Mycobacterial Phenolic Glycolipid Virulence Factor Biosynthesis: Mechanism and Small-Molecule Inhibition of Polyketide Chain Initiation

Ferreras, Julian A.; Stirrett, Karen; Lu, Xuequan; Ryu, Jae Sang; Soll, Clifford E.; Tan, Derek S.; Quadri, Luis

doi:10.1016/j.chembiol.2007.11.010

Cited by 76 publications

(80 citation statements)

References 52 publications

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“…8 Several FAAL functions have been characterized and shown to activate carboxylic acid substrates and load them onto cognate PKS proteins. For instance, FAAL22, which loads onto Pks15/1, was demonstrated to be essential for the synthesis of the virulence-conferring phenolic glycolipids (PGLs), 9 whose synthesis also involves FAAL29, which loads onto the polyketide synthases PpsA-E. 10 Similarly, FAAL26, which loads onto PpsA-E, and FAAL28, which loads onto Mas, are involved in the biosynthesis of the phthiocerol and mycocerosate, respectively, of the phthiocerol dimycocerosates (DIMs) virulence factor. Likewise, FAAL23, which loads onto Pks2, is implicated in the synthesis of sulfolipids, whereas FAAL33 (also called MbtM), which loads onto MbtL, is involved in mycobactin synthesis.…”

Section: Introductionmentioning

confidence: 99%

Assay Development for Identifying Inhibitors of the Mycobacterial FadD32 Activity

et al. 2013

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FadD32, a fatty acyl-AMP ligase (FAAL32) involved in the biosynthesis of mycolic acids, major and specific lipid components of the mycobacterial cell envelope, is essential for the survival of Mycobacterium tuberculosis, the causative agent of tuberculosis. The protein catalyzes the conversion of fatty acid to acyl-adenylate (acyl-AMP) in the presence of adenosine triphosphate and is conserved in all the mycobacterial species sequenced so far, thus representing a promising target for the development of novel antituberculous drugs. Here, we describe the optimization of the protein purification procedure and the development of a high-throughput screening assay for FadD32 activity. This spectrophotometric assay measuring the release of inorganic phosphate was optimized using the Mycobacterium smegmatis FadD32 as a surrogate enzyme. We describe the use of T m (melting temperature) shift assay, which measures the modulation of FadD32 thermal stability, as a tool for the identification of potential ligands and for validation of compounds as inhibitors. Screening of a selected library of compounds led to the identification of five novel classes of inhibitors.

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Section: Introductionmentioning

confidence: 99%

Assay Development for Identifying Inhibitors of the Mycobacterial FadD32 Activity

et al. 2013

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“…Nine unmarked in-frame deletion mbt mutants (M. smegmatis ⌬mbtX, where X is A, B, C, D, E, F, G, H, or T) were engineered by using the p2NIL/pGOAL19-based flexible-cassette method (32) as previously reported (3,10,30). A series of nine suicide vectors (p2NIL-GOALc-⌬mbtXc series) (see Table S1 in the supplemental material and below), each carrying an mbt gene-specific deletion cassette (⌬mbtXc), was used to generate the nine M. (3).…”

Section: Methodsmentioning

confidence: 99%

Mutational and Phylogenetic Analyses of the Mycobacterial mbt Gene Cluster

et al. 2011

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The mycobactin siderophore system is present in many Mycobacterium species, including M. tuberculosis and other clinically relevant mycobacteria. This siderophore system is believed to be utilized by both pathogenic and nonpathogenic mycobacteria for iron acquisition in both in vivo and ex vivo iron-limiting environments, respectively. Several M. tuberculosis genes located in a so-called mbt gene cluster have been predicted to be required for the biosynthesis of the core scaffold of mycobactin based on sequence analysis. A systematic and controlled mutational analysis probing the hypothesized essential nature of each of these genes for mycobactin production has been lacking. The degree of conservation of mbt gene cluster orthologs remains to be investigated as well. In this study, we sought to conclusively establish whether each of nine mbt genes was required for mycobactin production and to examine the conservation of gene clusters orthologous to the M. tuberculosis mbt gene cluster in other bacteria. We report a systematic mutational analysis of the mbt gene cluster ortholog found in Mycobacterium smegmatis. This mutational analysis demonstrates that eight of the nine mbt genes investigated are essential for mycobactin production. Our genome mining and phylogenetic analyses reveal the presence of orthologous mbt gene clusters in several bacterial species. These gene clusters display significant organizational differences originating from an intricate evolutionary path that might have included horizontal gene transfers. Altogether, the findings reported herein advance our understanding of the genetic requirements for the biosynthesis of an important mycobacterial secondary metabolite with relevance to virulence.The obligate human pathogen Mycobacterium tuberculosis, most opportunistic mycobacterial human pathogens (e.g., M. avium), and many nonpathogenic saprophytic mycobacteria (e.g., M. smegmatis) produce a structurally complex salicylic acid-derived siderophore known as mycobactin (MBT) (Fig. 1) (5, 33, 35). MBT has a core scaffold of a proposed nonribosomal peptide-polyketide origin consisting of a hydroxyphenylcapped (methyl)oxazoline moiety linked to an N ε -hydroxylysine residue, which is typically connected to a terminal cyclo-N ε -hydroxylysine by a 4-carbon linker. This core scaffold is decorated with a variable fatty acyl substituent on the N ε of the internal N ε -hydroxylysine residue. Structural variants (carboxymycobactins) with acyl substituents terminating in a carboxylate or a methyl ester are also produced. Interestingly, the core scaffold of MBT is remarkably similar to core scaffolds seen in several compounds-some with interesting pharmacological activities-produced by species of the genus Nocardia (Fig. 1). Nocardia is a saprophytic group of actinomycetes closely related to the mycobacteria and includes species that are increasingly recognized as opportunistic human pathogens (2, 25).Studies with cellular and animal models of mycobacterial infection have established the relevance of the M...

