Pathogenicity and virulence of Mycobacterium leprae.

Leprosy is caused by Mycobacterium leprae (M. leprae) and M. lepromatosis, an obligate intracellular organism, and over 200,000 new cases occur every year. M. leprae parasitizes histiocytes (skin macrophages) and Schwann cells in the peripheral nerves. Although leprosy can be treated by multidrug therapy, some patients relapse or have a prolonged clinical course and/or experience leprosy reaction. These varying outcomes depend on host factors such as immune responses against bacterial components that determine a range of symptoms. To understand these host responses, knowledge of the mechanisms by which M. leprae parasitizes host cells is important. This article describes the characteristics of leprosy through bacteriology, genetics, epidemiology, immunology, animal models, routes of infection, and clinical findings. It also discusses recent diagnostic methods, treatment, and measures according to the World Health Organization (WHO), including prevention. Recently, the antibacterial activities of anti-hyperlipidaemia agents against other pathogens, such as M. tuberculosis and Staphylococcus aureus have been investigated. Our laboratory has been focused on the metabolism of lipids which constitute the cell wall of M. leprae. Our findings may be useful for the development of future treatments.

Host-Related Laboratory Parameters for Leprosy Reactions.

Leprosy reactions are acute inflammatory episodes that complicate the course of a Mycobacterium leprae infection and are the major cause of leprosy-associated pathology. Two types of leprosy reactions with relatively distinct pathogenesis and clinical features can occur: type 1 reaction, also known as reversal reaction, and type 2 reaction, also known as erythema nodosum leprosum. These acute nerve-destructive immune exacerbations often cause irreversible disabilities and deformities, especially when diagnosis is delayed. However, there is no diagnostic test to detect or predict leprosy reactions before the onset of clinical symptoms. Identification of biomarkers for leprosy reactions, which impede the development of symptoms or correlate with early-onset, will allow precise diagnosis and timely interventions to greatly improve the patients' quality of life. Here, we review the progress of research aimed at identifying biomarkers for leprosy reactions, including its correlation with not only immunity but also genetics, transcripts, and metabolites, providing an understanding of the immune dysfunction and inflammation that underly the pathogenesis of leprosy reactions. Nevertheless, no biomarkers that can reliably predict the subsequent occurrence of leprosy reactions from non-reactional patients and distinguish type I reaction from type II have yet been found.

Essential Roles of PPARs in Lipid Metabolism during Mycobacterial Infection.

The mycobacterial cell wall is composed of large amounts of lipids with varying moieties. Some mycobacteria species hijack host cells and promote lipid droplet accumulation to build the cellular environment essential for their intracellular survival. Thus, lipids are thought to be important for mycobacteria survival as well as for the invasion, parasitization, and proliferation within host cells. However, their physiological roles have not been fully elucidated. Recent studies have revealed that mycobacteria modulate the peroxisome proliferator-activated receptor (PPAR) signaling and utilize host-derived triacylglycerol (TAG) and cholesterol as both nutrient sources and evasion from the host immune system. In this review, we discuss recent findings that describe the activation of PPARs by mycobacterial infections and their role in determining the fate of bacilli by inducing lipid metabolism, anti-inflammatory function, and autophagy.

Mycobacterium leprae promotes triacylglycerol de novo synthesis through induction of GPAT3 expression in human premonocytic THP-1 cells.

Mycobacterium leprae (M. leprae) is the etiological agent of leprosy, and the skin lesions of lepromatous leprosy are filled with numerous foamy or xanthomatous histiocytes that are parasitized by M. leprae. Lipids are an important nutrient for the intracellular survival of M. leprae. In this study, we attempted to determine the intracellular lipid composition and underlying mechanisms for changes in host cell lipid metabolism induced by M. leprae infection. Using high-performance thin-layer chromatography (HPTLC), we demonstrated specific induction of triacylglycerol (TAG) production in human macrophage THP-1 cells following M. leprae infection. We then used [14C] stearic acid tracing to show incorporation of this newly synthesized host cell TAG into M. leprae. In parallel with TAG accumulation, expression of host glycerol-3-phosphate acyltransferase 3 (GPAT3), a key enzyme in de novo TAG synthesis, was significantly increased in M. leprae-infected cells. CRISPR/Cas9 genome editing of GPAT3 in THP-1 cells (GPAT3 KO) dramatically reduced accumulation of TAG following M. leprae infection, intracellular mycobacterial load, and bacteria viability. These results together suggest that M. leprae induces host GPAT3 expression to facilitate TAG accumulation within macrophages to maintain a suitable environment that is crucial for intracellular survival of these bacilli.

