (Mtb), the causal agent of tuberculosis (TB) in humans, has a very complex lifestyle. The flexibility in its metabolism allows it to adapt and survive in the infected host. During this process, lipid metabolism is affected. Despite there is a lot of information about the biosynthesis, structure and biological function of the main lipids present in Mtb envelope, little is known about the mechanisms that allow the bacteria to modulate and adapt the biosynthesis of the components of the cell wall in response to changes in environment. Thus, the study of the processes involved in the regulation of the biosynthesis of lipids in Mtb represents a crucial step in the comprehension of the physiology of this pathogen, as well as to find potential drug targets and contribute to combat TB. The biosynthesis of fatty acids in Mtb involves two different systems of fatty acid synthases (FAS I and FAS II). Both synthases are involved in the biosynthesis of membrane fatty acids and several lipid components of the cell wall, like mycolic acids (essential for viability and pathogenesis). The multi domain single protein FAS I catalyses de novo biosynthesis of acyl-CoAs, which are used as primers by the FAS II multiprotein system for the synthesis of mycolic acids. FasR is a TetR-like transcriptional regulator that plays a key role in this process. FasR activates transcription of the fas-acpS operon by binding to the fas (Pfas) promoter in Mtb. The fas gene encodes the FAS I protein and the acpS gene is essential to produce functional ACP. We have previously shown that regulation of lipid biosynthesis mediated by FasR is critical for macrophage infection and essential for virulence in vivo using a mouse model of infection, suggesting that FasR would constitute an interesting drug target to identify antimycobacterial molecules with novel mechanisms of action. In this work, a bioluminescent reporter strain was designed in order to search for compounds capable of inhibiting FasR activity. For this, we constructed the plasmid pSD2, that carries the fas gene promoter region (Pfas) controlling the expression of the lux operon (luxCDABE). The construction of the reporter strain was carried out by electroporating the pSD2 integrative plasmid into Mtb H37Ra. In this strain, the expression of the lux operon is activated by FasR. We found that bioluminescence correlates with optical density (OD) in exponential phase growth and decreases in the presence of exogenously added fatty acids. We are currently performing assays in the presence of compound libraries available in our laboratory. We expect that those compounds that inhibit FasR activity will show reduced bioluminescence and will be further validated by electrophoretic mobility shift assays (EMSA). Thus, the high throughput screening of FasR inhibitors using our bioluminescent reporter strain will allow further validation of FasR as a new drug target for the design of new antimycobacterial agents.