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

Cyril Lefebvre,Mamadou Daffé,Stevie Jamet,Marie-Pierre Bousquet,Fabienne Bardou,Richard Boulon,Odile Burlet-Schiltz,Anne Lemassu,Sabine Gavalda,Kaymeuang Cam,Manuelle Ducoux,Françoise Laval,Nathalie Eynard,Annaïk Quémard

doi:10.1038/s41598-018-24380-5

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.

Highlights

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
With the aim of discovering novel partner proteins of the fatty acid synthase type II (FAS-II) system putatively involved in double bond formation, pull-down experiments were performed from M. smegmatis bacterial lysates using HadA as a bait
HadD is ubiquitous among mycobacteria but, unlike most fatty acid synthase (FAS)-II enzymes and HadAB dehydratase, has no ortholog in the other mycolic acid-producing genera

Summary

Introduction

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. 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. The MA-containing compounds are essential to the survival of mycobacteria and crucial for their physiology and fitness[7] They modulate the immune response to infection by pathogenic mycobacteria, thereby playing a role in their virulence and their persistence within the host[7,13,14]. With the aim of defining the role of HadD, its in vitro enzymatic activity, its structure and the impacts of its depletion on both the MA production and the physiology of mycobacteria were assessed

Methods

Results

Conclusion