Gerd Pluschke - Mycobacterium ulcerans: Methods and Protocols

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Cover Caption: Mycobacterium ulcerans bacilli in the context of infected tissue.

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The first description of chronic skin ulcers consistent with the pathology of Mycobacterium ulcerans infection dates back to the end of the nineteenth century, but the causative agent was described only 80 years ago in patients from southeast Australia. M. ulcerans disease is named Buruli ulcer (BU) after a geographic area in Uganda that was severely hit by a BU epidemic. Since then, cases of BU have been reported in 34 countries, with the main burden being observed in children in West and Central Africa. Recently, a steady increase in the incidence of BU in Victoria, Australia, and the occurrence of M. ulcerans infections in the local possum population demonstrated that control of this neglected disease, for which transmission pathways are still not fully understood, is difficult. In 1998, the WHO launched the Global Buruli Ulcer Initiative to coordinate BU control and research on the disease. Less than 200 scientific papers on BU had been published at that time, and intensified research since then is reflected by currently close to 2000 publications on BU.

Of key importance for our understanding of BU was the description of the cytotoxic macrolide toxin mycolactone by George et al. in 1999. Mycolactone destroys mammalian tissues by apoptosis and has immunosuppressive activities. Histopathological analysis of infected tissue has shown a striking lack of infiltrating immune cells in necrotic areas surrounding clusters of extracellularly replicating mycobacteria, indicating a radical mechanism of immune evasion. Recent advances in the synthesis, handling, and detection of mycolactone have opened up new possibilities to study the effects of this fascinating toxin. M. ulcerans carries the giant plasmid pMUM that encodes the polyketide synthases required for mycolactone synthesis. Acquisition of this plasmid was a key step in the emergence of M. ulcerans from the environmental M. marinum. Complete genome sequences of M. ulcerans isolates have revealed >98% nucleotide sequence identity with the genome of M. marinum. In addition to the acquisition of pMUM, M. ulcerans has accumulated multi-copy insertion sequences, many pseudogenes, and multiple DNA deletions. While the observed reductive evolution indicates that M. ulcerans is developing into a niche-adapted specialist, there isin spite of the insights given by comparative genome analysesstill no conclusive information on environmental reservoirs of the pathogen. Cultivation of M. ulcerans from environmental samples is hindered by the extremely slow growth rate of the pathogen, hampering also the generation of genetically modified M. ulcerans mutants for functional analyses. Two standardized multiplex real-time PCR assays for the detection of M. ulcerans are now widely used for clinical diagnosis and analysis of environmental samples. Genomic analyses have revealed that M. ulcerans has diverged into several ecovars and that local clonal complexes are found in individual BU endemic regions, which speaks against the existence of highly mobile reservoirs. BU control is currently focused on early diagnosis and rapid initiation of an 8-week course of combination antibiotic therapy, underscoring the importance of active BU surveillance. In remote areas of Africa that are most affected by the disease, efforts are made to facilitate decentralized diagnosis and treatment by the development of M. ulcerans antigen or mycolactone-based point-of-care diagnostic tests and faster and simpler drug treatment modalities.

We sincerely hope that this edition of methods and protocols for BU research will motivate scientists to develop research activities on BU, which is still an under-researched field of infection biology.