Studies on Mycobacterium ulcerans

Author

Terri Alford, University of Tennessee, Knoxville

Date of Award

8-2004

Degree Type

Thesis

Degree Name

Master of Science

Major

Microbiology

Major Professor

Pamela L. C. Small

Committee Members

Steven W. Wilhelm, Robert N. Moore

Abstract

Efforts to create a mycolactone-negative mutant of Mycobacterium ulcerans via transposon mutagenesis were undertaken to elucidate the genes involved in the production of this toxin. Though only five PRC-confirmed insertion mutants were produced, one of these, 1615:tnp6w, was confirmed to be deficient in mycolactone production via thin-layer chromatography (TLC) of acetone soluble lipids (ASLs), cytopathicity assay of culture filtrate on L929 fibroblasts, and mass spectroscopy of ASLs. Using nested-PRC, it was determined that the transposon insertion of 1615:tnp6w had occurred in a region of DNA containing a gene 97% identical to the M. marinum ferric uptake regulator homolog (FurA) gene and 89% identical to the fur A gene of M. tuberculosis. The region of M. ulcerans homologous to the already characterized M. marinum furA gener was cloned; and, the construct was used in complementation studies in an attempt to confirm the role of FurA in mycolactone production. Transformation of the 1615:tnp6w mutant with the construct containing furA failed to restore mycolactone production. The failure of the complementation of the furA gene to restore mycolactone production was explained via PCR studies which revealed a spontaneous deletion in the thioesterase II gene recently identified to be required for mycolactone production. Secondly, experiments aimed at developing an efficient transformation protocol for M. ulcerans were undertaken. In an effort to determine the effect of temperature on the transformation efficiency of M. ulcerans via electroporation, electroporation experiments were performed on M. ulcerans 1615 grown in Middlebrook 7H9 with 10% oleic acid-albumin-dextrose-complex supplement (M7HP + OADC). Log phase cultures were harvested, washed with 10% glycerol, and apportioned into 60 μL aliquots. Aliquots were briefly held at room temperature, 37˚C, 40˚C, or 45˚C before electroporation in the presence of 1μg of pPR27H1, a hygromycin B resistance conveying plasmid. After 8 weeks, colonies were counted to calculate transformation efficiencies. To determine if the presence of mycolactone affects electroporation efficiency, this experiment was repeated with M. ulcerans grown in Modified Reid’s synthetic medium and M. ulcerans grown in Sauton’s synthetic medium, both media which are thought to promote a decreased production of mycolactone; M. ulcerans grown in Middlebrook 7H9 with 20% oleic acid-albumin-dextrose-complex supplement (M7H9 + 2[OADC]), which is believed to foster an increased production of mycolactone; and, a polyketide synthase (PKS) knock-out mutant of M. ulcerans (mutant 115) which does not produce any mycolactone grown in M7H9 + OADC. Though electroporation of M. ulcerans grown in M7H9 + OADC produced transformants reliably (regardless of the temperature of the cell at the time of electroporation); the transformation efficiencies were not high and, there was no significant difference in the transformation efficiencies obtained by cells exposed to different temperatures before electroporation. However, an additional experiment, demonstrated that these cells exposed to 55˚C for the same amount of time prior to electroporation did not have a modest but significant increase in transformation efficiency. A similar increase in transformation efficiency was obtained when M. ulcerans grown in M7H9 + 2[OADC] was electroporated after incubation at the lower temperature of 45˚C. In an additional experiment, the fast growing M. fortuitum grown in M7H9 + OADC had the highest transformation efficiency when electroporated after incubation at 45˚C. Finally, though the natural reservoir of M. ulcerans is unknown, Buruli ulcer disease seems to be associated with water. For this reason, experiments aimed at investigation aquatic snails, also associated with water in Buruli ulcer endemic regions, as a component of the ecological niche of M. ulcerans were undertaken. Snails of the species Biomphalaria glabrata are intermediate hosts of the schistosome species Schistosoma mansoni; and, it is feasible that M. ulcerans could associate with cercariae, the schistosomal life stage infectious to humans, infecting humans through the wound caused by the cercaria entering its human host. Through various experiments, both healthy Biomphalaria glabrata and Schistosoma mansoni­-infected Biomphalaria glabrata were exposed to M. ulcerans and examined for M. ulcerans infection via PRC and plating of ground tissue. While PCR analysis demonstrated a possible association between M. ulcerans and healthy snails; M. ulcerans was not recovered by plating of the tissue of snails exposed to the bacteria during any of three infection experiments. Hystological sectioning of snail tissue yielded no reliable data.

Recommended Citation

Alford, Terri, "Studies on Mycobacterium ulcerans. " Master's Thesis, University of Tennessee, 2004.
https://trace.tennessee.edu/utk_gradthes/4667