Date of Degree

6-2016

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biology

Advisor(s)

Luis E. N. Quadri

Committee Members

Peter Lipke

James Nishiura

Derrick Brazill

G. Marcela Rodriguez

Subject Categories

Bacteriology | Life Sciences | Microbial Physiology

Keywords

Mycobacteria, Cell wall biosynthesis, Outer membrane lipids, Polyketide synthases, Fatty acyl AMP ligases, Pleiotropism, Dictyostelium, Biofilm, Antibiotic susceptibility

Abstract

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.

 
 

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