The sequence was annotated by using the software Artemis and has been deposited in the public databases with the ID MTBH37RV, or number NC_000962 .
Further information and services concerning M. tuberculosis
genes and predicted proteins can be obtained via the TubercuList
server at the Institut Pasteur, the Mycobacterium
tuberculosis project page at the Sanger
Centre, the NCBI genome brownser,
the Munich Information Centre for Protein Sequences, the KEGG: Kyoto Encyclopedia of Genes and Genomes or the Max Planck Institute for Infection Biology.
Information on finished and ongoing genome sequencing projects is also available via the web sites at Integrated Genomics, or the The Institute of Genomic Research.
Overview of Mycobacterial Genome Projects.
Evolution of the M. tuberculosis complex
Further reading:
A new evolutionary scenario for the Mycobacterium tuberculosiscomplex.
Proc Natl Acad Sci USA (2002) 99: 3684-3689.(pdf), Web-based supporting material:
(Primer-Table 1, RD-Table 2 ). (Science-Editor's choice commentary).
The evolution of mycobacterial pathogenicity: clues from comparative genomics.
Trends in Microbiology (2001) 9: 452-458, (pdf)
Mycobacterium bovis and Mycobacterium bovis BCG
Despite decades of use worldwide, the reason for the attenuation of M. bovis BCG (Bacille de Calmette et Guèrin) is still unknown although comparative genomics is beginning to shed light on some of the possible mechanisms involved. It has been shown recently in whole genome scans using either BAC arrays , (Gordon et al, 1999) , or DNA microarrays , (Behr et al, 1999) , that several genomic deletions and tandem duplications
can be found in BCG substrains relative to M. tuberculosis H37Rv.
In order to elucidate further genomic particularities of BCG, our laboratory,
in collaboration with the Sanger Institute , is currently
engaged in a project to determine the genome sequence of
Mycobacterium bovis BCG-Pasteur 1173P2 using clones from
the ordered BAC library and the physical map as tools (1,2,3).
This work is complementary to the Mycobacterium bovis
genome sequencing project that we are pursuing in collaboration with the Pathogen Genome Sequencing Unit at the Sanger Centre directed
by Bart Barrell and Glyn Hewinson at the United Kingdom's Veterinary Laboratories Agency.
The strain of M. bovis that is being studied (AF2122/97 spoligotype 9) was responsible for herd outbreaks in Devon in the UK and has been isolated from lesions in both cattle and badgers.
The sequence of M. bovis AF2122/97 was recently finished and is available either via the interactive database/search-tool
BoviList or the Sanger Centre
FTP site or BLAST server. The genome is with 4,345,492 bp about 66 kb smaller than the one from M. tuberculosis H37Rv.
Garnier, T., Eiglmeier, K., Camus, J.C., Medina, N., Mansoor, H.,
Pryor, M., Duthoy, S., Grondin, S., Lacroix, C., Monsempe, C., Simon, S.,
Harris, B., Atkin, R., Doggett, J., Mayes, R., Keating, L., Wheeler, P.R.,
Parkhill, J., Barrell, B.G., Cole, S.T., Gordon, S.V., and Hewinson, R.G.
(2003) The complete genome sequence of Mycobacterium bovis.
Proc. Natl. Acad. Sci. U S A. 2003 Jun 3
(Epub ahead of print), (fulltext).
Also see the news release in English or in French .
Comparison of the BCG genome sequence with those of virulent tubercle bacilli will highlight the genetic rearrangements that led to attenuation and gave rise to the world's most widely used live vaccine strain. The sequence analysis has recently been finished.
Sequence data comparison is now possible via the BCGList server or via the Sanger Center BLAST server.
Findings described in the articles listed below start to provide experimental evidence for the genetic background of the attenuation of BCG.
Parts of these studies were undertaken within the framework of the TB vaccine cluster
Pym, A. S, Brodin, P., Brosch, R., Huerre, M., Cole, S. T. (2002)
Loss of RD1 contributed to the attenuation of the live tuberculosis
vaccines Mycobacterium bovisBCG and Mycobacterium microti.
Mol Microbiol 46:709-717,
(full-text).
