Mycobacterium leprae Transcriptome During In Vivo Growth and Ex Vivo Stationary Phases.

Mycobacterium leprae, the causative agent of leprosy, is an obligate intracellular pathogen primarily residing within host macrophages and Schwann cells. Whole genome sequencing predicts a highly degraded genome with approximately one third of the coding capacity resulting in the loss of many catabolic pathways. Therefore, it can be assumed that M. leprae obtains many of the necessary metabolites for intracellular survival and growth from the host cells. In this study, global transcriptomic analyses were done on freshly harvested M. leprae growing in athymic mouse footpads for five months (MFP5) and compared to those held in axenic medium for 48 (ML48) and 96 (ML96) hours. Results show that all of the genes and pseudogenes were transcribed under both in vivo and in vitro conditions. 24% and 33% of gene transcript levels were significantly altered in ML48 and ML96 respectively, compared to MFP5. Approximately 45% (39/86) of lipid metabolism genes were significantly downregulated in ML96 compared to MFP5, majority of which are in the ß-oxidation pathway. Cholesterol oxidase, acyl-CoA dehydrogenase, and coenzyme F420-dependent oxidoreductase, were significantly upregulated in both ML48 and ML96 compared to MFP5. 30% of cell wall and cell processes functional category genes had altered gene transcription at 96hr compared to MFP5. 40% of 57 genes associated with mycobacterial virulence showed significantly altered transcript levels with 52% significantly downregulated in ML96, including most of the Pro-Glu/Pro-Pro-Glu genes. All 111 hypothetical protein genes with unknown function were expressed. Adenosine triphosphate (ATP) synthesis in M. leprae appears to be significantly downregulated under ex vivo conditions. This is the first study comparing M. leprae global gene expression during in vivo growth and ex vivo stationery phase in axenic medium confirming that during the growth phase in the footpads of experimentally infected mice, M. leprae is metabolically active and its primary source of energy production is probably lipids.

GSMN-ML- a genome scale metabolic network reconstruction of the obligate human pathogen Mycobacterium leprae.

Leprosy, caused by Mycobacterium leprae, has plagued humanity for thousands of years and continues to cause morbidity, disability and stigmatization in two to three million people today. Although effective treatment is available, the disease incidence has remained approximately constant for decades so new approaches, such as vaccine or new drugs, are urgently needed for control. Research is however hampered by the pathogen's obligate intracellular lifestyle and the fact that it has never been grown in vitro. Consequently, despite the availability of its complete genome sequence, fundamental questions regarding the biology of the pathogen, such as its metabolism, remain largely unexplored. In order to explore the metabolism of the leprosy bacillus with a long-term aim of developing a medium to grow the pathogen in vitro, we reconstructed an in silico genome scale metabolic model of the bacillus, GSMN-ML. The model was used to explore the growth and biomass production capabilities of the pathogen with a range of nutrient sources, such as amino acids, glucose, glycerol and metabolic intermediates. We also used the model to analyze RNA-seq data from M. leprae grown in mouse foot pads, and performed Differential Producibility Analysis to identify metabolic pathways that appear to be active during intracellular growth of the pathogen, which included pathways for central carbon metabolism, co-factor, lipids, amino acids, nucleotides and cell wall synthesis. The GSMN-ML model is thereby a useful in silico tool that can be used to explore the metabolism of the leprosy bacillus, analyze functional genomic experimental data, generate predictions of nutrients required for growth of the bacillus in vitro and identify novel drug targets.

Isolation of Mycobacterium lepromatosis and Development of Molecular Diagnostic Assays to Distinguish Mycobacterium leprae and M. lepromatosis.

