Doctoral Dissertations

Date of Award

12-2005

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Microbiology

Major Professor

Pamela Small

Committee Members

Robert N. Moore, Stephen P. Oliver, David A. Bemis

Abstract

Buruli ulcer is a necrotizing skin infection and is the third most important mycobacterial disease in immune competent individuals after tuberculosis and leprosy in humid tropical countries. The causative agent Mycobacterium ulcerans is unlike other mycobacterial pathogens in that it appears to maintain an extracellular location during infection. Another unusual feature of the bacterium is that it is the only mycobacterium known to produce a dermo-necrotic polyketide toxin called mycolactone. A single Buruli ulcer, which can cover 15% of a person's skin surface, contains huge numbers of extracellular bacteria. The infection is characterized by massive necrosis at the site of infection followed frequently by debilitating disfiguration. Despite their abundance and extensive tissue damage, there is a remarkable absence of acute inflammatory response to the bacteria, and lesions are often painless. Though there is extensive literature on interaction of other mycobacterial species with innate immune cells, information concerning interaction of M. ulcerans with macrophages and neutrophils is scarce and requires further investigation. Research in this dissertation was geared towards understanding the poor innate immune response generated following M. ulcerans infection.

One hallmark of most diseases caused by mycobacteria including M. tuberculosis, M. bovis, M. leprae, M. marinum, M. haemophilum is the ability of the bacilli to grow within host cells and cause granulomas. In contrast, M. ulcerans primarily forms extracellular microcolonies within necrotic tissues and is rarely found within host cells. The role of mycolactone in the extracellular location of the bacteria was investigated using a macrophage infection model. Experiments using a panel of mycolactone negative (myc-) and wild type (WT) M. ulcerans strains showed that presence or absence of mycolactone determines whether the bacteria are extracellular or intracellular. Exposure of macrophages to high concentrations of mycolactone interfered with their phagocytic ability. These observations that a mycolactone mutant is better phagocytized than the wild type strain is consistent with the presence of almost exclusively extracellular bacteria in Buruli ulcer patients.

Experiments studying the effect of mycolactone concentrations on fibroblast cell lines showed that mycolactone-mediated apoptosis and necrosis was concentration dependent. Mycolactone caused necrosis at high concentrations and apoptosis at low concentrations. Chemotaxis assays using human neutrophils showed that neutrophils do not respond to M. ulcerans (WT or myc-) or mycolactone. Mycolactone treatment resulted in rapid necrosis of human neutrophils in a dose dependent manner in vitro. These data could be relevant in vivo in human infections where toxin gradients produced by a pool of extracellular M ulcerans may cause apoptosis or necrosis of inflammatory cells trying to move into the focus of infection and clear the bacteria.

Lack of an inflammatory reaction during the necrotizing stage of Buruli ulcer could be due to abrogation of production of inflammatory cytokines (e.g. TNF-a, lL-l, lFN-y), chemokines (IL-8) and lowered expression of cell-adhesion molecules (e.g. lCAM-l, selectins, VCAM) which help inflammatory cells reach the site of infection. TNF-a and lL-8 are key players in immuno-inflammatory responses. Studies regarding TNF-a response to bacterial infection and mycolactone treatment in vitro showed that WT M. ulcerans and mycolactone did not induce TNF-a production while myc- M. ulcerans did. Interestingly, LPS mediated TNF-u production was inhibited by WT M. ulcerans and mycolactone. Both WT and myc- M. ulcerans as well as mycolactone did not induce IL-8 production. WT M. ulcerans and mycolactone induced expression of the cell adhesion molecule ICAM-I was less than that induced by myc- M. ulcerans or LPS.

Microarray analysis of genes modulated by mycolactone yielded interesting information. Genes that were significantly upregulated by mycolactone included those related to transcriptional repressors, cytoskeletal rearrangement, cell cycle control/proliferation, apoptosis, G-protein receptors, tumor supression and immune response. Genes downregulated by mycolactone included those related to DNA repair, inactivation of complement and metalloproteinases, immune response, leukotrienes production, receptors for collagen and laminin. Data from the present study provide new insight into the effect of mycolactone on macrophages, fibroblasts, neutrophils and host gene-expression pathways induced or repressed by mycolactone. Knowledge obtained from the present study can be expected to contribute to a better understanding of the role of mycolactone in host-pathogen interactions as well as pathophysiology of the disease.

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