The function of peroxisome proliferator-activated receptors PPAR-γ and PPAR-δ in Mycobacterium leprae-induced foam cell formation in host macrophages.

Leprosy is a chronic infectious disease caused by Mycobacterium leprae (M. leprae). In lepromatous leprosy (LL), skin macrophages, harboring extensive bacterial multiplication, gain a distinctive foamy appearance due to increased intracellular lipid load. To determine the mechanism by which M. leprae modifies the lipid homeostasis in host cells, an in vitro M. leprae infection system, using human macrophage precursor THP-1 cells and M. leprae prepared from the footpads of nude mice, was employed. RNA extracted from skin smear samples of patients was used to investigate host gene expressions before and after multidrug therapy (MDT). We found that a cluster of peroxisome proliferator-activated receptor (PPAR) target genes associated with adipocyte differentiation were strongly induced in M. leprae-infected THP-1 cells, with increased intracellular lipid accumulation. PPAR-δ and PPAR-γ expressions were induced by M. leprae infection in a bacterial load-dependent manner, and their proteins underwent nuclear translocalization after infection, indicating activation of PPAR signaling in host cells. Either PPAR-δ or PPAR-γ antagonist abolished the effect of M. leprae to modify host gene expressions and inhibited intracellular lipid accumulation in host cells. M. leprae-specific gene expressions were detected in the skin smear samples both before and after MDT, whereas PPAR target gene expressions were dramatically diminished after MDT. These results suggest that M. leprae infection activates host PPAR signaling to induce an array of adipocyte differentiation-associated genes, leading to accumulation of intracellular lipids to accommodate M. leprae parasitization. Certain PPAR target genes in skin lesions may serve as biomarkers for monitoring treatment efficacy.

Paleopathological evidence and detection of Mycobacterium leprae DNA from archaeological skeletal remains of Nabe-kaburi (head-covered with iron pots) burials in Japan.

The Nabe-kaburi is a unique burial method, the purpose of which is shrouded in mystery. The burials were performed during the 15(th) to 18(th) centuries in eastern Japan, and involved covering the heads of the deceased with iron pots or mortars. The identification of leprosy-specific osteological lesions among some of the excavated remains has led to the suggestion that Nabe-kaburi burials were a reflection of the social stigma against certain infectious diseases, such as leprosy, tuberculosis or syphilis. However, molecular evidence for the presence of disease has been lacking. The goal of this study was to detect Mycobacterium leprae (M. leprae) DNA in archaeological human skeletal remains from Nabe-kaburi burials. The paleopathological data from three Nabe-kaburi burials were re-evaluated before small samples were taken from affected and control areas. DNA was extracted and used as a template to target the M. leprae-specific DNA using a combination of whole genome amplification, PCR analysis and DNA sequencing. M. leprae DNA fragments were detected in the two sets of skeletal remains that had also shown paleopathological evidence of leprosy. These findings provide definitive evidence that some of the Nabe-kaburi burials were performed for people affected by leprosy. Demonstration of the presence of M. leprae DNA, combined with archeological and anthropological examinations, will aid in solving the mystery of why Nabe-kaburi burials were performed in medieval Japan.

Clofazimine modulates the expression of lipid metabolism proteins in Mycobacterium leprae-infected macrophages.

Mycobacterium leprae (M. leprae) lives and replicates within macrophages in a foamy, lipid-laden phagosome. The lipids provide essential nutrition for the mycobacteria, and M. leprae infection modulates expression of important host proteins related to lipid metabolism. Thus, M. leprae infection increases the expression of adipophilin/adipose differentiation-related protein (ADRP) and decreases hormone-sensitive lipase (HSL), facilitating the accumulation and maintenance of lipid-rich environments suitable for the intracellular survival of M. leprae. HSL levels are not detectable in skin smear specimens taken from leprosy patients, but re-appear shortly after multidrug therapy (MDT). This study examined the effect of MDT components on host lipid metabolism in vitro, and the outcome of rifampicin, dapsone and clofazimine treatment on ADRP and HSL expression in THP-1 cells. Clofazimine attenuated the mRNA and protein levels of ADRP in M. leprae-infected cells, while those of HSL were increased. Rifampicin and dapsone did not show any significant effects on ADRP and HSL expression levels. A transient increase of interferon (IFN)-ß and IFN-γ mRNA was also observed in cells infected with M. leprae and treated with clofazimine. Lipid droplets accumulated by M. leprae-infection were significantly decreased 48 h after clofazimine treatment. Such effects were not evident in cells without M. leprae infection. In clinical samples, ADRP expression was decreased and HSL expression was increased after treatment. These results suggest that clofazimine modulates lipid metabolism in M. leprae-infected macrophages by modulating the expression of ADRP and HSL. It also induces IFN production in M. leprae-infected cells. The resultant decrease in lipid accumulation, increase in lipolysis, and activation of innate immunity may be some of the key actions of clofazimine.