BACKGROUND: Mycobacterium leprae was thought to be the exclusive causative agent of leprosy until Mycobacterium lepromatosis was identified in a rare form of leprosy known as diffuse lepromatous leprosy (DLL). METHODS: We isolated M. lepromatosis from a patient with DLL and propagated it in athymic nude mouse footpads. Genomic analysis of this strain (NHDP-385) identified a unique repetitive element, RLPM, on which a specific real-time quantitative polymerase chain reaction assay was developed. The RLPM assay, and a previously developed RLEP quantitative polymerase chain reaction assay for M. leprae, were validated as clinical diagnostic assays according to Clinical Laboratory Improvement Amendments guidelines. We tested DNA from archived histological sections, patient specimens from the United States, Philippines, and Mexico, and US wild armadillos. RESULTS: The limit of detection for the RLEP and RLPM assays is 30 M. leprae per specimen (0.76 bacilli per reaction; coefficient of variation, 0.65%-2.44%) and 122 M. lepromatosis per specimen (3.05 bacilli per reaction; 0.84%-2.9%), respectively. In histological sections (n = 10), 1 lepromatous leprosy (LL), 1 DLL, and 3 Lucio reactions contained M. lepromatosis; 2 LL and 2 Lucio reactions contained M. leprae; and 1 LL reaction contained both species. M. lepromatosis was detected in 3 of 218 US biopsy specimens (1.38%). All Philippines specimens (n = 180) were M. lepromatosis negative and M. leprae positive. Conversely, 15 of 47 Mexican specimens (31.91%) were positive for M. lepromatosis, 19 of 47 (40.43%) were positive for M. leprae, and 2 of 47 (4.26%) contained both organisms. All armadillos were M. lepromatosis negative. CONCLUSIONS: The RLPM and RLEP assays will aid healthcare providers in the clinical diagnosis and surveillance of leprosy.

Dual RNA-Seq of Human Leprosy Lesions Identifies Bacterial Determinants Linked to Host Immune Response.

To understand how the interaction between an intracellular bacterium and the host immune system contributes to outcome at the site of infection, we studied leprosy, a disease that forms a clinical spectrum, in which progressive infection by the intracellular bacterium Mycobacterium leprae is characterized by the production of type I IFNs and antibody production. Dual RNA-seq on patient lesions identifies two independent molecular measures of M. leprae, each of which correlates with distinct aspects of the host immune response. The fraction of bacterial transcripts, reflecting bacterial burden, correlates with a host type I IFN gene signature, known to inhibit antimicrobial responses. Second, the bacterial mRNA:rRNA ratio, reflecting bacterial viability, links bacterial heat shock proteins with the BAFF-BCMA host antibody response pathway. Our findings provide a platform for the interrogation of host and pathogen transcriptomes at the site of infection, allowing insight into mechanisms of inflammation in human disease.

Dapsone Resistance in Leprosy Patients Originally from American Samoa, United States, 2010-2012.

Skin biopsies from US leprosy patients were tested for mutations associated with drug resistance. Dapsone resistance was found in 4 of 6 biopsies from American Samoa patients. No resistance was observed in patients from other origins. The high rate of dapsone resistance in patients from American Samoa warrants further investigation.

qPCR-High resolution melt analysis for drug susceptibility testing of Mycobacterium leprae directly from clinical specimens of leprosy patients.