Pym AS, Brodin P, Majlessi L, Brosch R, Demangel C, Williams A, Griffiths KE, Marchal G, Leclerc C, and Cole ST.(2003) Recombinant BCG exporting ESAT-6 confers enhanced protection against tuberculosis. Nat Med 2003 Apr 14, (pdf).
Further reading:
Philipp, W. J., S. Nair, G. Guglielmi, M. Lagranderie, B. Gicquel, and S. T.
Cole. 1996. Physical mapping of Mycobacterium bovis BCG Pasteur reveals differe
nces from the genome map of Mycobacterium tuberculosis H37Rv and from M. bovis.
Microbiology 142:3135-45
Brosch R, Gordon SV, Billault A, Garnier T, Eiglmeier K, Soravito
C, Barrell BG and Cole ST. Use of a Mycobacterium tuberculosis H37Rv
bacterial artificial chromosome library for genome mapping, sequencing, and comparative genomics. 1998
Infect Immun 66:2221-2229
Gordon, S.V., Brosch, R., Billault, A., Garnier, T., Eiglmeier, K. and Cole,
S.T. (1999) Identification of variable regions in the genomes of tubercle bacilli using bacterial artificial chromosome arrays.
Mol. Microbiol. 32: 643-656.
Brosch,R., Gordon, S.V., Pym, A.,and Cole S.T. (2001) The evolution of
mycobacterial pathogenicity: clues from comparative genomics. Trends in Microbiology 9:
452-458. (fulltext), (PDF)
Brosch, R., Gordon, S.V., Marmiesse, M., Brodin, P., Buchrieser, C., Eiglmeier, K., Garnier, T., Gutierrez, C, Hewinson, G., Kremer, K., Parsons, L.M., Pym, A.S., Samper, S., van Soolingen, D., and Cole, S.T. (2002)
A new evolutionary scenario for the Mycobacterium tuberculosis complex. Proc Natl Acad Sci USA 99: 3684-3689.(pdf),
Web-based supporting material:
(Table 1, Table 2
). (Science-Editor's choice commentary).
Mycobacterium microti
Mycobacterium microti, a member of the Mycobacterium
tuberculosis
complex, was originally isolated from voles in the UK in the 1930's
and causes tuberculosis in rodents . Several strains have been
isolated from voles and these have been found to be avirulent for both man and bovines.
Some of these OV strains (OV stands for Orkney Vole) have been used as
live vaccine by the Medical Research Council (MRC) in the UK in the 1960's
in controlled
clinical trials involving large human populations.
Another strain, OV166 has been used as live vaccine in former Czechoslovakia
in the 1960's. In total, about half a million new-borns were vaccinated with
this strain over an 18-year period. No particular health problems due to
the vaccination with M. microti were reported and immunisation with
M. microti conferred protection against tuberculosis equivalent to that
induced by BCG. These studies clearly show that
these strains of M. microti are not human pathogens.
However, strains that were classified as M. microti on the
basis of slow growth and their characteristic spoligotypes, that ressemble vole isolates, have recently been isolated from human infections.
To find out more of the common and divergent genomic characteristics of
these isolates, a whole genome screen based on BAC maps was undertaken in our
laboratory.
For further details, please refer to the article by Brodin et al.,
which also contains a extensive introduction to M. microti:
Brodin, P., Eiglmeier, K., Marmiesse, M., Billault, A., Garnier, T.,
Niemann, S., Cole, S.T., and Brosch, R. (2002)
Bacterial Artificial Chromosome-Based Comparative Genomic Analysis Identifies
Mycobacterium microtias
a Natural ESAT-6 Deletion Mutant.
Infect Immun. 70:5568-5578. (fulltext), (pdf).
Pym, A. S, Brodin, P., Brosch, R., Huerre, M., Cole, S. T. (2002)
Loss of RD1 contributed to the attenuation of the live tuberculosis
vaccines Mycobacterium bovisBCG and Mycobacterium microti.
Mol Microbiol 46:709-717,
(full-text).
In parallel, a whole genome sequencing project on strain OV254 was recently started and is being continued in collaboration with the Sanger Institute.
Mycobacterium leprae
The sequencing project of the complete genome of M. leprae is finished, (Nature 409: 1007-1011).
The annotation of the sequence was done using the software Artemis (Rutherford et al., 2000, Bioinformatics, 16, 944-5).
