From Marinum to Ulcerans: a Mycobacterial Human Pathogen Emerges Lateral gene transfers and genome reduction apparently remodeled a fish pathogen into the agent that causes Buruli ulcer in humans Tim Stinear and Paul D. R. Johnson ave you heard of Buruli ulcer? ulcer into the national disease surveillance sys- Probably not, unless you come tem” (http://www.who.int/topics/mycobacterium from equatorial Africa, Papua _ulcerans/en/). Growing numbers of researchers H New Guinea, the Daintree region and clinicians are committed to controlling the in tropical Northern Australia, or spread of Buruli ulcer disease. Under the leader- seaside towns in temperate South Eastern Aus- ship of Kingsley Asiedu at WHO in Geneva, the tralia. Other than sporadic cases among visitors Global Buruli Ulcer Initiative continues to expand. to these endemic zones, Buruli ulcer does not WHO now hosts an annual meeting, inviting occur in North American or European coun- those involved in Buruli ulcer treatment, research, tries. However, the surge and severity of this and disease control, and their combined effort is disease in Africa led the 57th General Assembly lifting the veil on this mysterious disease. of the World Health Organization (WHO) in WHO officials identified six priorities for re- 2004 to recognize Buruli ulcer as a global threat search, including mode of transmission; devel- to public health. WHO officials called for “re- opment of diagnostic tests; drug treatments and search to develop tools to diagnose, treat and new treatment modalities; development of vac- prevent the disease, as well as to integrate Buruli cines; social and economic studies; and studies to determine the incidence and prevailing mode of transmission. We also recommend investigating the environmental determi- Summary nants that led to emergence of Buruli ulcer. • Buruli ulcer, which is absent from North Amer- Despite progress, there is still much to be ica and Europe, is found in Papua New Guinea done before those who live in endemic areas and in tropical Northern as well as temperate are freed from fear of this disease. South Eastern Australia, but is surging in equa- Tim Stinear is a Re- torial Africa. search Fellow in • Buruli Ulcer in Humans Buruli ulcer arises through infections with My- the Department of cobacterium ulcerans from the environment What is Buruli ulcer, and what do we know Microbiology, Mo- that eventually produce deep skin ulcers. about the causative agent, Mycobacterium nash University, • Comparing the genome sequences of M. ulcer- ulcerans? Since the late 1980s, cases of Bu- Clayton, Victoria, ans and Mycobacterium marinum suggests that ruli ulcer have steadily increased through- the former emerged from the latter by acquiring Australia, and Paul genes for producing immunosuppressant myco- out West and Central Africa, including in D. R. Johnson is lactones. Ivory Coast, Ghana, Benin, and Cameroon. the Deputy Director • M. ulcerans apparently is adapted to a dark and Moreover, the incidence of Buruli ulcer now of the Department aerobic environment where its reduced antige- exceeds leprosy and, in some regions, tuber- of Infectious Dis- nicity, slow growth, and production of myco- culosis, that are caused by two other infa- eases, Austin Hos- lactones provide advantages for survival. mous mycobacterial pathogens. pital, Heidelberg, Like leprosy, Buruli ulcer is also a skin Victoria, Australia Volume 2, Number 4, 2007 / Microbe Y 187 FIGURE 1 some situations, particularly when it is diagnosed early, some patients with Buruli ulcer may avoid surgery altogether. The highly focal epidemiology and association with swamps and slow- flowing water are hallmarks of Buruli ulcer and have given rise to a plethora of regional names such as Kasongo ul- cer, Kakerifu ulcer, Cayenne ulcer, Ku- musu ulcer, Daintree ulcer, and Bairns- dale ulcer (Fig. 1). Bairnsdale is a small provincial town, 200 miles east of Melbourne in temperate South East Australia. An investigation of unusual skin ulcers among people from this dis- trict in the 1930s led Australian re- searchers to discover M. ulcerans. It was named Buruli ulcer later from Regions of the world where M. ulcerans and other mycolactone-producing mycobacteria have been reported. Yellow shading indicates regions where human cases of M. ulcerans an African epithet for a now-defunct disease (Buruli ulcer) have been reported. Red dots indicate some of the regional names county in Uganda (renamed the Naka- assigned to M. ulcerans disease. Green and blue shading indicates regions where M. songola District) where many hundreds ulcerans-like mycolactone-producing mycobacteria have been recovered from diseased fish and frogs respectively. of cases were studied during the 1950s and 1960s. In July 1998 WHO officials, alarmed at the dramatic rise of Buruli ulcer disease, but there the similarity ends. Buruli across sub-Saharan Africa, launched the Global ulcer typically starts with a small mobile lump Buruli Ulcer Initiative (GBUI) and also made the beneath the skin or a smaller raised lump at- name Buruli ulcer official for the