Mycobacteriophage L5, a temperate phage of mycobacteria, integrates site-specifically into the Mycobacterium smegmatis chromosome. We have identified the int gene and attP site of L5, characterized the chromosomal attachment site (attB), and constructed plasmid vectors that efficiently transform M. smegmatis through stable site-specific integration of the plasmid into the bacterial genome. These integration-proficient plasmids also efficiently transform slow-growing mycobacteria such as the pathogen Mycobacterium tuberculosis and the vaccine strain bacille Calmette-Guérin (BCG). The ability to easily generate stable recombinants in these slow-growing mycobacteria without the requirement for continual selection is of particular importance for the construction of recombinant BCG vaccines and for the isolation and characterization of mycobacterial pathogenic determinants in animal model systems. Integration vectors of this type should be of general use in a number of additional bacterial systems where temperate phages have been identified.

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Mycobacterium ulcerans is the causative agent of Buruli ulcer, a tropical ulcerative skin disease. One of the most intriguing aspects of this disease is the presence of extensive tissue damage in the absence of an acute inflammatory response. We recently purified and characterized a macrolide toxin, mycolactone, from M. ulcerans. Injection of this molecule into guinea pig skin reproduced cell death and lack of acute inflammatory response similar to that seen following the injection of viable bacteria. We also showed that mycolactone causes a cytopathic effect on mouse fibroblast L929 cells that is characterized by cytoskeletal rearrangements and growth arrest within 48 h. However, these results could not account for the extensive cell death which occurs in Buruli ulcer. The results presented here demonstrate that L929 and J774 mouse macrophage cells die via apoptosis after 3 to 5 days of exposure to mycolactone. Treatment of cells with a pan-caspase inhibitor can inhibit mycolactone-induced apoptosis. We demonstrate that injection of mycolactone into guinea pig skin results in cell death via apoptosis and that the extent of apoptosis increases as the lesion progresses. These results may help to explain why tissue damage in Buruli ulcer is not accompanied by an acute inflammatory response.Mycobacterium ulcerans is the causative agent of a tropical skin disease called Buruli ulcer (18), which has recently been recognized as an emerging infection in West Africa (12). Buruli ulcer patients present with indolent, necrotizing ulcerative lesions. The ulcers are characterized by extensive necrosis of the skin and underlying fat. Erythema and focal necrosis, including vascular erosion, are also present. In contrast to other pathogenic mycobacterial diseases such as tuberculosis, there is little evidence of an early acute inflammatory response to the infection, and the bacteria are primarily extracellular. Although the lesions may be quite extensive, covering up to 15% of a patient's skin surface, they are relatively painless.We have recently purified and characterized a polyketide toxin from M. ulcerans and termed this mycolactone (10). When added to the mouse fibroblast cell line L929, the toxin causes 90 to 100% of the adherent cells to undergo cytoskeletal rearrangement, subsequently rounding up and detaching from the tissue culture plate within 24 to 36 h of treatment. This has been termed the cytopathic effect (CPE) (9,15,25). The toxin also causes an arrest in the G 0 /G 1 phase of the cell cycle within 48 h (9, 10). More importantly, mycolactone, when injected intradermally into guinea pigs, is capable of causing a lesion similar to that produced by whole organisms (10). An isogenic mutant which does not produce the toxin is not virulent in the guinea pig model. Taken together, these data provide strong evidence that mycolactone plays a pivotal role in Buruli ulcer pathogenesis.Necrotic areas in infected guinea pig skin contain many pyknotic nuclei, a finding suggestive of apoptosis. It is impossible to determine ...

An integrated map of the genome of the tubercle bacillus, Mycobacterium tuberculosis, was constructed by using a twin-pronged approach. Pulsed