Interactive tool for gene and/or protein analyses in the M. leprae genome: Leproma.
The genome sequence of a strain of Mycobacterium
leprae,originally isolated in Tamil Nadu (TN), has been completed
recently, thus meeting one of the highest priorities defined for
leprosy research and control programmes at the joint WHO/Sasakawa
Memorial Health Fund meeting held in Bangkok in 1995. The project was
undertaken in collaboration with the Pathogen Genome Sequencing Unit
at the Sanger Centre directed by Dr. Bart Barrell.
The sequence was obtained by a combined approach employing automated DNA
sequence analysis of selected cosmids and whole genome shotgun clones.
After the finishing process, the genome sequence of the TN strain of the
leprosy bacillus was found to contain 3,268,203 bp, and to have an average
G+C content of 57.8%.
These values are much lower than those seen in Mycobacterium tuberculosis which were 4,441, 529 bp and 65.6% G+C.
There are an estimated 4,000 genes encoding proteins in the tubercle bacillus but only 1,605 functional genes were identified in M. leprae. From the combined results of BLASTX and BLASTN searches, using filters to reduce noise, it was established that M. leprae contains at least 1,114 pseudogenes, with two or more mutations which should prevent their expression, and that a further 1,686 genes have been "deleted" from the genome. The latter conclusion is based upon the not unreasonable assumption that both mycobacteria are derived from a common ancestor and had gene pools of similar size at one stage, as suggested by comparative analysis. Downsizing from a 4.41 Mb genome such as that of M. tuberculosis to one of 3.26 Mb would account for the loss of some 1,200 protein-coding sequences.
There is clear evidence from inspection of the genomic context and from the presence of extensively truncated coding sequences that many of these genes were once present in the M. leprae genome and have truly been lost. It is also certain that M. tuberculosis harbors a substantial number of genes that were never present in the leprosy bacillus and that M. leprae contains >100 genes that have no counterpart in the genome of the present tubercle bacillus. From the results of highly sensitive dot-matrix comparisons, it seems probable that the extensively-decayed coding sequences of around 450 once-common genes remain within the M. leprae genome but that these have mutated so much that they are now below the threshold used in the BLAST analysis. The corresponding genes may have been lost at an early stage of the genome degeneration process and thus been under lower selective pressure.
Information about the functional classification of both the remaining
active genes, and the pseudogenes, of M. leprae can be found on
this website
ML_gene_list together with the complete EMBL entry ML.embl.
If we are to develop a specific immunological test for the early
diagnosis of leprosy, in particular the tuberculoid form of the
disease, it is essential to define the protein repertoire accurately
through a combination of genomic and proteomic studies, and to
identify those proteins that are confined to M. leprae or show
extensive diversity from their counterparts in other mycobacteria. At
the present time, the results of preliminary in silico proteome
comparisons are available and these are particularly
informative. Roughly 10% of the M. tuberculosis genome encodes 167
proteins belonging to the novel, glycine-rich PE and PPE
families. There are only about 10 of these proteins present in
M. leprae and this may account for the significant difference in the
respective genome sizes. Of interest is the finding that one of the
PPE proteins, the serine-rich antigen, is well recognised by sera from
leprosy patients and strikingly different from the equivalent
M. tuberculosis protein.
Consistent with this downsizing trend, the
class of proteins referred to as "conserved hypotheticals"
i.e. present in two or more bacteria but of unknown function, is
two-thirds smaller in the leprosy bacillus (~300) than in
M. tuberculosis (915). Likewise of the 606 proteins predicted
to be present in the proteome of the tubercle bacillus, that
previously had no counterparts elsewhere, 130 were also found in M. leprae, and some of these show extensive sequence divergence. These polypeptides could be exploited for diagnostic purposes for the identification of intracellular mycobacteria.
In the framework of the X-TB
structural and functional genomics project, sponsored by the
European Union 5th Framework programme, many of these proteins will be
produced and used for biochemical, immunological and structural studies.
Funding for the M. leprae genome project was provided
by the Heiser Program for Research in Leprosy and Tuberculosis of
The New York Community Trust, L'Association Raoul Follereau
and the Groupement de Recherches et des Etudes des Genomes (GIP-GREG).
The sequences of previously sequenced M. leprae cosmids and shotgun reads are
available via
FTP.
