Understanding Immune Response in \u3cem\u3eMycobacterium ulcerans\u3c/em\u3e Infection

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\u27s 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

Molecular Interactions that Enable Movement of the Lyme Disease Agent from the Tick Gut into the Hemolymph

Borrelia burgdorferi, the causative agent of Lyme disease, is transmitted to humans by bite of Ixodes scapularis ticks. The mechanisms by which the bacterium is transmitted from vector to host are poorly understood. In this study, we show that the F(ab)2 fragments of BBE31, a B.burgdorferi outer-surface lipoprotein, interfere with the migration of the spirochete from tick gut into the hemolymph during tick feeding. The decreased hemolymph infection results in lower salivary glands infection, and consequently attenuates mouse infection by tick-transmitted B. burgdorferi. Using a yeast surface display approach, a tick gut protein named TRE31 was identified to interact with BBE31. Silencing tre31 also decreased the B. burgdorferi burden in the tick hemolymph. Delineating the specific spirochete and arthropod ligands required for B. burgdorferi movement in the tick may lead to new strategies to interrupt the life cycle of the Lyme disease agent

Mycolactone-Mediated Inhibition of Tumor Necrosis Factor Production by Macrophages Infected with Mycobacterium ulcerans Has Implications for the Control of Infection▿

The pathogenicity of Mycobacterium ulcerans, the agent of Buruli ulcer, depends on the cytotoxic exotoxin mycolactone. Little is known about the immune response to this pathogen. Following the demonstration of an intracellular growth phase in the life cycle of M. ulcerans, we investigated the production of tumor necrosis factor (TNF) induced by intramacrophage bacilli of diverse toxigenesis/virulence, as well as the biological relevance of TNF during M. ulcerans experimental infections. Our data show that murine bone marrow-derived macrophages infected with mycolactone-negative strains of M. ulcerans (nonvirulent) produce high amounts of TNF, while macrophages infected with mycolactone-positive strains of intermediate or high virulence produce intermediate or low amounts of TNF, respectively. These results are in accordance with the finding that TNF receptor P55-deficient (TNF-P55 KO) mice are not more susceptible than wild-type mice to infection by the highly virulent strains but are more susceptible to nonvirulent and intermediately virulent strains, demonstrating that TNF is required to control the proliferation of these strains in animals experimentally infected by M. ulcerans. We also show that mycolactone produced by intramacrophage M. ulcerans bacilli inhibits, in a dose-dependent manner, but does not abrogate, the production of macrophage inflammatory protein 2, which is consistent with the persistent inflammatory responses observed in experimentally infected mice

Globally Distributed Mycobacterial Fish Pathogens Produce a Novel Plasmid-Encoded Toxic Macrolide, Mycolactone F

Mycobacterium ulcerans and Mycobacterium marinum are closely related pathogens which share an aquatic environment. The pathogenesis of these organisms in humans is limited by their inability to grow above 35°C. M. marinum causes systemic disease in fish but produces localized skin infections in humans. M. ulcerans causes Buruli ulcer, a severe human skin lesion. At the molecular level, M. ulcerans is distinguished from M. marinum by the presence of a virulence plasmid which encodes a macrolide toxin, mycolactone, as well as by hundreds of insertion sequences, particularly IS2404. There has been a global increase in reports of fish mycobacteriosis. An unusual clade of M. marinum has been reported from fish in the Red and Mediterranean Seas and a new mycobacterial species, Mycobacterium pseudoshottsii, has been cultured from fish in the Chesapeake Bay, United States. We have discovered that both groups of fish pathogens produce a unique mycolactone toxin, mycolactone F. Mycolactone F is the smallest mycolactone (molecular weight, 700) yet identified. The core lactone structure of mycolactone F is identical to that of M. ulcerans mycolactones, but a unique side chain structure is present. Mycolactone F produces apoptosis and necrosis on cultured cells but is less potent than M. ulcerans mycolactones. Both groups of fish pathogens contain IS2404. In contrast to M. ulcerans and conventional M. marinum, mycolactone F-producing mycobacteria are incapable of growth at above 30°C. This fact is likely to limit their virulence for humans. However, such isolates may provide a reservoir for horizontal transfer of the mycolactone plasmid in aquatic environments