Inactivation of tesA Reduces Cell Wall Lipid Production and Increases Drug Susceptibility in Mycobacteria

Abstract

Phthiocerol dimycocerosates (PDIMs) and phenolic glycolipids (PGLs) are structurally related lipids noncovalently bound to the outer cell wall layer of Mycobacterium tuberculosis, Mycobacterium leprae, and several opportunistic mycobacterial human pathogens. PDIMs and PGLs are important effectors of virulence. Elucidation of the biosynthesis of these complex lipids will not only expand our understanding of mycobacterial cell wall biosynthesis, but it may also illuminate potential routes to novel therapeutics against mycobacterial infections. We report the construction of an in-frame deletion mutant of tesA (encoding a type II thioesterase) in the opportunistic human pathogen Mycobacterium marinum and the characterization of this mutant and its corresponding complemented strain control in terms of PDIM and PGL production. The growth and antibiotic susceptibility of these strains were also probed and compared with the parental wild-type strain. We show that deletion of tesA leads to a mutant that produces only traces of PDIMs and PGLs, has a slight growth yield increase and displays a substantial hypersusceptibility to several antibiotics. We also provide a robust model for the three-dimensional structure of M. marinum TesA (TesAmm) and demonstrate that a Ser-to-Ala substitution in the predicted catalytic Ser of TesAmm renders a mutant that recapitulates the phenotype of the tesA deletion mutant. Overall, our studies demonstrate a critical role for tesA in mycobacterial biology, advance our understanding of the biosynthesis of an important group of polyketide synthase-derived mycobacterial lipids, and suggest that drugs aimed at blocking PDIM and/or PGL production might synergize with antibiotic therapy in the control of mycobacterial infections. Mycobacterium tuberculosis (Mtb),3 Mycobacterium leprae, and several opportunistic mycobacterial human pathogens (e.g. M. marinum (Mm)) produce two related groups of diesters of ␤-glycol-containing aliphatic polyketides (e.g. phenolphthiocerols and phthiocerols) and polyketide synthase-derived multimethyl-branched fatty acids (e.g. mycocerosic acids) (Fig. 1). One of these groups is represented by phthiocerol dimycocerosates (PDIMs). The other group is represented by phenolphthiocerol dimycocerosates, which are glycosylated compounds generally known as phenolic glycolipids (PGLs). These complex lipids, which are believed to be noncovalently bound constituents of the outer leaflet of the unique mycobacterial outer membrane, are known important effectors of virulence (for review, see Ref. 1).Cox et al. (3) and Camacho et al.(2) independently demonstrated in 1999 that loss of PDIMs in PGL-deficient Mtb strains correlates with attenuation in animal models of tuberculosis. It has also been documented that production of PGLs confers a hyperlethality phenotype to PDIM-producing Mtb in murine disease models (4). Since these seminal reports, an overwhelming body of evidence has accumulated demonstrating that PDIMs and PGLs play key roles in virulence and host-pathogen interaction ...

The mycobacterial acyltransferase PapA5 is required for biosynthesis of cell wall-associated phenolic glycolipids

“…[ 14 C]Propionate was added to each well at 0.2 mCi ml 21 (7.4 MBq ml 21 ) and the plates were incubated at 30 uC with rotary agitation (170 r.p.m.) for 24 h. After incubation, the OD 595 of the cultures was measured in a DTX Plate Reader (Beckman Coulter), and the cells were harvested for apolar lipid fraction extraction with a biphasic mixture of methanolic saline and petroleum ether, as reported previously (Chavadi et al, 2011b;Ferreras et al, 2008). The lipid extracts were subjected to radio-TLC for analysis of 14 C-labelled PGLs (using CHCl 3 /CH 3 OH, 95 : 5) and PDIMs (using petroleum ether/diethyl ether, 9 : 1) as described previously (Ferreras et al, 2008).…”

“…Thus, dissecting the molecular logic of PGL and PDIM biosynthesis not only will expand our general understanding of cell wall biosynthesis in mycobacteria of clinical significance, but also may reveal potential avenues for exploring the development of alternative therapeutics against selected mycobacterial infections (Ferreras et al, 2008;Quadri, 2007). These views have directed our previous studies of the biosynthesis of PGLs and PDIMs Chavadi et al, 2011b;Ferreras et al, 2008;He et al, 2009;Onwueme et al, 2004Onwueme et al, , 2005a, which include the development of the first PGL biosynthesis inhibitor (Ferreras et al, 2008). This inhibitor drastically reduces PGL production in several Mycobacterium species (Ferreras et al, 2008) using a mechanistic principle analogous to that of the first reported inhibitor of mycobacterial siderophore (iron chelator) production (Ferreras et al, 2005;Quadri, 2007).…”

“…Moreover, production of these compounds has been shown to correlate with decreased antimicrobial drug susceptibility (Alibaud et al, 2011;Chavadi et al, 2011b). PGL production has also been suggested as a trait that predisposes M. tuberculosis strains of the W-Beijing family to their characteristic epidemic spread and increased likelihood of developing drug resistance (Reed et al, Abbreviations: GPPOL, glycosyl-phenolphthiocerol; PDIM, phthiocerol dimycocerosate; PGL, phenolic glycolipid; SOE, splicing by overlap extension; WT, wild-type.…”

“…A suicide delivery vector (p2NIL-GOALc-DpapA5c, see below) carrying a papA5 (MMAR_1768) deletion cassette (DpapA5c) was used to generate M. marinum DpapA5. Electroporation of p2NIL-GOALc-DpapA5c into M. marinum and selection of potential single-and double-crossover mutants were conducted as previously reported (Chavadi et al, 2011b). The papA5 deletion in potential double-crossover mutants was screened for and confirmed by PCR using two independent primer pairs (papA5OF and papA5OR, papA5F and papA5R).…”

“…Bacterial culturing and recombinant DNA manipulations. M. marinum (strain M; ATCC BAA-535) was cultured at 30 uC in Middlebrook 7H9 medium (Difco) supplemented with 10 % ADN (5 % BSA, 2 % glucose, 0.85 % NaCl) (Difco) and 0.05 % Tween-80 (supplemented 7H9) or Middlebrook 7H11 (Difco) supplemented with ADN (supplemented 7H11) (Chavadi et al, 2011b). Escherichia coli DH5a (Invitrogen) was cultured in Luria-Bertani media under standard conditions (Sambrook et al, 1989).…”