Further reading:
Eiglmeier et al.; Use of an ordered cosmid library to deduce the genomic
organization of Mycobacterium leprae. Mol. Microbiol. (1993) 7(2):197-206.
Brosch et al.; Comparative genomics of the leprosy and tubercle bacilli.
Res. Microbiol. 151: 135-142.
(pdf)
Please also find more information on leprosy on the web-pages of the World Health Organization (WHO) .
Mycobacterium ulcerans
|
Mr Glen Buckle, Australian
microbiologist, who in 1948 with Ms Jean Tolhurst co-discovered M.
ulcerans (86).
|
|
Sequelae of Buruli ulcer caused by M.
ulcerans. |
Génopole Institut Pasteur et l'Unité de Génétique Moléculaire Bactérienne,
Institut Pasteur, 28 Rue du Docteur Roux, 75724 Paris Cedex 15, France
Mycobacterium ulcerans is an emerging pathogen that causes Buruli ulcer, a chronic, necrotic skin lesion in
humans, and has rapidly emerged as an important cause of morbidity around the world. The prevalence of Buruli ulcer throughout West Africa
appears to have increased dramatically since the late 1980s (Marston and al., 1995).
The following African countries have reported cases to date: Angola, Benin, Burkina Faso, Cameroon, Congo, Côte d’Ivoire,
Democratic Republic of Congo, Equatorial Guinea, Gabon, Ghana, Guinea, Liberia, Nigeria, Sierra Leone, Togo, Uganda, Sudan. The disease is
also common in Asia: China, India, Indonesia, Malaysia, Sumatra and the Western Pacific: Australia, Kiribati, Papua New Guinea (Anonymus,
2000).
The significant increase in disease in many parts of the world caused by Mycobacterium ulcerans, particularly throughout
rural West Africa where the disease imposes a severe economic and social burden, has renewed interest in the study of the pathogenesis and
ecology of this organism.
Buruli ulcer is considered the third most common mycobacterial disease of non-immunocompromised persons after
tuberculosis and leprosy.
M. ulcerans is unlike other mycobacterial pathogens in that it appears to maintain an extracellular location during infection (Hayman and McQueen,
1985) and produces a macrolide toxin,
mycolactone
(George and al., 1999).
Treatment of the disease is usually by surgical excision of infected and surrounding tissue, as the organism in situ is unresponsive to
drug therapy . Possible explanations for the increased occurrence of this disease include environmental changes that have led to proliferation
of the organism followed by increased human contact or adaptation of the organism to a changed environment and coincidental acquisition of
increased virulence.
Despite several extensive investigations over the past 30 years, the mode of transmission of M. ulcerans has not been determined .
However, in a recent article by Marsollier et al., AEM (2002) 68: 4623-8, a transmission chain involving aquatic insects has been proposed.
Recent detection of M. ulcerans-specific DNA sequences in water from swamps in SE Australia and aquatic insects in Benin have
confirmed that it is an environmental organism .
It has been suggested that M. ulcerans is derived from M. marinum, an intracellular pathogen of fish and
humans, commonly isolated from aquatic environments worldwide . Indeed, according to one hypothesis, M. ulcerans may have diverged
recently from M. marinum by the recruitment of foreign DNA from the environment (Stinear
et al., 2000b).(Stinear et al., 2004).
Genome sequence analysis of Mycobacterium ulcerans
In the framework of the Génopole programme, the Institut Pasteur is sequencing the 4.4 Mb genome of an
epidemic strain of M. ulcerans. About 40,000 reads from a whole genome shotgun library will be obtained using PE3700 automated
sequencers then assembled into contigs using the programs Phrap and GAP4. Gap closure will then be undertaken using primer walking and
an ordered BAC library, and the resultant contiguous sequence subjected to bioinformatic analysis and exhaustive comparisons with the
genome sequences of other mycobacteria.
This approach has been successful and the genome sequence was described and published at the beginning of the year 2007 in Genome Res., and the access to the data can be easily obtained via the BuruList server
In parallel with the genome project, PFGE and sequence analysis has led to the identification of a large plasmid that is required for the production of mycolactone Proc Natl Acad Sci U S A. (2004), 101: 1345-49, Blaise Pascale lecture.