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“…The oligonucleotides used in this study are listed in Tables S3 and S4 in the supplemental material, and they Construction of mycobacterial mutants. The mutants were engineered using the p2NIL/pGOAL19-based flexible cassette method (47) as reported previously (34,35,48). A gene-specific mutagenesis cassette delivery vector (see below) was used to construct each mutant.…”

Section: Methodsmentioning

confidence: 99%

“…After incubation, the OD 595 of the cultures was measured in a plate reader (Beckman Coulter, Inc.) and the cells were harvested for apolar lipid fraction extraction with a biphasic mixture of methanolic saline and petroleum ether as reported previously (35,50). Lipid extracts were subjected to radiometric thin-layer chromatography (radio-TLC) for analysis of 14 C-labeled PGLs and 14 C-labeled PDIMs as described earlier (35,50). Developed TLC plates were exposed to phosphor screens, which were scanned using a Cyclone Plus Storage Phosphor System (PerkinElmer, Inc.).…”

Section: Methodsmentioning

confidence: 99%

“…Our previous studies of the PGL biosynthetic pathway have revealed a functional cooperation between the M. marinum proteins FadD22 (p-hydroxybenzoate-AMP ligase/initiation module) and Pks15/1 (iterative type I polyketide synthase [PKS]) for production of p-hydroxyphenylalkanoate (PHPA) intermediates required for PGL biosynthesis (35,36). The PHPA intermediates synthesized by the M. marinum FadD22-Pks15/1 iterative system, which is conserved in PGL producers, are thought to be further extended to form the phenolphthiocerol moiety of PGLs (Fig.…”

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Biosynthesis of Cell Envelope-Associated Phenolic Glycolipids in Mycobacterium marinum

et al. 2015

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dPhenolic glycolipids (PGLs) are polyketide synthase-derived glycolipids unique to pathogenic mycobacteria. PGLs are found in several clinically relevant species, including various Mycobacterium tuberculosis strains, Mycobacterium leprae, and several nontuberculous mycobacterial pathogens, such as M. marinum. Multiple lines of investigation implicate PGLs in virulence, thus underscoring the relevance of a deep understanding of PGL biosynthesis. We report mutational and biochemical studies that interrogate the mechanism by which PGL biosynthetic intermediates (p-hydroxyphenylalkanoates) synthesized by the iterative polyketide synthase Pks15/1 are transferred to the noniterative polyketide synthase PpsA for acyl chain extension in M. marinum. Our findings support a model in which the transfer of the intermediates is dependent on a p-hydroxyphenylalkanoyl-AMP ligase (FadD29) acting as an intermediary between the iterative and the noniterative synthase systems. Our results also establish the p-hydroxyphenylalkanoate extension ability of PpsA, the first-acting enzyme of a multisubunit noniterative polyketide synthase system. Notably, this noniterative system is also loaded with fatty acids by a specific fatty acyl-AMP ligase (FadD26) for biosynthesis of phthiocerol dimycocerosates (PDIMs), which are nonglycosylated lipids structurally related to PGLs. To our knowledge, the partially overlapping PGL and PDIM biosynthetic pathways provide the first example of two distinct, pathwaydedicated acyl-AMP ligases loading the same type I polyketide synthase system with two alternate starter units to produce two structurally different families of metabolites. The studies reported here advance our understanding of the biosynthesis of an important group of mycobacterial glycolipids. Mycobacterial infections are responsible for devastating morbidity and mortality worldwide (1-5). A critical player in the ability of the mycobacteria to produce disease is a formidable cell envelope believed to be responsible for the intrinsic resilience of the mycobacteria to inhospitable environments, antimicrobial agents, and host immune defenses (6-12). Among the unique components found in the cell envelope of several pathogenic mycobacteria are two structurally related families of glycosylated and nonglycosylated lipids commonly referred to as phenolic glycolipids (PGLs) and phthiocerol dimycocerosates (PDIMs), respectively (for a review, see reference 13). PGLs and PDIMs have unusual lipid scaffolds consisting of ␤-diol-containing, long-chain, aliphatic polyketides esterified with long-chain, multimethylbranched fatty acids onto the diol functionality (Fig. 1). PGLs and PDIMs are found in several mycobacterial pathogens (e.g., Mycobacterium tuberculosis strains, Mycobacterium bovis, Mycobacterium leprae, and Mycobacterium marinum) and thought to be constituents of the characteristic mycobacterial outer membrane. Multiple lines of investigation have provided considerable support for the idea that PGLs and PDIMs are implicated in virulence via co...

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Mycobacterial Phenolic Glycolipid Virulence Factor Biosynthesis: Mechanism and Small-Molecule Inhibition of Polyketide Chain Initiation

Cited by 76 publications

References 52 publications

Assay Development for Identifying Inhibitors of the Mycobacterial FadD32 Activity

Assay Development for Identifying Inhibitors of the Mycobacterial FadD32 Activity

Mutational and Phylogenetic Analyses of the Mycobacterial mbt Gene Cluster

Biosynthesis of Cell Envelope-Associated Phenolic Glycolipids in Mycobacterium marinum

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