disease caused tached to the skin. How M. ulcerans is intro- by M. ulcerans. It remains a neglected disease of duced into the skin of humans remains un- the rural poor of Africa, despite the efforts of known, but it is acquired directly or indirectly dedicated researchers. from the environment, not from contact with Key questions such as identifying the environ- other patients. The disease progresses slowly mental reservoir of this bacterium, explaining over weeks to months, with little pain and none focal outbreaks, and establishing how M. ulcer- of the usual telltale signs of an infection such as ans is transmitted remain unanswered. How- fever or loss of weight and appetite. ever, the GBUI is helping to address these short- Eventually, the affected area breaks down to comings. For instance, the genome sequence of produce a deep skin ulcer, usually on a limb, but M. ulcerans was recently completed, which is any part of the body can be involved. If left leading to detailed comparisons with the closely untreated, these ulcers may become massive. related Mycobacterium marinum and could Although host immunity can arrest the process, help to explain how evolutionary forces shaped allowing scars to form, the aftermath can be this enigmatic pathogen. devastating because scarring can cause major disabilities, including loss of limb or destruction of eyes or skin appendages, including the geni- M. marinum and M. ulcerans talia. Are Phenotypically Distinct At first, treatment typically depended pri- marily on surgical excision and reconstructive In the 1990s, when mycobacteriologists began plastic surgery. However, more recently, Bu- compiling and comparing DNA sequences of the ruli ulcer patients are being treated early with 16S rRNA gene from different members of the potent antimycobacterial drugs, such as ri- genus, they discovered that a few phenotypically fampicin combined with streptomycin. In distinct pairs of mycobacteria had identical se- 188 Y Microbe / Volume 2, Number 4, 2007 FIGURE 2 Multi-locus sequence analysis 16SrRNA gene (2853 bp crtB, adk, fbpA, aroE, groEL, ppk, sod) M. terrae M. hiberniae <0.6% nucleotide variation M. nonchromogenicum M. cookii M. ulc erans Africa M. celatum M. ulculc eransns VictoriaVictoria M. xenopi M. ulculc eransns SESE AsiaAsia M. shimoidei M. ulc erans China/Japan M. asiaticum M. ulculc eransns MexicoMexico M. gordonae M. ulc erans Surinam M. marinum M. marinumrinum ST4ST4 M. ulc erans M. marinumrinum ST5ST5 M. marinumrinum ST3ST3 M. tub erculosis compl ex M. marinum ST1 M. leprae MM.. sszulgaizulgai M. marinum ST2 MM.. mmalmoensealmoense M. haemophilum MM.. ggastrii/kansasiiastrii/kansasii M. scofulaceum M. paraffinicum <3% nucleotide variation M. intracellulare M. avium M. avium subsp. paratuberculosis <3% nucleotide variation Phylogenetic relationship between: (left-side) some slow growing mycobacteria as revealed by analysis of the 16SrRNA gene and (right-side) Mycobacterium ulcerans and Mycobacterium marinum as revealed my multi-locus sequence analysis. quences. One of these pairs was M. marinum ent strains of M. ulcerans with those of M. and M. ulcerans (Fig. 2). marinum, she learned that the intraspecies rela- Their phenotypes are strikingly different. M. tive binding ratio (RBR) is more than 85%, marinum grows relatively rapidly, doubling whereas the interspecies RBR is less than 40%. within 6–11 hours, produces photochromoge- These findings suggest that, despite the apparent nic pigments, and causes an intracellular, gran- identity on the basis of 16SrRNA sequences, ulomatous disease in fish, frogs, and other ecto- there are other major genetic differences be- therms such as reptiles. It also occasionally tween these two species that can help to explain causes self-limiting skin lesions in humans, usu- their very different phenotypes. ally on the cooler extremities of the body such as Our scheme for multilocus sequence analysis the hands. Meanwhile, M. ulcerans grows (MLSA) provides another means to probe the slowly, taking more than 48 hours to double, is genetic relationship between these species. Se- nonpigmented, and causes a progressive, ulcer- quences for seven particular genes from 18 ative disease in humans, in which extracellular strains of M. ulcerans and 22 strains of M. bacteria collect in pools of necrotic subcutane- marinum show more than 97% nucleotide iden- ous fat. This pathology is largely attributed to tity, which is about the same as the 16SrRNA mycolactone, a polyketide produced by M. ul- gene comparisons. cerans that has cytotoxic and immunosuppres- However, when we infer phylogeny by align- sive properties. M. marinum does not make ing these sequences, we see that all the M. ulcer- mycolactone. ans strains fall into a single cluster that could be The relationship between contrasting pheno- further separated into unique genotypes based type and identical 16S rRNA gene sequences on one or two nucleotide polymorphisms that piqued the curiosity of Tone Tonjum from the correlate with the geographic origin of a strain Institute of Microbiology at the University of (Fig.