Essential role of hormone-sensitive lipase (HSL) in the maintenance of lipid storage in Mycobacterium leprae-infected macrophages.

Mycobacterium leprae (M. leprae), the causative agent of leprosy, parasitizes within the foamy or enlarged phagosome of macrophages where rich lipids accumulate. Although the mechanisms for lipid accumulation in the phagosome have been clarified, it is still unclear how such large amounts of lipids escape degradation. To further explore underlying mechanisms involved in lipid catabolism in M. leprae-infected host cells, we examined the expression of hormone-sensitive lipase (HSL), a key enzyme in fatty acid mobilization and lipolysis, in human macrophage THP-1 cells. We found that infection by live M. leprae significantly suppressed HSL expression levels. This suppression was not observed with dead M. leprae or latex beads. Macrophage activation by peptidoglycan (PGN), the ligand for toll-like receptor 2 (TLR2), increased HSL expression; however, live M. leprae suppressed this increase. HSL expression was abolished in the slit-skin smear specimens from patients with lepromatous and borderline leprosy. In addition, the recovery of HSL expression was observed in patients who experienced a lepra reaction, which is a cell-mediated, delayed-type hypersensitivity immune response, or in patients who were successfully treated with multi-drug therapy. These results suggest that M. leprae suppresses lipid degradation through inhibition of HSL expression, and that the monitoring of HSL mRNA levels in slit-skin smear specimens may be a useful indicator of patient prognosis.

Chimpanzees used for medical research shed light on the pathoetiology of leprosy.

Leprosy is a chronic infectious disorder caused by Mycobacterium leprae, which mainly affects skin and peripheral nerves. It is classified as either paucibacillary or multibacillary based upon clinical manifestations and slit-skin smear results. It is speculated that leprosy develops after a long latency period following M. leprae infection. However, the actual time of infection and the duration of latency have never been proven in human patients. To date, four cases of spontaneous leprosy have been reported in chimpanzees who were caught in West Africa in infancy and used for medical research in the USA and Japan. One of these chimpanzees was extensively studied in Japan, and single-nucleotide polymorphism analysis for the M. leprae genome was conducted. This analysis revealed that the chimpanzee was infected with M. leprae during infancy in West Africa and the pathognomonic signs of leprosy appeared after at least 30 years of incubation. Analysis of leprosy in chimpanzees can contribute not only to medical research but also to the understanding of the pathoetiology of leprosy.

[Leprosy in a chimpanzee].

Leprosy is suspected to develop after a long period of latency following infection with Mycobacterium leprae (M. leprae) during infancy, but definitive proof has been lacking. We found a rare case of leprosy in a chimpanzee (Pan troglodytes) born in West Africa (Sierra Leone) and brought to Japan around 2 years of age. At 31, the ape started exhibiting pathognomic signs of leprosy. Pathological diagnosis, skin smear, serum anti-phenolic glycolipid-I (PGL-I) antibody, and by PCR analysis demonstrated lepromatous leprosy. Single-nucleotide polymorphism (SNP) analysis verified the West African origin of the bacilli. This occurrence suggests the possibility of leprosy being endemic among wild chimpanzees in West Africa, potentially posing a zoonotic risk.

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.

Infection during infancy and long incubation period of leprosy suggested in a case of a chimpanzee used for medical research.

The length of the incubation period of leprosy following Mycobacterium leprae infection has never been conclusively determined, owing to the lack of a method to demonstrate the presence of an asymptomatic infection. We report a rare case of leprosy in a chimpanzee in which a 30-year incubation period was strongly suggested by single nucleotide polymorphism (SNP) analysis.

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.