BACKGROUND: Real-Time PCR-High Resolution Melting (qPCR-HRM) analysis has been recently described for rapid drug susceptibility testing (DST) of Mycobacterium leprae. The purpose of the current study was to further evaluate the validity, reliability, and accuracy of this assay for M. leprae DST in clinical specimens. METHODOLOGY/PRINCIPAL FINDINGS: The specificity and sensitivity for determining the presence and susceptibility of M. leprae to dapsone based on the folP1 drug resistance determining region (DRDR), rifampin (rpoB DRDR) and ofloxacin (gyrA DRDR) was evaluated using 211 clinical specimens from leprosy patients, including 156 multibacillary (MB) and 55 paucibacillary (PB) cases. When comparing the results of qPCR-HRM DST and PCR/direct DNA sequencing, 100% concordance was obtained. The effects of in-house phenol/chloroform extraction versus column-based DNA purification protocols, and that of storage and fixation protocols of specimens for qPCR-HRM DST, were also evaluated. qPCR-HRM results for all DRDR gene assays (folP1, rpoB, and gyrA) were obtained from both MB (154/156; 98.7%) and PB (35/55; 63.3%) patients. All PCR negative specimens were from patients with low numbers of bacilli enumerated by an M. leprae-specific qPCR. We observed that frozen and formalin-fixed paraffin embedded (FFPE) tissues or archival Fite's stained slides were suitable for HRM analysis. Among 20 mycobacterial and other skin bacterial species tested, only M. lepromatosis, highly related to M. leprae, generated amplicons in the qPCR-HRM DST assay for folP1 and rpoB DRDR targets. Both DNA purification protocols tested were efficient in recovering DNA suitable for HRM analysis. However, 3% of clinical specimens purified using the phenol/chloroform DNA purification protocol gave false drug resistant data. DNA obtained from freshly frozen (n = 172), formalin-fixed paraffin embedded (FFPE) tissues (n = 36) or archival Fite's stained slides (n = 3) were suitable for qPCR-HRM DST analysis. The HRM-based assay was also able to identify mixed infections of susceptible and resistant M. leprae. However, to avoid false positives we recommend that clinical specimens be tested for the presence of the M. leprae using the qPCR-RLEP assay prior to being tested in the qPCR-HRM DST and that all specimens demonstrating drug resistant profiles in this assay be subjected to DNA sequencing. CONCLUSION/SIGNIFICANCE: Taken together these results further demonstrate the utility of qPCR-HRM DST as an inexpensive screening tool for large-scale drug resistance surveillance in leprosy.

qPCR-High resolution melt analysis for drug susceptibility testing of Mycobacterium leprae directly from clinical specimens of leprosy patients

BACKGROUND: Real-Time PCR-High Resolution Melting (qPCR-HRM) analysis has been recently described for rapid drug susceptibility testing (DST) of Mycobacterium leprae. The purpose of the current study was to further evaluate the validity, reliability, and accuracy of this assay for M. leprae DST in clinical specimens. METHODOLOGY/PRINCIPAL FINDINGS: The specificity and sensitivity for determining the presence and susceptibility of M. leprae to dapsone based on the folP1 drug resistance determining region (DRDR), rifampin (rpoB DRDR) and ofloxacin (gyrA DRDR) was evaluated using 211 clinical specimens from leprosy patients, including 156 multibacillary (MB) and 55 paucibacillary (PB) cases. When comparing the results of qPCR-HRM DST and PCR/direct DNA sequencing, 100% concordance was obtained. The effects of in-house phenol/chloroform extraction versus column-based DNA purification protocols, and that of storage and fixation protocols of specimens for qPCR-HRM DST, were also evaluated. qPCR-HRM results for all DRDR gene assays (folP1, rpoB, and gyrA) were obtained from both MB (154/156; 98.7%) and PB (35/55; 63.3%) patients. All PCR negative specimens were from patients with low numbers of bacilli enumerated by an M. leprae-specific qPCR. We observed that frozen and formalin-fixed paraffin embedded (FFPE) tissues or archival Fite's stained slides were suitable for HRM analysis. Among 20 mycobacterial and other skin bacterial species tested, only M. lepromatosis, highly related to M. leprae, generated amplicons in the qPCR-HRM DST assay for folP1 and rpoB DRDR targets. Both DNA purification protocols tested were efficient in recovering DNA suitable for HRM analysis. However, 3% of clinical specimens purified using the phenol/chloroform DNA purification protocol gave false drug resistant data. DNA obtained from freshly frozen (n = 172), formalin-fixed paraffin embedded (FFPE) tissues (n = 36) or archival Fite's stained slides (n = 3) were suitable for qPCR-HRM DST analysis. The HRM-based assay was also able to identify mixed infections of susceptible and resistant M. leprae. However, to avoid false positives we recommend that clinical specimens be tested for the presence of the M. leprae using the qPCR-RLEP assay prior to being tested in the qPCR-HRM DST and that all specimens demonstrating drug resistant profiles in this assay be subjected to DNA sequencing. CONCLUSION/SIGNIFICANCE: Taken together these results further demonstrate the utility of qPCR-HRM DST as an inexpensive screening tool for large-scale drug resistance surveillance in leprosy.

