| dc.description.abstract | Within the bacterial genus Burkholderia, the Bptm group is made up of three closely related species: Burkholderia pseudomallei, B. thailandensis, and B. mallei. This group contains both pathogenic and non-pathogenic species each with distinctly different lifestyles. B. mallei is a host-adapted equine pathogen, B. thailandensis is a non-pathogenic soil saprophyte, and B. pseudomallei is an opportunistic human pathogen that causes the disease melioidosis, which can be fatal up to 40% of the time. The Bptm species possess many conserved biosynthetic gene clusters coding for secondary metabolites. The conservation of secondary metabolites across this group affords opportunities to learn how these metabolites are useful among each member’s diverse biological niches. Secondary metabolites can be underappreciated contributors to bacterial fitness and also have roles in pathogenicity: the regulation and chemical biology of two examples, malleilactone and methylated hydroxy-alkylquinolones (HMAQs), encoded by the mal and hmq operons, respectively, are the focus of this work. Herein, we characterize B. pseudomallei malleilactone and show that it is important for virulence in nematode hosts, as well as cytotoxic to eukaryotic cells and some Gram-positive bacteria. Studies on its regulation revealed that malleilactone can be induced by treatment with various antibiotics, notably by the antibiotic trimethoprim via the activator MalR, and that a global secondary metabolite regulator ScmR, which is activated by acyl-homoserine lactone-dependent quorum sensing, strongly suppresses malleilactone production in later growth stages, likely by blocking its activation by MalR. Further contained within this work are the novel findings that efflux is important for malleilactone export and for mitigating malleilactone self-toxicity. We show that the B. pseudomallei BpeEF-OprC efflux pump system, encoded adjacent to the mal gene cluster, exports endogenous malleilactone into the extracellular environment. Notably, our results show that BpeEF-OprC protects mal-expressing cells from self-toxic effects of mal biosynthesis, especially under oxidative stress and in conditions where the recently-described malleicyprol-family malleilactone isomers, which were recently reported in B. thailandensis to have increased toxicity relative to malleilactone, are likely favored. We demonstrate that BpeT and BpeS, known regulators of the bpeEF-oprC operon, also play minor roles in mal cluster regulation. Additionally, studies within reveal that the mal genes are strongly upregulated under iron starvation conditions, in which we see that malleilactone provides a fitness benefit to wild-type cells, though in a siderophore-dependent manner. Our B. pseudomallei-purified malleilactone can bind to ferric iron. Taken all together, these findings all point to a notable and previously under-characterized contribution of malleilactone to B. pseudomallei fitness in situations of iron limitation. The new evidence from this dissertation suggests that malleilactone promotes fitness under stress, including growth- inhibiting antibiotics, host immune responses, and nutrient (iron) limitation, and that B. pseudomallei possesses an innate mal resistance mechanism. Regarding the HMAQ-family compounds, a transposon mutant screen performed by collaborators helped to identify HMAQs as B. thailandensis-produced antimicrobial compounds. Our results demonstrate using both liquid and plate co-culture assays that HMAQs are important for B. thailandensis to compete with another soil species, Bacillus subtilis. We also show that previously-uncharacterized HmqL catalyzes the formation of N-oxide derivatives of HMAQs in B. thailandensis, which have further increased antimicrobial potency against B. subtilis. Altogether, in this work we provide new insights into the biology of B. thailandensis and B. pseudomallei malleilactone and HMAQ secondary metabolites and their roles in survival in the host and in polymicrobial soil communities, and information that could be important for developing new therapeutics to treat melioidosis and other diseases. | |
