Whole-genome expression analysis of Mycobacterium leprae and its clinical application.

The whole-genome sequence analysis of Mycobacterium leprae, which was completed in 2001, revealed the characteristics of this microbe's genomic structure. Half of the M. leprae genome consists of a limited number of protein-coding genes and the rest comprises non-coding regions and pseudogenes. We performed membrane array and tiling array analyses to analyze the gene-expression profile of the M. leprae genome and found that pseudogenes and non-coding regions were expressed similarly to coding regions at the RNA level. The RNA expressions were confirmed by real-time PCR analysis. Expression of these RNAs in clinical samples showed varying patterns among patients, thus indicating that the analysis of RNA expression patterns, including non-coding regions and pseudogenes, may be useful for understanding the pathological state, prognosis, and assessment of therapeutic progress in leprosy.

Detection of Mycobacterium leprae DNA from archaeological skeletal remains in Japan using whole genome amplification and polymerase chain reaction.

BACKGROUND: Identification of pathogen DNA from archaeological human remains is a powerful tool in demonstrating that the infectious disease existed in the past. However, it is very difficult to detect trace amounts of DNA remnants attached to the human skeleton, especially from those buried in a humid atmosphere with a relatively high environmental temperature such as in Asia. METHODOLOGY/PRINCIPAL FINDINGS: Here we demonstrate Mycobacterium leprae DNA from archaeological skeletal remains in Japan by polymerase chain reaction, DNA sequencing and single nucleotide polymorphism (SNP) analysis. In addition, we have established a highly sensitive method of detecting DNA using a combination of whole genome amplification and polymerase chain reaction, or WGA-PCR, which provides superior sensitivity and specificity in detecting DNA from trace amounts of skeletal materials. CONCLUSION/SIGNIFICANCE: We have detected M. leprae DNA in archaeological skeletal remains for the first time in the Far East. Its SNP genotype corresponded to type 1; the first detected case worldwide of ancient M. leprae DNA. We also developed a highly sensitive method to detect ancient DNA by utilizing whole genome amplification.

Analysis of Mycobacterium leprae gene expression using DNA microarray.

Mycobacterium leprae, the causative agent of leprosy, does not grow under in vitro condition, making molecular analysis of this bacterium difficult. For this reason, bacteriological information regarding M. leprae gene function is limited compared with other mycobacterium species. In this study, we performed DNA microarray analysis to clarify the RNA expression profile of the Thai53 strain of M. leprae grown in footpads of hypertensive nude rats (SHR/NCrj-rnu). Of 1605 M. leprae genes, 315 showed signal intensity twofold higher than the median. These genes include Acyl-CoA metabolic enzymes and drug metabolic enzymes, which might be related to the virulence of M. leprae. In addition, consecutive RNA expression profile and in silico analyses enabled identification of possible operons within the M. leprae genome. The present results will shed light on M. leprae gene function and further our understanding of the pathogenesis of leprosy.

Detection of RNA expression from pseudogenes and non-coding genomic regions of Mycobacterium leprae.

We have previously reported that some pseudogenes are expressed in Mycobacterium leprae (M. leprae), the causative agent of leprosy, and that their expression levels alter upon infection of macrophages. We attempted to further examine the expression of pseudogene and non-coding genomic region in M. leprae, in this study. 19 Pseudogenes, 17 non-coding genomic regions, and 21 coding genes expression in M. leprae maintained in the footpads of the hypertensive nude rat (SHR/NCrj-rnu) were examined by reverse transcriptase polymerase chain reaction (RT-PCR). The expression of some of these pseudogenes, non-coding genomic regions and coding genes were also examined in M. leprae from skin smear specimens obtained from patients with lepromatous leprosy by RT-PCR. Transcripts from pseudogenes, non-coding genomic regions and coding genes examined in this study were clearly observed in M. leprae. The expression patterns of some of these transcripts vary greatly among different leprosy patients. These results indicate that some of pseudogenes and non-coding genomic regions are transcribed in M. leprae and analysis of RNA expression patterns including pseudogene and non-coding genomic region in M. leprae may be useful in understanding the pathological states of infected patients.

Whole-genome tiling array analysis of Mycobacterium leprae RNA reveals high expression of pseudogenes and noncoding regions.

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 homology 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.

[Detection of RNA expression on whole genome analysis of Mycobacterium leprae by tiling array].

Completion of Mycobacterium leprae genome sequence revealed that there are many pseudogenes and non-coding regions, but rather small numbers of protein-coding genes. Although it was thought that pseudogenes and non-coding regions were silent and junk, our previous studies indicated that RNA expression was detected from these regions. To elucidate comprehensive RNA expression pattern on M. leprae whole genome, tiling array was designed and total RNA of M. leprae Thai-53 strain was analyzed. As a result, highly expressed regions were detected among not only the gene regions but also pseudogenes and non-coding regions. Since some of the RNA expression levels were modulated by MDT, evaluation of RNA expression pattern might be a good indicator for the treatment of leprosy.

[Comprehensive analysis of RNA expression of Mycobacterium leprae and clinical and biological significance].

Completion of Mycobacterium leprae genome sequence revealed that there are many pseudogenes and non-coding regions, but rather small numbers of protein-coding genes. This result indicates that M. leprae is a very unique organism, and this future is important to understand the biological nature and/or pathogenicity of M. leprae, which remain unclear. We attempted to find the biological nature of M. leprae by detecting the gene and pseudogene regions transcribed at high level. We detected the genomic regions including pseudogenes and demonstrated that six out of twelve high expression regions were pseudogenes. In addition, its transcription level was changed when M. leprae infects macrophage. RNA was detected from genes, pseudogenes and non-coding regions. The expression levels of these regions were different among patients and a part of them is disappeared just after treatment. These results suggested that RNA derived from pseudogene and non-coding region have some function concerning the infection and/or intracellular parasitism and that the analysis of pseudogene and non-coding region expression pattern of M. leprae is available as a criterion for therapeutic effect and disease type of leprosy, and a prognostic marker.

Expression of adipose differentiation-related protein (ADRP) and perilipin in macrophages infected with Mycobacterium leprae.

Mycobacterium leprae survives and replicates within a lipid droplet stored in the enlarged phagosome of histiocytes, a typical feature of lepromatous leprosy that is thought to be an important nutrient source for the bacillus. However, the underlying mechanisms by which lipids accumulate within phagosomes remain unclear. Recently, it was revealed that the lipid droplet-associated proteins, including ADRP and perilipin, play essential roles in lipid accumulation in adipocytes or macrophages. Therefore, we attempted to examine the role of these proteins in leprosy pathogenesis. ADRP and perilipin localized to the phagosomal membrane, which contains M. leprae in skin biopsy specimens of lepromatous leprosy. ADRP expression was transiently increased after phagocytosis in THP-1 cells. However, high levels of ADRP expression persisted only when live M. leprae, but not dead bacilli or latex beads, was added. Furthermore, although peptidoglycan, a Toll-like receptor 2 ligand, suppressed the expression levels of ADRP and perilipin, M. leprae infection inhibited this suppression. These results suggest that live M. leprae has the ability to actively induce and support ADRP/perilipin expression to facilitate the accumulation of lipids within the phagosome and to further maintain a suitable environment for the intracellular survival within the macrophage.

[Molecular mechanisms for intracellular parasitisation and exclusion in macrophage infected with Mycobacterium leprae].

It was previously demonstrated that TLR2 and CORO1A (TACO, Coronin 1, p57) localize phagosome membrane of macrophage. However, the functional relationship between TLR2 and CORO1A was not known. We show here that there is a functional counteraction between TLR2 and CORO1A.