Long-term survival and virulence of Mycobacterium leprae in amoebal cysts.

Leprosy is a curable neglected disease of humans caused by Mycobacterium leprae that affects the skin and peripheral nerves and manifests clinically in various forms ranging from self-resolving, tuberculoid leprosy to lepromatous leprosy having significant pathology with ensuing disfiguration disability and social stigma. Despite the global success of multi-drug therapy (MDT), incidences of clinical leprosy have been observed in individuals with no apparent exposure to other cases, suggestive of possible non-human sources of the bacteria. In this study we show that common free-living amoebae (FLA) can phagocytose M. leprae, and allow the bacillus to remain viable for up to 8 months within amoebic cysts. Viable bacilli were extracted from separate encysted cocultures comprising three common Acanthamoeba spp.: A. lenticulata, A. castellanii, and A. polyphaga and two strains of Hartmannella vermiformis. Trophozoites of these common FLA take up M. leprae by phagocytosis. M. leprae from infected trophozoites induced to encyst for long-term storage of the bacilli emerged viable by assessment of membrane integrity. The majority (80%) of mice that were injected with bacilli extracted from 35 day cocultures of encysted/excysted A. castellanii and A. polyphaga showed lesion development that was similar to mice challenged with fresh M. leprae from passage mice albeit at a slower initial rate. Mice challenged with coculture-extracted bacilli showed evidence of acid-fast bacteria and positive PCR signal for M. leprae. These data support the conclusion that M. leprae can remain viable long-term in environmentally ubiquitous FLA and retain virulence as assessed in the nu/nu mouse model. Additionally, this work supports the idea that M. leprae might be sustained in the environment between hosts in FLA and such residence in FLA may provide a macrophage-like niche contributing to the higher-than-expected rate of leprosy transmission despite a significant decrease in human reservoirs due to MDT.

Semi-automated protocol for purification of Mycobacterium leprae from tissues using the gentleMACS™ Octo Dissociator.

Mycobacterium leprae, etiologic agent of leprosy, is propagated in athymic nude mouse footpads (FPs). The current purification protocol is tedious and physically demanding. A simpler, semi-automated protocol was developed using gentleMACS™ Octo Dissociator. The gentleMACS protocol provided a very effective means for purification of highly viable M. leprae from tissue.

Drug resistance in patients with leprosy in the United States.

Molecular drug susceptibility testing was performed on 39 US patients with leprosy. Of these, 2 had dapsone-resistant Mycobacterium leprae and 1 of these patients also had rifampin-resistant M. leprae. Even though antileprosy drug resistance occurs in this leprosy population, resistance does not appear to be a major problem.

Molecular assays for determining Mycobacterium leprae viability in tissues of experimentally infected mice.

BACKGROUND: The inability of Mycobacterium leprae to grow on axenic media has necessitated specialized techniques in order to determine viability of this organism. The purpose of this study was to develop a simple and sensitive molecular assay for determining M. leprae viability directly from infected tissues. METHODOLOGY/PRINCIPLE FINDINGS: Two M. leprae-specific quantitative reverse transcription PCR (qRT-PCR) assays based on the expression levels of esxA, encoding the ESAT-6 protein, and hsp18, encoding the heat shock 18 kDa protein, were developed and tested using infected footpad (FP) tissues of both immunocompetent and immunocompromised (athymic nu/nu) mice. In addition, the ability of these assays to detect the effects of anti-leprosy drug treatment on M. leprae viability was determined using rifampin and rifapentine, each at 10 mg/kg for 1, 5, or 20 daily doses, in the athymic nu/nu FP model. Molecular enumeration (RLEP PCR) and viability determinations (qRT-PCR) were performed via Taqman methodology on DNA and RNA, respectively, purified from ethanol-fixed FP tissue and compared with conventional enumeration (microscopic counting of acid fast bacilli) and viability assays (radiorespirometry, viability staining) which utilized bacilli freshly harvested from the contralateral FP. Both molecular and conventional assays demonstrated growth and high viability of M. leprae in nu/nu FPs over a 4 month infection period. In contrast, viability was markedly decreased by 8 weeks in immunocompetent mice. Rifapentine significantly reduced bacterial viability after 5 treatments, whereas rifampin required up to 20 treatments for the same efficacy. Neither drug was effective after a single treatment. In addition, host gene expression was monitored with the same RNA preparations. CONCLUSIONS: hsp18 and esxA qRT-PCR are sensitive molecular indicators, reliably detecting viability of M. leprae in tissues without the need for bacterial isolation or immediate processing, making these assays applicable for in vivo drug screening and promising for clinical and field applications.

