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Whole-genome sequence analysis of Mycobacterium leprae has revealed a limited number of protein-coding genes, with half of the genome composed of pseudogenes and noncoding regions. We previously showed that some M. leprae pseudogenes are transcribed at high levels and that their expression levels change following infection. In order to clarify the RNA expression profile of the M. leprae genome, a tiling array in which overlapping 60-mer probes cover the entire 3.3-Mbp genome was designed. The array was hybridized with M. leprae RNA from the SHR/NCrj-rnu nude rat, and the results were compared to results from an open reading frame array and confirmed by reverse transcription-PCR. RNA expression was detected from genes, pseudogenes, and noncoding regions. The signal intensities obtained from noncoding regions were higher than those from pseudogenes. Expressed noncoding regions include the M. leprae unique repetitive sequence RLEP and other sequences without any homoiogy to known functional noncoding RNAs. Although the biological functions of RNA transcribed from M. leprae pseudogenes and noncoding regions are not known, RNA expression analysis will provide insights into the bacteriological significance of the species. In addition, our study suggests that M. leprae will be a useful model organism for the study of the molecular mechanism underlying the creation of pseudogenes and the role of microRNAs derived from noncoding regions.

Activation of extracellular signal-regulated kinase (Erk) 1/2, which plays a critical role in diverse cellular processes, including cell proliferation, is known to be mediated by the canonical Rafmitogen- activated protein kinase kinase (MEK) kinase cascade. Alternative MEK-independent signaling pathways for Erk1/2 activation in mammalian cells are not known. During our studies of human primary Schwann cell response to long-term infection of Mycobacterium leprae, the causative organism of leprosy, we identified that intracellular M. leprae activated Erk1/2 directly by lymphoid cell kinase (p56Lck), a Src family member, by means of a PKC[epsilon]-dependent and MEK-independent signaling pathway. Activation of this signaling induced nuclear accumulation of cyclin D1, [G.sub.1]/S-phase progression, and continuous proliferation, but without transformation. Thus, our data reveal a previously unknown signaling mechanism of glial cell proliferation, which might play a role in dedifferentiation as well as nerve regeneration and degeneration. Our findings may also provide a potential mechanism by which an obligate intracellular bacterial pathogen like M. leprae subverts nervous system signaling to propagate its cellular niche for colonization and long-term bacterial survival. cell signaling | cell cycle | peripheral nerve | human | Mycobacterium leprae

Research has been conducted on the recombinant hsp65 from Mycobacterium leprae. Results indicate that this recombinant exhibits proteolytic activity toward polypeptides and that this activity is characterized by the fluorometric assay.

Nerve damage is the hallmark of Mycobacterium leprae infection, which results from M. leprae invasion of the Schwann cell of the peripheral nervous system. We have recently shown that the laminin-2 isoform, specially the G domain of laminin [Alpha]2 chain, on the Schwann cell-axon unit serves as an initial neural target for M. leprae. However, M. leprae surface molecules that mediate bacterial invasion of peripheral nerves are entirely unknown. By using human [Alpha]2 laminins as a probe, a major 28-kDa protein in the M. leprae cell wall fraction that binds [Alpha]2 laminins was identified. After N-terminal amino acid sequence analysis, PCR-based strategy was used to clone the gene that encodes this protein. Deduced amino acid sequence of this M. leprae laminin-binding protein predicts a 21-kDa molecule (ML-LBP21), which is smaller than the observed molecular size in SDS/PAGE. Immunofluorescence and immunoelectron microscopy on intact M. leprae with mAbs against recombinant (r) ML-LBP21 revealed that the protein is surface exposed, rML-LBP21 avidly bound to [Alpha]2 laminins, the rG domain of the laminin-[Alpha]2 chain, and the native peripheral nerve laminin-2. The role of ML-LBP21 in Schwann cell adhesion and invasion was investigated by using fluorescent polystyrene beads coated with rML-LBP21. Although beads coated with rML-LBP21 alone specifically adhered to and were ingested by primary Schwann cells, these functions were significantly enhanced when beads were preincubated with exogenous [Alpha]2 laminins. Taken together, the present data suggest that ML-LBP21 may function as a critical surface adhesin that facilitates the entry of M. leprae into Schwann cells.

The Mycobacterium leprae katG gene encodes a heme-containing catalase-peroxidase. The DNA region of the Mycobacterium leprae katG gene is was highly homologous to other bacterial katG genes. The presence of termination codons in three frames of katG region encodes a catalase-peroxidase that contains two related domains that exhibited a strong similarity to yeast cytochrome c peroxidase protein.

Nucleotide sequencing of the 75 627 bp segment of the Mycobacterium leprae chromosome with the oriC gene indicated the presence of a gene order that is similar to the replication origin region of prokaryotes. The 75 627 bp segment also contains gyrB, dnaA, dnaB and gidB genes which mediate deoxyribonucleotide metabolism, cell wall synthesis and cell division. Furthermore, the protein products of the open reading frames that are located in the region mediate the generation of serine/threonine protein kinases and protein phosphatase.

An integrated map of the genome of the tubercle bacillus, Mycobacterium tuberculosis, was constructed by using a twin-pronged approach. Pulsed-field gel electrophoretic analysis enabled cleavage sites for Asn I and Dra I to be positioned on the 4.4-Mb circular chromosome, while, in parallel, clones from two cosmid libraries were ordered into contigs by means of fingerprinting and hybridization mapping. The resultant contig map was readily correlated with the physical map of the genome via the landmarked restriction sites. Over 165 genes and markers were localized on the integrated map, thus enabling comparisons with the leprosy bacillus, Mycobacterium leprae, to be undertaken. Mycobacterial genomes appear to have evolved as mosaic structures since extended segments with conserved gene order and organization are interspersed with different flanking regions. Repetitive sequences and insertion elements are highly abundant in M. tuberculosis, but the distribution of IS6110 is apparently nonrandom.