The M. ulcerans genome project will deal with following points:
- Definition of complete sets of genes, enzymes, proteins and antigens of M. ulcerans
- Identification of species-specific functions that could serve as novel drug targets
- Identification of species-specific functions that could serve as novel drug targets
- Identification of species-specific proteins for use as immunodiagnostic reagents
- Leads for developing therapy for Buruli ulcer
- Tools for detecting infection
- Improved molecular epidemiology
- Insight into the process by which an environmental bacterium becomes a pathogen
A Brief Background to Mycobacterium ulcerans
Based on accurate clinical descriptions, the first recorded report of ulcers caused by M. ulcerans was probably in 1897 by Sir Albert
Cook, a physician working in Uganda (27). However the aetiologic agent was not identified from this
early work. In 1938, two medical practitioners in the Bairnsdale area of Victoria in rural southeast Australia reported unusual skin ulcers in
several patients from the region (4).
Areas of necrotic tissue with extensive undermining of the dermis characterized the ‘Bairnsdale ulcer’. Ten years later a seminal
series of articles was published by MacCallum, Tolhurst, Buckle and Sissons (86) describing in detail the clinical and pathological aspects of the Bairnsdale ulcer but also a new
mycobacteria, Mycobacterium ulcerans, which they had identified as the causative agent of this disease. Shortly after publication of this
work, reports of skin ulcers caused by a new mycobacteria, similar to those caused by M. ulcerans, began to emerge from the Congo-
Kinshasa in Africa (71), (152). Drug sensitivity and immunodiffusion
studies subsequently showed this organism to be a strain of M. ulcerans (131), (137). Then, a few years later in 1953, cases were recognized in the Buruli County of Uganda where
the disease was given the epithet Buruli ulcer (21). In the ensuing years several hundred patients were diagnosed with Buruli ulcer in many other
districts of Uganda and all were in close proximity to the River Nile. The Uganda Buruli Group was established at this time and they conducted
comprehensive clinical and epidemiological studies of the disease. They studied in detail a refugee settlement that had nearly 300 cases among
a population of 2500 over a three-year period at the Kinyara refugee settlement in the Bunyoro District of Uganda. Their work, described in 3
articles published between 1969 and 1971, established the partial efficacy of the BCG vaccine (147),
and described the range of clinical manifestations of the disease and treatment options (148). They also provided convincing epidemiological evidence that M. ulcerans was an
environmental mycobacteria that was not spread by direct person-to-person transmission (149). At this time hundreds of environmental samples were collected from endemic areas of Uganda
and tested for M. ulcerans, however the organism was not detected in any samples (14), (127), (138). By the early 1970s over 1500 cases of Buruli ulcer had been reported from
Uganda (Barker, 1973).
Unfortunately M. ulcerans research in Uganda was halted because of civil war but the
research effort, and the disease, continued in the Congo-Kinshasa (90). In another attempt to identify the environmental source of M. ulcerans, mycobacteria were
cultured from thousands of environmental specimens collected in the Congo between 1973 and 1977 but all were negative for M. ulcerans
(111). From the work of researchers in Uganda, the Congo and
other African countries it became clear that the greatest burden of M. ulcerans disease was central and West Africa (109). However cases were also being reported from
many other countries including, Mexico, Malaysia, Sumatra, Papua New Guinea, Australia and French Guyana (110). The major scientific finding during this decade was that
M. ulcerans elaborated a toxin (124). Also of note was the first International Mycobacterium ulcerans Conference (IMUCI) held
at Middlesex Hospital, London in 1973.
During the 1980s there were only a handful of scientists conducting M. ulcerans research and publications concerning this organism
were scarce although ad hoc reports of new foci of the disease continued. However towards the end of the 1980s in rural West Africa
there was a dramatic increase in the prevalence of M. ulcerans disease (6), (Marston and al., 1995), (95). This trend continued through the 1990s to the present day. The reasons for the increase remain
unknown.
