HadD, a novel fatty acid synthase type II protein, is essential for alpha- and epoxy-mycolic acid biosynthesis and mycobacterial fitness

Abstract

Mycolic acids (MAs) have a strategic location within the mycobacterial envelope, deeply influencing its architecture and permeability, and play a determinant role in the pathogenicity of mycobacteria. The fatty acid synthase type II (FAS-II) multienzyme system is involved in their biosynthesis. A combination of pull-downs and proteomics analyses led to the discovery of a mycobacterial protein, HadD, displaying highly specific interactions with the dehydratase HadAB of FAS-II. In vitro activity assays and homology modeling showed that HadD is, like HadAB, a hot dog folded (R)-specific hydratase/dehydratase. A hadD knockout mutant of Mycobacterium smegmatis 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 altered the bacterial fitness and capacities to aggregate, assemble into colonies or biofilms and spread by sliding motility, and conferred a hypersensitivity to the firstline antimycobacterial drug rifampicin. This showed that the cell surface properties and the envelope integrity were greatly affected. With the alarmingly increasing case number of nontuberculous mycobacterial diseases, HadD appears as an attractive target for drug development.

Discovery of a novel dehydratase of the fatty acid synthase type II critical for ketomycolic acid biosynthesis and virulence of Mycobacterium tuberculosis

“…1C). The viability of the resulting mutant strain showed that hadD Mtb is not essential for the survival of Mtb in axenic culture, similar to the situation seen for hadD Msm in M. smegmatis 16 . This is consistent with the prediction of non-essentiality of hadD Mtb made from a microarray-based study 20 , but in discrepancy with a more recent survey using high-resolution phenotypic profiling where hadD Mtb was predicted to be essential 21 .…”

“…In a recent survey, a combination of pull-downs, using the dehydratase HadAB of FAS-II as a bait, and proteomic analyses led to the discovery of a novel (R)-specific dehydratase, HadD Msm , of the FAS-II system from M. smegmatis, a non-tuberculous mycobacterium 16 . HadD displays highly specific interactions with HadAB.…”

“…HadD displays highly specific interactions with HadAB. Bioinformatic analyses showed that, unlike HadAB, there is no HadD Msm ortholog in the genera of the Corynebacteriales order, such as Nocardia, Rhodococcus, and Gordonia, that produce medium-chain mycolic acids 16 . This suggested that HadD Msm might have a role during the late FAS-II elongation cycles.…”

“…Thus, HadD Msm is most likely involved in building the third meromycolic segment leading to the synthesis of the full-size αand epoxy-MAs. Importantly, hadD Msm inactivation induced an upheaval of both the bacterial cell surface and envelope properties of M. smegmatis, strongly altering the bacterial fitness and capacities to aggregate, assemble into colonies or biofilms and spread by sliding motility, critical for mycobacterial survival under stress and hostile conditions and for colonization 16 . It also conferred a hypersensitivity to the first line TB drug rifampicin.…”

“…Interestingly, a putative ortholog of HadD Msm was found in all of the sequenced mycobacterial genomes 16 , including those of the pathogen Mtb. Yet, Mtb produces a MA combination distinct from that of M. smegmatis, i.e.…”