Worldwide Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is the second leading infectious killer and multidrug resistance severely hampers disease control. Additionally, the number of diseases" cases caused by other nontuberculous mycobacteria is alarmingly increasing. Thanks to the nontuberculous and nonpathogenic effects, mycobacteria such as Mycobacterium smegmatis (Msm), can be used routinely and easily in the laboratory.

Mycolic acids (MA) are a unique category of lipids that are essential for viability, virulence, and persistence of mycobacteria. They are synthetized by enzymes of the the fatty acid synthase type II system (FAS-II), which thus represent an important class of drug targets.

In order to identify new therapeutic targets, we used a combination of affinity purification experiments and proteomics analyses to explore the composition and organization of the FasII system in mycobacteria. We expressed tagged version of HadA, member of the Had family of (3R)-hydroxyacyl-ACP dehydratase proteins, taking part in the FAS-II system from either Msm or Mtb, purified the associated complexes, and characterized them by mass spectrometry analysis.

We discovered a mycobacterial protein, HadD, displaying highly specific interaction with the dehydratases HadA and HadB of FAS-II, and investigated its function in Msm and Mtb.

In Msm, a hadD knockout mutant produced only the medium-size alpha"-MAs. Data strongly suggest that HadD is involved in building the third meromycolic segment during the late FAS-II elongation cycles, leading to the synthesis of the full-size alpha- and epoxy-MAs. The change in the envelope composition induced by hadD inactivation strongly alters the bacterial fitness and capacities to aggregate and to assemble into colonies or biofilms.

In Mtb, we showed that the HadD protein is dedicated mainly to the production of keto-mycolic acids and plays a determinant role in Mtb biofilm formation and virulence. We discovered that HAD activity requires the formation of a tight heterotetramer between HadD and HadB. Using biochemical, structural, and cell-based analyses, we showed that HadB is the catalytic subunit, whereas HadD is involved in substrate binding. Our study shows that the HadB-HadD, and not HadD alone, is the biologically relevant functional unit.

These results suggest that the target to consider is not an isolated subunit, but the whole HadB-HadD complex, either in Mtb or Msm, and have important implications for designing innovative antivirulence molecules to fight tuberculosis as well as other pathologies caused by mycobacteria.