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

Cyril Lefebvre,Anne Lemassu,Patricia Constant,Françoise Lauzeral-Vizcaino,Annaïk Quémard,Roland Brosch,Tanya Parish,Nathalie Eynard,Mamadou Daffé,Nawel Slama,Wafa Frigui

doi:10.1038/s41598-020-58967-8

Abstract

The fatty acid synthase type II (FAS-II) multienzyme system builds the main chain of mycolic acids (MAs), important lipid pathogenicity factors of Mycobacterium tuberculosis (Mtb). Due to their original structure, the identification of the (3 R)-hydroxyacyl-ACP dehydratases, HadAB and HadBC, of Mtb FAS-II complex required in-depth work. Here, we report the discovery of a third dehydratase protein, HadDMtb (Rv0504c), whose gene is non-essential and sits upstream of cmaA2 encoding a cyclopropane synthase dedicated to keto- and methoxy-MAs. HadDMtb deletion triggered a marked change in Mtb keto-MA content and size distribution, deeply impacting the production of full-size molecules. Furthermore, abnormal MAs, likely generated from 3-hydroxylated intermediates, accumulated. These data strongly suggest that HadDMtb catalyzes the 3-hydroxyacyl dehydratation step of late FAS-II elongation cycles during keto-MA biosynthesis. Phenotyping of Mtb hadD deletion mutant revealed the influence of HadDMtb on the planktonic growth, colony morphology and biofilm structuration, as well as on low temperature tolerance. Importantly, HadDMtb has a strong impact on Mtb virulence in the mouse model of infection. The effects of the lack of HadDMtb observed both in vitro and in vivo designate this protein as a bona fide target for the development of novel anti-TB intervention strategies.

Highlights

Tuberculosis (TB) is the top infectious killer worldwide[1]
The present study reports the existence in Mycobacterium tuberculosis (Mtb) of a putative ortholog, which we named HadDMtb, of the recently discovered (3R)-hydroxyacyl-acyl carrier protein (ACP) dehydratase HadDMsm of the fatty acid synthase type II (FAS-II) system from M. smegmatis[16]
The in-depth analysis of the distribution and fine structure of the mycolic acids (MAs) produced by Mtb hadD deletion mutant revealed that HadDMtb is involved in the oxygenated MA biosynthesis, and more dedicated to the keto-MA pathway

Summary

Introduction

Tuberculosis (TB) is the top infectious killer worldwide[1]. The control of this disease has been challenged by the emergence of multidrug and extensively drug-resistant Mycobacterium tuberculosis (Mtb) strains. The mycolic acid (MA)-containing lipids, which play a strategic role in the envelope architecture and permeability, are essential to the mycobacterial survival and constitute important pathogenicity factors As a consequence, their biosynthesis pathway represents one of the Achilles’ heels of the tubercle bacillus. MAs, extremely long-chain α-alkylated β-hydroxylated fatty acids (FAs), are the major components of a highly efficient permeability barrier, the mycobacterial outer membrane (called mycomembrane)[5], where they are covalently linked to the arabinogalactan layer or to polyol molecules such as trehalose[6] Their production requires three distinct multienzyme systems, including the acyl carrier protein (ACP)-dependent fatty acid www.nature.com/scientificreports synthase type II (FAS-II) that is responsible for the synthesis of their main chain called ‘meromycolic chain’[6]. Given the importance of HadDMsm for M. smegmatis physiology, we generated a Rv0504c knock-out Mtb mutant and examined the impact of Rv0504c inactivation on different physiological properties of Mtb in axenic cultures as well as on its virulence in the mouse model of infection

Methods

Results

Conclusion