A conditional mutant of the fatty acid synthase unveils unexpected cross talks in mycobacterial lipid metabolism

Abstract

Unlike most bacteria, mycobacteria rely on the multi-domain enzyme eukaryote-like fatty acid synthase I (FAS I) to make fatty acids de novo. These metabolites are precursors of the biosynthesis of most of the lipids present both in the complex mycobacteria cell wall and in the storage lipids inside the cell. In order to study the role of the type I FAS system in Mycobacterium lipid metabolism in vivo, we constructed a conditional mutant in the fas-acpS operon of Mycobacterium smegmatis and analysed in detail the impact of reduced de novo fatty acid biosynthesis on the global architecture of the cell envelope. As expected, the mutant exhibited growth defect in the non-permissive condition that correlated well with the lower expression of fas-acpS and the concomitant reduction of FAS I, confirming that FAS I is essential for survival. The reduction observed in FAS I provoked an accumulation of its substrates, acetyl-CoA and malonyl-CoA, and a strong reduction of C12 to C18 acyl-CoAs, but not of long-chain acyl-CoAs (C19 to C24). The most intriguing result was the ability of the mutant to keep synthesizing mycolic acids when fatty acid biosynthesis was impaired. A detailed comparative lipidomic analysis showed that although reduced FAS I levels had a strong impact on fatty acid and phospholipid biosynthesis, mycolic acids were still being synthesized in the mutant, although with a different relative species distribution. However, when triacylglycerol degradation was inhibited, mycolic acid biosynthesis was significantly reduced, suggesting that storage lipids could be an intracellular reservoir of fatty acids for the biosynthesis of complex lipids in mycobacteria. Understanding the interaction between FAS I and the metabolic pathways that rely on FAS I products is a key step to better understand how lipid homeostasis is regulated in this microorganism and how this regulation could play a role during infection in pathogenic mycobacteria.

Complete auxotrophy for unsaturated fatty acids requires deletion of two sets of genes in Mycobacterium smegmatis

“…Given these results, further work to address the role of MSMEG_1741‐1743 in NTM is warranted. However, a note of caution is given by a recent publication describing that a M. smegmatis fasI‐acpS conditional mutant – thus incapable of making fatty acids – was able to make mycolic acids possibly at the expense of triglycerides (Cabruja et al ., ); this is a humbling demonstration that mycobacterial fatty acid synthesis and regulation is yet to be deciphered in full and that will require the identification and exhaustive studies of the players involved. Our report provides a working frame for the identification of the regulatory mechanisms underlying the expression of the M. smegmatis des BCT operon and its links to other parts of the mycobacterial fatty acid metabolism.…”

FasR regulates fatty acid biosynthesis and is essential for virulence ofMycobacterium tuberculosis

“…The expression of fas, fasR and acpS was quantified after normalization of RNA levels to the expression of the sigA gene as previously described (Cabruja et al, 2017). qPCR data are presented as relative expression values normalized to sigA and to the wild type strain H37Rv.…”

“…Cell-associated lipid for lipidomic analyses were prepared as previously described (Cabruja et al, 2017). Briefly, cell pellets of cultures grown at 37 ºC in Middlebrook 7H9 broth (DIFCO) supplemented with 0.2 % glycerol, 0.03 % Tyloxapol and 10 % OADC were washed with 50 mM ammonium acetate, pH 7.8 and transferred to glass tubes containing 5 ml CHCl 3 :CH 3 OH (2:1, v/v).…”

“…Previous characterization of M. smegmatis fasR (Mondino et al, 2013) and fas (Cabruja et al, 2017) conditional mutant strains revealed that reduced FAS I levels had a strong impact on fatty acid and phospholipid biosynthesis. Interestingly, MA remained being synthesized in the FAS I mutant for several hours after addition of ATc (non-permissive conditions), although with different abundance of the various species (Cabruja et al, 2017). Considering the relevance that lipids have in M. tuberculosis survival and virulence, the impact of the absence of FasR on the global lipid profile of M. tuberculosis was further studied.…”

“…We performed comparative lipidomic analyses between MtbH37Rv, MtbΔfasR and the complemented strain. Briefly, we extracted cell-associated lipids as previously described (Cabruja et al, 2017) and analyzed the lipid content by high-performance liquid chromatography-mass spectrometry with data-dependent tandem MS (HPLC-MS/MS).…”