Post-genomic research on Mycobacterium ulcerans

understanding pathogenesis and addressing public health research priorities for the control of Buruli ulcer.

Tim Stinear

Head of Research Group: Dr Tim Stinear
email: tim.stinear@med.monash.edu.au

Research staff (as of July 2007)
Dr Grant Jenkin (Senior Research Fellow), grant.jenkin@med.monash.edu.au
Jessica Porter (Research Assistant), jessica.porter@med.monash.edu.au
Sacha Pidot (PhD student), sacha.pidot@med.monash.edu.au
Nicholas Tobias (PhD student), nicholas.tobias@med.monash.edu.au
Bahram Ravadgar (Visiting scientist), bahram.ravadgar@med.monash.edu.au
Tasnim Zakir (Honours student), tzak3@student.monash.edu.au

People who have recently left the laboratory:
Marcus Yip
Steffen Falgner
Delan Adakari

Research Interests

Mycobacterium ulcerans is an environmental mycobacterium that causes Buruli ulcer, a serious skin disease characterized by chronic ulceration of subcutaneous fat that can leave victims with debilitating, life-long deformity and disability (Fig. 1). Surgery that is often extensive is the only recommended treatment. M. ulcerans is related to Mycobacterium tuberculosis and Mycobacterium leprae the causative agents of tuberculosis and leprosy respectively. However, there is no vaccine and no recommended antibiotic therapy against Buruli ulcer. The disease is found throughout the world, including Australia where recent outbreaks in Victoria have coincided with an ongoing epidemic across West and Central Africa. In rural Central and West Africa the prevalence of Buruli ulcer now exceeds leprosy and, in some instances, tuberculosis. In 1998 the World Health Organisation launched the Global Buruli Ulcer Initiative with the principal aim of trying to control the spread of this debilitating disease (http://www.who.int/gtb-buruli/).

M. ulcerans produces an unusual lipid toxin called mycolactone (Fig. 2). This molecule is thought to be the major determinant of virulence because injection of purified mycolactone into the skin of guinea pigs provokes the appearance of ulcers. Mycolactone has been shown to have in vitro cytotoxic and immunosuppressive properties however little more is known of its biological attributes and the precise role it plays in pathogenesis is unclear. It is also possible that M. ulcerans may elaborate other factors - as yet unknown - that play a role in ulcer formation.

In collaboration with the Institut Pasteur we have recently determined the genome sequence of M. ulcerans (http://genolist.pasteur.fr/BuruList/). A major finding from the genome project was the discovery of a virulence plasmid and three polyketide synthase (PKS) genes on this plasmid required for mycolactone biosynthesis. The 12-membered core of mycolactone is produced by two giant type I modular PKS, whereas its side chain is synthesized by a third PKS (Fig. 3).

Fig 3: Biosynthetic pathway and module and domain organisation of the two type I polyketide synthases (MlsA1 and MlsA2) required for the sythesis of the mycolactone core. A similar PKS (MlsB) produces the fatty acyl side-chain. (Click on image for High Resolution image)

The availability of the complete genome sequence will provide an important resource for international research efforts aimed at controlling the spread of Buruli ulcer. In this respect the research within our group is focussed on:

• Using genome data to identify and characterise M. ulcerans specific antigens for use in the development of a rapid diagnostic tests and future vaccine studies.
• Using genome data to identify potential determinants of virulence other than mycolactone.
• Developing systems for heterologous expression of mycolactone so that a reliable source of mycolactone and derivatives is available for:
  • determining the role of mycolactone in the bacterium
  • establishing the relationship between mycolactone structure and function in the eukaryotic host

Collaborators and some useful WWW links:

We also study the human immune response to Mycobacterium ulcerans and collaborate with:

Dr Kumar Visanathan at the Centre for Inflammatory Diseases at the Monash Institute of Medical Research
(http://www.monashinstitute.org/cid-innate-immunity.html),

Prof. Gerd Pluschke at the Swiss Tropical Institute in Basel
(http://www.sti.ch/nc/about-us/staff/ detail-single-all/staff/209/gerd-pluschke.html )

Prof. Geoff Pietersz and Prof. Ian MacKenzie at the Burnet Research Institute
(http://www.ari.unimelb.edu.au/research/biolab.html )

For more information regarding polyketide synthases visit the site of one of our collaborators:
http://www.bio.cam.ac.uk/~pflgroup/

For more information on Mycobacterium ulcerans and the disease it causes visit:
http://www.health.vic.gov.au/ideas/diseases/myco_facts.htm

For access to the complete M. ulcerans genome sequence visit: http://genopole.pasteur.fr/Mulc/BuruList.html

Publications from the research group

1.      Rondini S, Käser M, Stinear T, Tessier M, Mangold C, Dernick G, Naegelli M, Portaels F, Certa U and Plushcke G. 2007. Ongoing genome reduction in Mycobacterium ulcerans. Emerg Infect Dis. In press

2.      Fyfe JA, Lavender CJ, Johnson PD, Globan M, Sievers A, Azuolas J and Stinear TP. 2007. Development and application of two multiplex real-time PCR assays for the detection of Mycobacterium ulcerans in clinical and environmental samples. Appl Environ Microbiol. 73: 4733-4740

3.      Hilty M, Kaser M, Zinsstag J, Stinear T and  Pluschke G. 2007. Analysis of the Mycobacterium ulcerans genome sequence reveals new loci for variable number tandem repeats (VNTR) typing. Microbiol. 153:1483-1487.