The armadillo: a model for the neuropathy of leprosy and potentially other neurodegenerative diseases.

Leprosy (also known as Hansen's disease) is an infectious peripheral neurological disorder caused by Mycobacterium leprae that even today leaves millions of individuals worldwide with life-long disabilities. The specific mechanisms by which this bacterium induces nerve injury remain largely unknown, mainly owing to ethical and practical limitations in obtaining affected human nerve samples. In addition to humans, nine-banded armadillos (Dasypus novemcinctus) are the only other natural host of M. leprae, and they develop a systemically disseminated disease with extensive neurological involvement. M. leprae is an obligate intracellular parasite that cannot be cultivated in vitro. Because of the heavy burdens of bacilli they harbor, nine-banded armadillos have become the organism of choice for propagating large quantities of M. leprae, and they are now advancing as models of leprosy pathogenesis and nerve damage. Although armadillos are exotic laboratory animals, the recently completed whole genome sequence for this animal is enabling researchers to undertake more sophisticated molecular studies and to develop armadillo-specific reagents. These advances will facilitate the use of armadillos in piloting new therapies and diagnostic regimens, and will provide new insights into the oldest known infectious neurodegenerative disorder.

Increased CXC ligand 10 levels and gene expression in type 1 leprosy reactions.

Type 1 reaction (T1R) is a systemic inflammatory syndrome causing substantial morbidity in leprosy. T1R results from spontaneously enhanced cellular immunity in borderline types of leprosy, but there are no established laboratory markers for the reaction. Preliminary studies have identified elevated circulating CXC ligand 10 (CXCL10) during T1R. Correlation of CXCL10 with clinical T1R was studied in repeated serum specimens obtained before, during, and after T1R. CXCL10 gene expression was assessed in biopsy specimens taken before and during T1R, and sections were stained for the cytokine using monoclonal antibodies. Sequential serum specimens revealed elevation of circulating CXCL10 associated with episodes of T1R (P = 0.0001) but no evidence of an earlier, predictive change in the level of the chemokine. Reverse transcriptase (RT)-PCR revealed elevated expression of CXCL10 transcripts during T1R, but not in patients who did not have T1R. No significant correlation between CXCL10 and gamma interferon (IFN-γ) mRNA levels was observed. Immunohistochemical staining of the skin biopsy specimens suggested an overall increase in CXCL10 but did not identify a particular strongly staining population of leukocytes. Increased CXCL10 in lesions and serum is characteristic of T1R. CXCL10 measurement offers new possibilities for laboratory diagnosis and monitoring of T1R. Studies of the regulation of CXCL10 may provide insight into the mechanisms of T1R and identify potential new drug targets for treatment.

Implications of high level pseudogene transcription in Mycobacterium leprae.

