Presentation on Current Issues and Workshop Reports
Causative Organism and Host Response
C. THEME： CAUSATIVB ORGANISM AND HOST RESPONSES
Moderator： Dr. J. Krahenbuhl
A) PRESENTATION ON CURRENT ISSUES
1) The Mycobacterium Leprae Genome Project
Dr. Stewart T Cole
Everything that we need to know about the leprosy bacillus, from its biology to its behavior, is encoded in its genome. Through the concerted use of DNA sequencing and bioinformatics, a vast body of information can be deduced from the genes and this will further all aspects of biomedical research on Mycobacterium leprae. The startpoint for the genome project was the collection of cosmid clones (3) from the Institut Pasteur that covered most of the -3 Mb chromosome. In the first phase of sequencing, -1.7 Mb of genomic sequences were generated in conjunction with the Genome Therapeutics Corporation of Waltham, USA (4, 8) , and this dataset was later extended by the Sanger Centre, Hinxton, England. At the time of writing, an estimated 0.3 Mb of sequence remains to be generated to achieve completion.
When the first cosmid carrying M. leprae DNA was analysed, it was immediately apparent that only 50% of the potential coding sequence was actually used (5) . As more cosmid sequences became available, this atypical trend was confirmed. The situation in M. leprae is thus radically different from that seen in all other bacteria where ＞90% of the genome codes for proteins. This raised the possibility that the chromosome may have mutated on a large scale resulting in some of the unusual properties of M. leprae, such as its remarkably long generation time and lack of growth in the laboratory.
Interpreting the features of the non-coding regions of the genome was initially difficult as no suitable comparisons could be performed. However, a strong indication that gene inactivation had occurred was provided by scrutiny of the katG Ioci (2, 6) of Mycobacterium tuberculosis and M. leprae. In M. tuberculosis, katG encodes catalase-per-oxidase, a heme-containing enzyme that potentiates the toxic effect of isoniazid. Comparison of the corresponding regions revealed the presence of numerous mutations in the M. leprae katG gene that abolish its activity. This undoubtedly explains why the leprosy bacillus produces no catalase-peroxidase and displays high level resistance to isoniazid.
The recent completion of the genome sequence of M. tuberculosis, comprising 4,411,529 bp (1) , now enables us to perform systematic, genome-wide comparisons that will be of immense value for interpreting the genetic organisation and function of M. leprae. Preliminary studies reveal extensive relatedness and conservation of blocks of genes of very similar sequence and arrangement. However, in M. leprae these are often separated by DNA segments that appear to have contracted, consistent with the smaller genome size, and to have degenerated in sequence. These show lower levels of similarity and correspond to pseudogenes, the vestiges of genes that still function in M. tuberculosis. Yet again, M. leprae differ radically from other bacteria since few pseudogenes have been found in microbial genomes. While adapting to its intracellular niche, the leprosy bacillus apparently dispensed with genes conferring a limited competitive advantage and this downsizing may have led to the loss of one, or more, metabolic functions that affect growth rate.
Comparison of the genes found in these two mycobacterial genomes has led to the identification of coding sequences in M. leprae that have no counterparts in M. tuberculosis. The corresponding proteins offer great potential as reagents for use in diagnostic skin tests and may even be involved in neuropathy (7).
