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

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, HadD Mtb (Rv0504c), whose gene is non-essential and sits upstream of cmaA2 encoding a cyclopropane synthase dedicated to keto-and methoxy-MAs. HadD Mtb 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 HadD Mtb 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 HadD Mtb on the planktonic growth, colony morphology and biofilm structuration, as well as on low temperature tolerance. importantly, HadD Mtb has a strong impact on Mtb virulence in the mouse model of infection. The effects of the lack of HadD Mtb observed both in vitro and in vivo designate this protein as a bona fide target for the development of novel anti-TB intervention strategies. 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. According to the WHO, the development of a new generation of drugs effective against these strains is urgently needed 1. The very thick lipid-rich envelope of the tubercle bacillus provides a vital protective coat against the attacks of the infected host. In particular, 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. It is the primary target of several anti-TB antibiotics, including the first line drug isoniazid 2,3. Furthermore, recently discovered small molecules, that are showing great promise as TB therapeutics, affect their metabolism 4. 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

1,3-Diarylpyrazolyl-acylsulfonamides Target HadAB/BC Complex in Mycobacterium tuberculosis

“…While most acyl sulfonamides ( 2 , 3 ) did not inhibit HadBC enzyme activity at 50 μM, acyl sulfonylureas ( 11 and 14 ) and oxadiazolone 12 showed moderate inhibition of HadBC at 50 μM (Table ). These results suggest that inhibition of HadAB rather than HadD likely accounts for the bactericidal activity of the compounds since HadD is nonessential . This is in agreement with a K157R mutation in HadC providing resistance to the compounds as it permits HadBC to functionally compensate for HadAB while being a lot less susceptible to the inhibition by these compounds …”

“…The HadD Mtb (Rv0504c) displayed a sequence identity of 100% with BCG_0547c (not shown); hence BCG_0547c will be referred to as HadD in the following text. HadD Mtb appears to catalyze the 3-hydroxyacyl dehydration step of late FAS-II elongation cycles during keto-MA biosynthesis . MSMEG_0948 (HadD) protein from M .…”

Pathogenomic analyses of Mycobacterium microti, an ESX-1-deleted member of the Mycobacterium tuberculosis complex causing disease in various hosts

“…microti strains (OV254, ATCC 35782, 94-2272 and Maus IV) relative to M. tuberculosis H37Rv and BCG Pasteur control strains, we used an infection assay of SCID mice, as this model provides a rapid and sensitive method for evaluating the in vivo growth characteristics of mycobacterial strains during the acute phase of infection [31, 87–90]. This approach showed that all four tested M.…”

Multi-Omics Technologies Applied to Tuberculosis Drug Discovery

“…Subsequently, the authors showed that hadD deletion in M.tb resulted in a 63% reduction in keto-mycolic acids, while overexpression of hadD induced an 87% increase in keto-mycolic acids compared to wild type. Knockout mutants of hadD were attenuated in a murine model of infection, confirming hadD as a new target for drug discovery [107].…”