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.
Highlights
Erols and phthiocerols) and polyketide synthase-derived multimethyl-branched fatty acids (Fig. 1)
Our analysis reported conclusively demonstrates that deletion of tesA leads to a drastic reduction in Phthiocerol dimycocerosates (PDIMs) and phenolic glycolipids (PGLs) production and suggests that drugs aimed at blocking PDIM and/or PGL production might synergize with antibiotic therapy in the control of mycobacterial infections
The TesA Orthologs Are Highly Conserved—We previously reported an analysis of the organization of the genes in the chromosomal locus involved in PDIM and PGL production of several species [1], yet the sequence similarity of TesA orthologs, each encoded at the 5Ј-end of its cognate PDIM/PGL biosynthetic gene cluster, has not been highlighted
Summary
These observations have led to the hypothesis that TesA is a thioesterase required for release of polyketide products from PpsE [24]. Two Mtb tesA mutants, each with a transposon-based disruption of tesA, have been reported to be PDIM-deficient [25, 26]. The possibility of polar effect on genes located downstream of tesA and needed for PDIM production has not been ruled out in either of these insertional mutants, precluding a conclusive determination as to the requirement of tesA for PDIM production. Mtb is known to spontaneously and irreversibly lose its PDIM production capacity with high frequency during propagation in vitro [27, 28], and the possibility that the PDIM deficiency of the transposon mutants is due to spontaneous loss cannot be conclusively dismissed either. Our analysis reported conclusively demonstrates that deletion of tesA leads to a drastic reduction in PDIM and PGL production and suggests that drugs aimed at blocking PDIM and/or PGL production might synergize with antibiotic therapy in the control of mycobacterial infections
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