Mycobacterium abscessus Mutants with a Compromised Functional Link between the Type VII ESX-3 System and an Iron Uptake Mechanism Reliant on an Unusual Mycobactin Siderophore.

The opportunistic pathogen Mycobacterium abscessus subsp. abscessus (Mab) has become an emerging public health threat due to the increasing number of Mab-associated chronic pulmonary disease cases. Treatment requires multiple drug courses and is often combined with surgical resection. Cure rates are only ~50% due to treatment failure and comorbidities. Deeper understanding of the biology of Mab is required to illuminate potential avenues for the development of better therapeutics against Mab infections. The ESX-3 type VII protein secretion system of Mab has an important role in host inflammatory and pathological responses during infection. In this work, we demonstrate a functional link between ESX-3 and an iron uptake system based on an unusual mycobactin-type siderophore (designated MBT Ab) and exploit this link to implement a large screen for transposon mutants with an impaired ESX-3. Most mutants we identified carry insertions in genes encoding predicted ESX-3 secretion machinery components or potential ESX-3 substrates. The mutants overproduce MBT Ab, a trait consistent with an iron uptake defect. Our characterization of MBT Ab revealed structural features reminiscent of nocardial mycobactin-like compounds with cytotoxicity. This finding raises the possibility that MBT Ab may play roles in pathogenesis unlinked to iron homeostasis. The mutants generated herein will facilitate research to better understand the role of ESX-3 and its interplay with the siderophore system.

Kinetic Analyses of the Siderophore Biosynthesis Inhibitor Salicyl-AMS and Analogues as MbtA Inhibitors and Antimycobacterial Agents.

There is a paramount need for expanding the drug armamentarium to counter the growing problem of drug-resistant tuberculosis. Salicyl-AMS, an inhibitor of salicylic acid adenylation enzymes, is a first-in-class antibacterial lead compound for the development of tuberculosis drugs targeting the biosynthesis of salicylic-acid-derived siderophores. In this study, we determined the Ki of salicyl-AMS for inhibition of the salicylic acid adenylation enzyme MbtA from Mycobacterium tuberculosis (MbtAtb), designed and synthesized two new salicyl-AMS analogues to probe structure-activity relationships (SAR), and characterized these two analogues alongside salicyl-AMS and six previously reported analogues in biochemical and cell-based studies. The biochemical studies included determination of kinetic parameters ( Kiapp, konapp, koff, and tR) and analysis of the mechanism of inhibition. For these studies, we optimized production and purification of recombinant MbtAtb, for which Km and kcat values were determined, and used the enzyme in conjunction with an MbtAtb-optimized, continuous, spectrophotometric assay for MbtA activity and inhibition. The cell-based studies provided an assessment of the antimycobacterial activity and postantibiotic effect of the nine MbtAtb inhibitors. The antimycobacterial properties were evaluated using a strain of nonpathogenic, fast-growing Mycobacterium smegmatis that was genetically engineered for MbtAtb-dependent susceptibility to MbtA inhibitors. This convenient model system greatly facilitated the cell-based studies by bypassing the methodological complexities associated with the use of pathogenic, slow-growing M. tuberculosis. Collectively, these studies provide new information on the mechanism of inhibition of MbtAtb by salicyl-AMS and eight analogues, afford new SAR insights for these inhibitors, and highlight several suitable candidates for future preclinical evaluation.

Mycobacteria Encode Active and Inactive Classes of TesB Fatty-Acyl CoA Thioesterases Revealed through Structural and Functional Analysis.

Mycobacteria contain a large number of highly divergent species and exhibit unusual lipid metabolism profiles, believed to play important roles in immune invasion. Thioesterases modulate lipid metabolism through the hydrolysis of activated fatty-acyl CoAs; multiple copies are present in mycobacteria, yet many remain uncharacterized. Here, we undertake a comprehensive structural and functional analysis of a TesB thioesterase from Mycobacterium avium (MaTesB). Structural superposition with other TesB thioesterases reveals that the Asp active site residue, highly conserved across a wide range of TesB thioesterases, is mutated to Ala. Consistent with these structural data, the wild-type enzyme failed to hydrolyze an extensive range of acyl-CoA substrates. Mutation of this residue to an active Asp residue restored activity against a range of medium-chain length fatty-acyl CoA substrates. Interestingly, this Ala mutation is highly conserved across a wide range of Mycobacterium species but not found in any other bacteria or organism. Our structural homology analysis revealed that at least one other TesB acyl-CoA thioesterase also contains an Ala residue at the active site, while two other Mycobacterium TesB thioesterases harbor an Asp residue at the active site. The inactive TesBs display a common quaternary structure that is distinct from that of the active TesB thioesterases. Investigation of the effect of expression of either the catalytically active or inactive MaTesB in Mycobacterium smegmatis exposed, to the best of our knowledge, the first genotype-phenotype association implicating a mycobacterial tesB gene. This is the first report that mycobacteria encode active and inactive forms of thioesterases, the latter of which appear to be unique to mycobacteria.

Design, synthesis, and biological evaluation of α-hydroxyacyl-AMS inhibitors of amino acid adenylation enzymes.

Biosynthesis of bacterial natural-product virulence factors is emerging as a promising antibiotic target. Many such natural products are produced by nonribosomal peptide synthetases (NRPS) from amino acid precursors. To develop selective inhibitors of these pathways, we have previously described aminoacyl-AMS (sulfamoyladenosine) macrocycles that inhibit NRPS amino acid adenylation domains but not mechanistically-related aminoacyl-tRNA synthetases. To improve the cell permeability of these inhibitors, we explore herein replacement of the α-amino group with an α-hydroxy group. In both macrocycles and corresponding linear congeners, this leads to decreased biochemical inhibition of the cysteine adenylation domain of the Yersina pestis siderophore synthetase HMWP2, which we attribute to loss of an electrostatic interaction with a conserved active-site aspartate. However, inhibitory activity can be regained by installing a cognate ß-thiol moiety in the linear series. This provides a path forward to develop selective, cell-penetrant inhibitors of the biosynthesis of virulence factors to probe their biological functions and potential as therapeutic targets.

Pleiotropic consequences of gene knockouts in the phthiocerol dimycocerosate and phenolic glycolipid biosynthetic gene cluster of the opportunistic human pathogen Mycobacterium marinum.

Phthiocerol dimycocerosates (PDIMs) and phenolic glycolipids (PGLs) contribute to the pathogenicity of several mycobacteria. Biosynthesis of these virulence factors requires polyketide synthases and other enzymes that represent potential targets for the development of adjuvant antivirulence drugs. We used six isogenic Mycobacterium marinum mutants, each with a different gene knockout in the PDIM/PGL biosynthetic pathway, to probe the pleiotropy of mutations leading to PDIM(-) PGL(-), PDIM(+) PGL(-) or PDIM(-) PGL(+) phenotypes. We evaluated the M. marinum mutants for changes in antibiotic susceptibility, cell envelope permeability, biofilm formation, surface properties, sliding motility and virulence in an amoeba model. The analysis also permitted us to begin exploring the hypothesis that different gene knockouts rendering the same PDIM and/or PGL deficiency phenotypes lead to M. marinum mutants with equivalent pleiotropic profiles. Overall, the results of our study revealed a complex picture of pleiotropic patterns emerging from different gene knockouts, uncovered unexpected phenotypic inequalities between mutants, and provided new insight into the phenotypic consequences of gene knockouts in the PDIM/PGL biosynthetic pathway.