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Biogenesis of the mycobacterial envelope and search for novel targets

Mycolic acid metabolism and Regulation

Although recent work has allowed drawing a more comprehensive scheme of both biosynthesis and export pathways of the mycolate-containing compounds, many important issues remain unresolved. How mycobacteria control and modulate the content and the fine structure of their MAs? What are the compositions and structures of the metabolic systems responsible for the production and degradation of MAs and MA-containing compounds? How this metabolism responds to environmental conditions and what regulatory mechanisms are involved? Elucidating these open questions is of paramount importance for understanding the intricate network of events governing the host–pathogen dialogue involving the MAs; they constitute the main aim of this topic.

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Figure 2. Model of mycolic acid biogenesis. A simplified model of MA biosynthesis : the very-long (C40-60) meromycoloyl chain produced by the fatty acid synthase (FAS)-II system are activated by FadD32 prior to their condensation with the C22-24 fatty acids produced by FAS-I catalyzed by Pks13 to yield MA chains linked to trehalose (Tre).

1. MA biosynthesis

MAs are the products of a mixed fatty acid synthase (FAS)/polyketide synthase (PKS) biosynthesis pathway (Fig. 2). The formation of the main very long 'meromycolic' (meroMA) chain of MAs is dependent upon an original Fatty Acid Synthase type II (FAS-II) elongation system, which contains two dehydratases, namely the HadAB and HadBC heterodimers we discovered a decade ago. Recently, within the ANR project FASMY, we developed a novel experimental strategy based on the affinity purification of protein complexes in tandem with proteomics analysis applied to mycobacteria, in collaboration with the team of O. Schiltz (IPBS). Thanks to this approach, we discovered a third dehydratase from the mycobacterial FAS-II, HadD, which physically interacts with HadAB and belongs to the same structural family. We showed that the three dehydratases are important for the physiology of mycobacteria (bacterial fitness, colony and biofilm formation, spreading, tolerance to stresses...). We also found that HadBC and HadD are specifically required for the late elongation cycles necessary to the biosynthesis of the longest MAs.

Another enzymatic machinery, the mycolic condensation system, catalyzes the ultimate stage of the MA biosynthesis pathway. Dependent upon the polyketide synthase Pks13, it condenses a meroMA chain with a long carboxylated acyl chain, generating the mycolic motif. Within an ECOS-Sud Cooperation program, we also brought evidences in collaboration with the team of H. Gramajo (CONICET, Rosario, Argentina) that a long-chain acyl-CoA carboxylase supercomplex (AccA3-AccD4-AccD5-AccE5), displaying a protein composition specific to mycobacteria, activates the fatty acyl chain to be condensed to the meroMAs. We have previously deciphered the sequential reaction steps catalyzed by Pks13 and the associated FadD32 enzyme that activates the meroMAs produced by FAS-II. Unlike the classical PKS-associated fatty acyl-AMP ligases (FAAL), FadD32 possesses an additional fatty acyl-ACP ligase function. Within a collaboration with the team of L. Mourey (IPBS), the structural bases of this unprecedented dual function were determined through crystallography analyses of FadD32 orthologs. In contrast to the commonly accepted biosynthesis model, we demonstrated that the thioesterase-like domain of Pks13 catalyzes itself the release of the neosynthesized condensation products and, in an original manner for a PKS thioesterase domain, their transfer onto trehalose, in collaboration with the teams of L. Mourey and C. Guilhot (IPBS). This work highlights a unique mechanism of transfer of PKS products in mycobacteria. Importantly, it allowed elucidation of a long-sought step of the MA-containing compound biosynthesis, i.e. the covalent binding of the MA chains to trehalose to form trehalose monomycolate (TMM), the precursor of a lipid pathogenicity factor, the trehalose dimycolate (TDM).


2. Regulatory mechanisms of mycolic acid metabolism

It is known that genes involved in MA biosynthesis in Mtu are downregulated in the macrophage infection model, and several transcription regulators of this pathway, FasR, MabR and FadR, have been identified. Accordingly, our group recently observed that nutrient scarcity, encountered during infection, leads to downregulation of the hadABC genes encoding FAS-II dehydratases along with most of the genes required for the synthesis and transport of mycolates. This adaptation phenomenon partly relies on the bacterial stringent response. Post-translational modifications and notably signaling through Ser/Thr phosphorylation have also recently emerged as a key regulatory mechanism in pathogenic mycobacteria. The activity of important or essential enzymes of MA synthesis pathway was found to be negatively regulated by phosphorylation. In particular, our group demonstrated that FadD32 activity is controlled by serine/threonine protein kinases (STPK), in collaboration with the teams of O. Schiltz (IPBS), L. Mourey (IPBS), P. Alzari (Institut Pasteur, Paris) and V. Molle (DIMN, Montpellier). Moreover, we demonstrated the impact of phosphorylation on other enzymes both involved in the synthesis and transport of MA.