BACKGROUND: The Mycobacterium leprae genome has less than 50% coding capacity and 1,133 pseudogenes. Preliminary evidence suggests that some pseudogenes are expressed. Therefore, defining pseudogene transcriptional and translational potentials of this genome should increase our understanding of their impact on M. leprae physiology. RESULTS: Gene expression analysis identified transcripts from 49% of all M. leprae genes including 57% of all ORFs and 43% of all pseudogenes in the genome. Transcribed pseudogenes were randomly distributed throughout the chromosome. Factors resulting in pseudogene transcription included: 1) co-orientation of transcribed pseudogenes with transcribed ORFs within or exclusive of operon-like structures; 2) the paucity of intrinsic stem-loop transcriptional terminators between transcribed ORFs and downstream pseudogenes; and 3) predicted pseudogene promoters. Mechanisms for translational "silencing" of pseudogene transcripts included the lack of both translational start codons and strong Shine-Dalgarno (SD) sequences. Transcribed pseudogenes also contained multiple "in-frame" stop codons and high Ka/Ks ratios, compared to that of homologs in M. tuberculosis and ORFs in M. leprae. A pseudogene transcript containing an active promoter, strong SD site, a start codon, but containing two in frame stop codons yielded a protein product when expressed in E. coli. CONCLUSION: Approximately half of M. leprae's transcriptome consists of inactive gene products consuming energy and resources without potential benefit to M. leprae. Presently it is unclear what additional detrimental affect(s) this large number of inactive mRNAs has on the functional capability of this organism. Translation of these pseudogenes may play an important role in overall energy consumption and resultant pathophysiological characteristics of M. leprae. However, this study also demonstrated that multiple translational "silencing" mechanisms are present, reducing additional energy and resource expenditure required for protein production from the vast majority of these transcripts.

Molecular determination of Mycobacterium leprae viability by use of real-time PCR.

Mycobacterium leprae, the etiological agent of leprosy, is noncultivable on axenic media. Therefore, the viability of M. leprae for clinical or experimental applications is often unknown. To provide new tools for M. leprae viability determination, two quantitative reverse transcriptase PCR (RT-PCR) assays were developed and characterized. M. leprae sodA mRNA and 16S rRNA were used as RNA targets, and M. leprae repetitive element (RLEP) DNA was used to determine relative bacterial numbers in the same purified bacterial preparations or from crude biological specimens. Results demonstrated that both assays were good predictors of M. leprae viability during short-term experiments (48 h) involving rifampin (rifampicin) treatment in axenic medium, within rifampin-treated murine macrophages (MPhi), or within immune-activated MPhi. Moreover, these results strongly correlated those of other M. leprae viability assays, including radiorespirometry-based and Live/Dead BacLight viability assays. The 16S rRNA/RLEP assay consistently identified the presence of M. leprae in eight multibacillary leprosy patient biopsy specimens prior to multidrug therapy (MDT) and demonstrated a decline in viability during the course of MDT. In contrast, the sodA/RLEP assay was able to detect the presence of M. leprae in only 25% of pretreatment biopsy specimens. In conclusion, new tools for M. leprae viability determination were developed. The 16S rRNA/RLEP RT-PCR M. leprae viability assay should be useful both for short-term experimental purposes and for predicting M. leprae viability in biopsy specimens to monitor treatment efficacy, whereas the sodA/RLEP RT-PCR M. leprae viability assay should be limited to short-term experimental research purposes.

Molecular basis of the defective heat stress response in Mycobacterium leprae.

Mycobacterium leprae, a major human pathogen, grows poorly at 37 degrees C. The basis for its inability to survive at elevated temperatures was investigated. We determined that M. leprae lacks a protective heat shock response as a result of the lack of transcriptional induction of the alternative sigma factor genes sigE and sigB and the major heat shock operons, HSP70 and HSP60, even though heat shock promoters and regulatory circuits for these genes appear to be intact. M. leprae sigE was found to be capable of complementing the defective heat shock response of mycobacterial sigE knockout mutants only in the presence of a functional mycobacterial sigH, which orchestrates the mycobacterial heat shock response. Since the sigH of M. leprae is a pseudogene, these data support the conclusion that a key aspect of the defective heat shock response in M. leprae is the absence of a functional sigH. In addition, 68% of the genes induced during heat shock in M. tuberculosis were shown to be either absent from the M. leprae genome or were present as pseudogenes. Among these is the hsp/acr2 gene, whose product is essential for M. tuberculosis survival during heat shock. Taken together, these results suggest that the reduced ability of M. leprae to survive at elevated temperatures results from the lack of a functional transcriptional response to heat shock and the absence of a full repertoire of heat stress response genes, including sigH.

