Biosynthesis and Roles of Virulence Conferring Cell Wall Associated Dimycocerosate Esters in \u3cem\u3eMycobacterium marinum\u3c/em\u3e

Mycobacterial species include a variety of obligate and opportunistic pathogens that cause several important diseases affecting mankind such as tuberculosis and leprosy. The most unique feature of these bacteria is their intricate cell wall that poses a permeability barrier to antibiotics and contributes to their pathogenicity and persistence within the host. The cell wall hosts several complex lipids such as dimycocerosate esters (DIMs), which are found in many clinically relevant pathogenic species of mycobacteria. DIMs have been implicated in the virulence of mycobacteria and play a major role in helping the bacteria evade host immune responses. It is therefore crucial to define the biosynthesis and role of DIMs in mycobacteria, to better understand these organisms and identify new drug target candidates. DIMs consist of two structurally related groups: phthiocerol dimycocerosates (PDIMs) and phenolic glycolipids (PGLs). PDIMs and PGLs share part of a biosynthetic pathway that consists of two enzyme families: polyketide synthases (PKSs) and fatty acyl AMP ligases (FAALs). This dissertation has investigated the roles of PKSs and FAALs during PGL biosynthesis in the pathogenic nontuberculous mycobacterium; Mycobacterium marinum. More specifically, it is focused on mutational studies that probed the mechanism by which intermediates synthesized by an iterative PKS, Pks15/1 are transferred to a non-iterative PKS, PpsA during PGL biosynthesis. Our findings specified the role of the loading acyl carrier protein domain of PpsA, in the capture of intermediates from Pks15/1 during PGL biosynthesis. We also provided the first evidence supporting a model in which the transfer of intermediates during PGL biosynthesis is dependent on a novel FAAL enzyme (FadD29) that acts as an intermediary between Pks15/1 and PpsA, within a nontuberculous mycobacterial species. This dissertation has also explored the hypothesis that different gene knockouts that render the same PDIM and/or PGL deficiency phenotypes lead to strains with equivalent pleiotropic profiles. The availability of six M. marinum mutants, each with a different gene knockout in the PDIM/PGL biosynthetic pathway, provided an opportunity to probe for the pleiotropic consequences of gene knockouts leading to PDIMˉ PGLˉ, PDIM+ PGLˉ, or PDIMˉ PGL+ phenotypes. We evaluated the mutants for changes in cell surface properties, cell envelope permeability, antimicrobial drug susceptibility, biofilm formation virulence in an amoeba model system, sliding motility and in vitro growth assays. Our results revealed that the pleiotropic patterns emerging from the different gene knockouts lead to: altered cell surface properties, weakened cell envelope permeability barrier, increased antibiotic susceptibility, reduced biofilm formation and different attenuation levels in an amoeba model. No notable differences were observed in sliding motility and in vitro growth of the different mutants. Our findings also advocate that, different enzymes of the pathway whose elimination equally leads to PDIM and PGL deficiency might not be equivalent drug target candidates

Production of mycobacterial cell wall glycopeptidolipids requires a member of the MbtH-like protein family

Background Glycopeptidolipids (GPLs) are among the major free glycolipid components of the outer membrane of several saprophytic and clinically-relevant Mycobacterium species. The architecture of GPLs is based on a constant tripeptide-amino alcohol core of nonribosomal peptide synthetase origin that is N-acylated with a 3-hydroxy/methoxy acyl chain synthesized by a polyketide synthase and further decorated with variable glycosylation patterns built from methylated and acetylated sugars. GPLs have been implicated in many aspects of mycobacterial biology, thus highlighting the significance of gaining an understanding of their biosynthesis. Our bioinformatics analysis revealed that every GPL biosynthetic gene cluster known to date contains a gene (referred herein to as gplH) encoding a member of the MbtH-like protein family. Herein, we sought to conclusively establish whether gplH was required for GPL production. Results Deletion of gplH, a gene clustered with nonribosomal peptide synthetase-encoding genes in the GPL biosynthetic gene cluster of Mycobacterium smegmatis, produced a GPL deficient mutant. Transformation of this mutant with a plasmid expressing gplH restored GPL production. Complementation was also achieved by plasmid-based constitutive expression of mbtH, a paralog of gplH found in the biosynthetic gene cluster for production of the siderophore mycobactin of M. smegmatis. Further characterization of the gplH mutant indicated that it also displayed atypical colony morphology, lack of sliding motility, altered capacity for biofilm formation, and increased drug susceptibility. Conclusions Herein, we provide evidence formally establishing that gplH is essential for GPL production in M. smegmatis. Inactivation of gplH also leads to a pleiotropic phenotype likely to arise from alterations in the cell envelope due to the lack of GPLs. While genes encoding MbtH-like proteins have been shown to be needed for production of siderophores and antibiotics, our study presents the first case of one such gene proven to be required for production of a cell wall component. Furthermore, our results provide the first example of a mbtH-like gene with confirmed functional role in a member of the Mycobacterium genus. Altogether, our findings demonstrate a critical role of gplH in mycobacterial biology and advance our understanding of the genetic requirements for the biosynthesis of an important group of constituents of the mycobacterial outer membrane

<i>Mycobacterium abscessus</i> 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