In southeast Australia a small cluster of cases occurred between 1993 and 1995 at a small holiday village near Melbourne where there had been
no previous cases of disease (73). Local interest in M. ulcerans was renewed and a Melbourne-based research group was
established. Using new molecular methods, they developed a PCR test that dramatically improved the clinical diagnosis of M. ulcerans
infection (130). By using the PCR on water samples collected near the centre of the case cluster they
were also able to demonstrate for the first time that water was an environmental source of the organism (129). In April 1998, fifty years after the first description of the organism
by MacCallum et al., the second International Conference on Mycobacterium ulcerans (IMUCII) was held at Box Hill Hospital. This
meeting was followed shortly after in July of that year by the launch of the Global Buruli Ulcer Initiative (GBUI)(put in WHO webpage link). The
GBUI was an initiative of the World Health Organization with the purpose of controlling the spread of Buruli ulcer by increasing public awareness
and by identifying key research objectives for disease prevention. In 1999 the chemical structure of a polyketide-derived lipid toxin produced
by M. ulcerans was determined George, K. M., D. Chatterjee, G. Gunawardana, D. Welty, J. Hayman, R. Lee, and P. L. Small. 1999.
Mycolactone: a polyketide toxin from Mycobacterium ulcerans required for virulence. (George,1999) and, using the same PCR as outlined above, aquatic
insects were implicated as an environmental source of the organism (114). The hypothesis drawn from the most recent molecular and environmental studies is that M.
ulcerans is a host-adapted, ecotype of M. marinum (Stinear
and al., 2000b). It appears that M. ulcerans may have a niche
role within organisms such as fish, frogs and aquatic insects.
Research Priorities
Despite these significant advances, they are only the beginning of the research effort required to be able to control M. ulcerans
disease. More data is required concerning effective drug treatments, vaccine options, and environmental ecology. Research priorities should
now be directed towards microenvironment studies that seek to replicate and model potential M. ulcerans habitats and test environmental
habitat hypotheses. Continuing comprehensive environmental surveys in endemic regions that are supported with good epidemiological data
would also be worthwhile. It is expected that combinations of microenvironment and comparative/functional genomic approaches should
answer many of the questions regarding the pathogenesis and environmental ecology of M. ulcerans. The determination of the complete
genome sequence of M. ulcerans will provide the basis for many of the studies.
More information and services concerning M. ulcerans
genes and predicted proteins can be obtained via the BuruList server and on
the web-page of the Institut Pasteur
and on the site of the World Health Organization (WHO).
Further reading
Anon. 2000. Report of WHO ad hoc advisory group meeting on Buruli ulcer.
Dailloux, M., C. Laurain, R. Weber, and P. Hartemann. 1999. Water and nontuberculous mycobacteria. Water Res. 33:2219-2228.
Dobos, K. M., F. D. Quinn, D. A. Ashford, C. R. Horsburgh, and C. H. King. 1999. Emergence of a unique group of necrotizing mycobacterial diseases.
Emerg. Infect. Dis. 5:367-
78.
Hayman, J., and A. McQueen. 1985. The pathology of Mycobacterium ulcerans infection. Pathol. 17:594-600.
Hayman, J. 1991. Postulated epidemiology of Mycobacterium ulcerans infection. Int J Epidemiol. 20:1093-1098.
Horsburgh, C. R., Jr. 1996. Epidemiology of disease caused by nontuberculous mycobacteria. Semin Respir Infect. 11:244-251.
Johnson, P. D. R., T. P. Stinear, and J. A. Hayman. 1999. Mycobacterium ulcerans - a mini review. J Med Microbiol. 48:511-513.
Marston, B. J., M. O. Diallo, C. R. Horsburgh, Jr., I. Diomande, M. Z. Saki, J. M. Kanga, G. Patrice, H. B. Lipman, S. M. Ostroff, and R. C. Good. 1995.
Emergence of Buruli ulcer disease in the Daloa region of Cote d'Ivoire. Am J Trop Med Hyg. 52:219-224
Stinear, T. P., J. K. Davies, G. A. Jenkin, J. A. Hayman, F. Oppedesano, and P. D. R. J. Johnson. 2000a. The identification of Mycobacterium
ulcerans in the environment from regions in which it is endemic in south eastern Australia with sequence capture-PCR. Appl. Environ. Microbiol. 66:3206-13.
Please address questions and comments concerning M. ulceransto Tim STINEAR.
We thank Florence MARTIN for her help in setting up the M. ulceranswebpages and the Burulist server.
End of webpage - Mycobacterial Genomics.
| | Please address questions and comments to Roland BROSCH or to Stewart COLE |
|---|
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