Identification of specific proteins and peptides in Mycobacterium leprae suitable for the selective diagnosis of leprosy.

Diagnosis of leprosy is a major obstacle to disease control and has been compromised in the past due to the lack of specific reagents. We have used comparative genome analysis to identify genes that are specific to Mycobacterium leprae and tested both recombinant proteins and synthetic peptides from a subset of these for immunological reactivity. Four unique recombinant proteins (ML0008, ML0126, ML1057, and ML2567) and a panel of 58 peptides (15 and 9 mer) were tested for IFN-gamma responses in PBMC from leprosy patients and contacts, tuberculosis patients, and endemic and nonendemic controls. The responses to the four recombinant proteins gave higher levels of IFN-gamma production, but less specificity, than the peptides. Thirty-five peptides showed IFN-gamma responses only in the paucibacillary leprosy and household contact groups, with no responses in the tuberculosis or endemic control groups. High frequencies of IFN-gamma-producing CD4+ and CD8+ T cells specific for the 15- and 9-mer peptides were observed in the blood of a paucibacillary leprosy patient. 9-mer peptides preferentially activated CD8+ T cells, while the 15-mer peptides were efficient in inducing responses in both the CD4+ and CD8+ T cell subsets. Four of the six 9-mer peptides tested showed promising specificity, indicating that CD8+ T cell epitopes may also have diagnostic potential. Those peptides that provide specific responses in leprosy patients from an endemic setting could potentially be developed into a rapid diagnostic test for the early detection of M. leprae infection and epidemiological surveys of the incidence of leprosy, of which little is known.

Postgenomic approach to identify novel Mycobacterium leprae antigens with potential to improve immunodiagnosis of infection.

Early detection of Mycobacterium leprae infection is considered an important component of strategies aiming at reducing transmission of infection, but currently available diagnostic tools often lack sufficient sensitivity and specificity to reach this goal. Recent comparative genomics have revealed the presence of 165 M. leprae genes with no homologue in M. tuberculosis. We selected 17 of these genes for further study. All 17 genes were found to be expressed at the mRNA level in M. leprae from infected mice and from a multibacillary leprosy patient. Additional comparative genomic analyses of all currently available mycobacterial genome databases confirmed 12 candidate genes to be unique to M. leprae, whereas 5 genes had homologues in mycobacteria other than M. tuberculosis. Evaluation of the immunogenicity of all 17 recombinant proteins in PBMC from 127 Brazilians showed that five antigens (ML0576, ML1989, ML1990, ML2283, and ML2567) induced significant gamma interferon levels in paucibacillary leprosy patients, reactional leprosy patients, and exposed healthy controls but not in most multibacillary leprosy patients, tuberculosis patients, or endemic controls. Importantly, among exposed healthy controls 71% had no detectable immunoglobulin M antibodies to the M. leprae-specific PGL-I but responded to one or more M. leprae antigen(s). Collectively, the M. leprae proteins identified are expressed at the transcriptome level and can efficiently activate T cells of M. leprae-exposed individuals. These proteins may provide new tools to develop tests for specific diagnosis of M. leprae infection and may enhance our understanding of leprosy and its transmission.

On the origin of leprosy.

Leprosy, a chronic human disease with potentially debilitating neurological consequences, results from infection with Mycobacterium leprae. This unculturable pathogen has undergone extensive reductive evolution, with half of its genome now occupied by pseudogenes. Using comparative genomics, we demonstrated that all extant cases of leprosy are attributable to a single clone whose dissemination worldwide can be retraced from analysis of very rare single-nucleotide polymorphisms. The disease seems to have originated in Eastern Africa or the Near East and spread with successive human migrations. Europeans or North Africans introduced leprosy into West Africa and the Americas within the past 500 years.