4.      Lavender CJ, Senanayake SN, Fyfe JA, Buntine JA, Globan M, Stinear TP, Hayman JA, Johnson PD.       2007. First case of Mycobacterium ulcerans disease (Bairnsdale or Buruli ulcer) acquired inNew South Wales . Med J Aust. 186:62-63.

5.      Stinear TP, Seemann T, Pidot S, Frigui W, Reysset G, Garnier T, Meurice G, Simon D, Bouchier C, Ma L, Tichit M, Porter JL, Ryan J, Johnson PD, Davies JK, Jenkin GA, Small PL, Jones LM, Tekaia F, Laval F, Daffe M, Parkhill J, Cole ST. 2007. Reductive evolution and niche adaptation inferred from the genome of Mycobacterium ulcerans, the causative agent of Buruli ulcer. Genome Res. 17: 192-200.

6.      Yip MJ, Porter JL, Fyfe JAM, Lavender CJ, Portaels F, Rhodes M, Kator H, Colorni A, Jenkin GA and Stinear T. 2006. Evolution of Mycobacterium ulcerans and other mycolactone-producing mycobacteria from a common Mycobacterium marinum progenitor. J. Bacteriol.  185: 2021-2029.

7.      Howden B, Johnson PDR, Ward PB, Stinear TP and Davies JKD. 2006. Isolates with low-level vancomycin resistance associated with persistent methicillin-resistant Staphylococcus aureus bacteremia. Antimicrob Agents Chemother. 50:3039-3047.

8.      Hong H, Stinear TP, Skelton P, Spencer JB, Leadlay PF. 2005. Structure elucidation of a novel family of mycolactone toxins from the frog pathogen Mycobacterium sp. MU128FXT by mass spectrometry. Chem Commun (Camb). 2005 34:4306-4308.

9.      Stinear TP, Hong H, Frigui W, Pryor MJ, Brosch R, Garnier T, Leadlay PF and Cole ST. 2005. A common evolutionary origin for the unstable virulence plasmid pMUM found in geographically diverse strains of Mycobacterium ulcerans. J Bacteriol. 187: 1668-1676.

10.  Hong H, Spencer JB, Porter JL, Leadlay PF, and Stinear TP. 2005. A novel mycolactone from a clinical isolate of Mycobacterium ulcerans provides evidence for additional toxin heterogeneity as a result of specific changes in the modular polyketide synthase. ChemBioChem. 6:1-5.

11.  Stinear TP, Pryor MJ, Porter JL and Cole ST. 2005. Functional analysis and annotation of the virulence plasmid pMUM001 from Mycobacterium ulcerans. Microbiol. 151:683-692.

12.  Marsollier L, Stinear TP, Aubry J, Robert R, Legras P, Manceau A, Audrain A, Kouakou H and Carbonnelle B. 2004. Aquatic plants stimulate the growth of and biofilm formation by Mycobacterium ulcerans in axenic culture and harbour these bacteria in the environment. Appl Environ Microbiol 70:1097-1103.

13.  Stinear TP, Mve-Obiang A, Small PLC, Frigui W, Pryor MJ, Brosch R, Jenkin GA, Johnson PD, Davies JK, Lee RE, Adusumilli S, Garnier T, Haydock SF, Leadlay PF, and Cole ST. 2004. Giant plasmid-encoded polyketide synthases produce the macrolide toxin of Mycobacterium ulcerans. Proc Natl Acad Sci U S A 101:1345-1349.

14.  Hong H, Gates P J, Staunton J, Stinear T, Cole ST, Leadlay PF and Spencer JB. 2003. Identification using LC-MSn of Co-metabolites in the Biosynthesis of the Polyketide Toxin Mycolactone from a Clinical Isolate of Mycobacterium ulcerans. Chem Commun (Camb) 22:2822-2823.

15.  Jenkin, GA, Stinear T, Johnson PD, and Davies JK. 2003. Subtractive hybridization reveals a type I polyketide synthase locus specific to Mycobacterium ulcerans. J Bacteriol 185:6870-6882.

16.  Stinear T, Olden DC, Johnson PD, Davies JK, Grayson ML. Enterococcal vanB resistance locus in anaerobic bacteria in human faeces. Lancet. 2001 357(9259):855-856.

17.  Stinear T, Jenkin GA, Johnson PDR, and Davies JK. Comparative genetic analysis of Mycobacterium ulcerans and Mycobacterium marinum reveals evidence of recent divergence. J Bacteriol. 2000 182:6322-6330.

18.  Stinear T, Davies JK, Jenkin GA, Hayman JA, Oppedisano F, and Johnson PDR. 2000. The identification of Mycobacterium ulcerans in the environment from endemic regions in south east Australia with sequence capture-PCR. Appl Environ Microbiol. 2000 66:3206-3213.

19.  Stinear T, Davies JK, Jenkin GA, Portaels F, Ross BC, Oppedisano F, Purcell M, Hayman JA, and Johnson PDR. 2000. A simple PCR method for rapid genotype analysis of Mycobacterium ulcerans. J Clin Microbiol 38:1482-1487.