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Previous Article | Next Article 
Clinical Microbiology Reviews, April 2006, p. 338-381, Vol. 19, No. 2
0893-8512/06/$08.00+0 doi:10.1128/CMR.19.2.338-381.2006
Laboratory Research Branch, National Hansen's Disease Programs, Louisiana State University-SVM, Baton Rouge, Louisiana 70803
SUMMARY INTRODUCTION Basic Clinical and Immunopathological Features of Leprosy Lepromin Test Leprosy in Immunocompromised Individuals Laboratory Tests for the Diagnosis of Leprosy MYCOBACTERIUM LEPRAE, THE ETIOLOGIC AGENT OF LEPROSY Basic Characteristics Cellular morphology. Growth. Metabolism. Genome, Transcriptome, and Proteome Genome. Transcriptome. Proteome. Molecular Identification by PCR Epidemiology and Strain Identification EXPERIMENTAL MODELS OF LEPROSY Overcoming Obstacles to Leprosy Research Mouse Footpads and Nude Mice Gene Knockout Mice Nine-Banded Armadillo HOST RESPONSE TO M. LEPRAE Genetic Influences on Leprosy in Humans Genetic influences on innate resistance to M. leprae. (i) PARK2/PACRG. (ii) NRAMP1. Genetic influences on acquired immune responses in leprosy. (i) HLA. (ii) Chromosome 10p13. (iii) TAP. (iv) TNFA. (v) TLRs. (vi) VDR. Development of the Immune Response Innate immunity. (i) Antigen-presenting cells and dendritic cells. (ii) Pattern recognition receptors. (iii) C-type lectin receptors. (iv) Toll-like receptors. (v) C' receptors. Adaptive immunity: development of cell-mediated immunity. (i) Protective and destructive effects of cell-mediated immunity. (ii) T-lymphocyte populations. (iii) Cytotoxic cells. (iv) Macrophages. (v) Cytokines in leprosy. LEPROSY REACTIONS Clinical Presentation, Diagnosis, and Histopathology Immunological Features of Reactions Evidence regarding the mechanisms of leprosy reactions. (i) Type 1 (reversal) reactions. (ii) Type 2 leprosy reaction (erythema nodosum leprosum). Evidence from therapeutic trials regarding mechanisms of reactions. MECHANISMS OF NERVE INJURY Localization of M. leprae to Peripheral Nerves M. leprae Interactions with Schwann Cells Adhesion to Schwann cells. Ingestion by SCs. Effects of SCs on M. leprae. Effects of M. leprae on SCs. Immune responses and SCs. CHEMOTHERAPY Current Therapies and Drug Resistance Molecular Mechanisms of Drug Resistance Development of Drug Resistance in M. leprae Detection of Drug-Resistant Leprosy PCR-direct DNA sequencing. PCR-SSCP. PCR-solid-phase hybridization. PCR-heteroduplex analysis. PREVENTION: THE QUEST FOR A LEPROSY VACCINE SUMMARY AND CONCLUSIONS Future of Leprosy Treatment and Research ACKNOWLEDGMENTS REFERENCES
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| INTRODUCTION |
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Leprosy is not going to disappear anytime soon. Effective multidrug regimens are now used worldwide, and the infection in individuals is curable. However, although the reported number of registered cases worldwide has declined in the last two decades, the reported number of new cases registered each year has remained the same (at 500,000 to 700,000) over the same interval (42, 237). In some countries where leprosy is endemic the number of new cases actually appears to be increasing, while in others decreasing trends are reported. Great caution must be used in reaching conclusions from these observations, however, because they are based entirely on operational data which reflect the intensity of ongoing work more than the extent of any given problem (112). Mathematical modeling of the potential decline in leprosy incidence and prevalence, using various premises regarding efficacy of treatment and prevention, suggests that the disease will remain a major public health problem for at least several decades (259).
The precise mechanism of transmission of Mycobacterium leprae is unknown. No highly effective vaccine has yet been developed, and extensive laboratory efforts have not yet produced any practical tools for early diagnosis of clinically unapparent disease.
The full genome of M. leprae was among the first to be sequenced, and this new knowledge is beginning to bear fruit. Molecular microbiology has begun to explain, for example, M. leprae's fastidious nature and predilection for an intracellular lifestyle. Similarly, recent human genetic studies have been highly informative, indicating that immunity to M. leprae is controlled at two fundamental levels: first, genetic determinants of overall susceptibility and resistance to this organism have now been described, and second, a range of HLA-D-related immune responses have been demonstrated among individuals who are infected.
Only recently has the probable mechanism of intracellular killing of M. leprae been identified. The regulation of cell-mediated immunity to M. leprae by cellular and cytokine interactions continues to be unraveled. The major animal models available are the nine-banded armadillo and footpad infection of normal or immunologically crippled (nu–/–) mice. These models, however, are seriously flawed in their ability to recapitulate many aspects of the human disease and are exceptionally slow, difficult, and expensive to employ. Leprosy therefore remains a medical and scientific challenge of the first order, even though support for research on this disease has declined substantially as other conditions have assumed greater global priority.
A great deal of important new information has been generated by recent research. Brief, authoritative overviews on progress in leprosy have been published in recent years, notably those of Jacobson and Krahenbuhl (167) and Britton and Lockwood (42). Specialized reviews of narrower scope are cited in the appropriate sections below. Here, we have attempted to provide a critical summary of current knowledge from basic and clinical research, focusing particularly on developments from 1990 to the present.
The five-part Ridley-Jopling classification identifies, at one extreme, patients with a high degree of cell-mediated immunity and delayed hypersensitivity, presenting with a single, well-demarcated lesion with central hypopigmentation and hypoesthesia. Biopsies of these reveal well-developed granulomatous inflammation and rare acid-fast bacilli demonstrable in the tissues; this is termed the polar tuberculoid (TT) (Fig. 1). At the other extreme, patients have no apparent resistance to M. leprae. These patients present with numerous, poorly demarcated, raised or nodular lesions on all parts of the body, biopsies of which reveal sheets of foamy macrophages in the dermis containing very large numbers of bacilli and microcolonies called globi. This nonresistant, highly infected form of the disease is termed polar lepromatous (LL). The majority of patients, however, fall into a broad borderline category between these two polar forms; this is subdivided into borderline lepromatous (BL), mid-borderline (BB), and borderline tuberculoid (BT).
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In spite of nearly three decades of intensive research into the immunology of leprosy, the mechanism by which M. leprae is able to elicit the entire range of human cellular immune responses has still not been explained. Most clinical immunological inquiries have focused on the "immunologic defect" of lepromatous patients, i.e., their apparently specific anergy to M. leprae. The broad research efforts of recent years have, however, provided an increasingly detailed description of the immunological components in skin lesions across the leprosy spectrum, detailed below under Development of the Immune Response.
Leprosy bacilli, derived from different sources and subjected to different purification procedures, are the basis for different types of preparations used for intradermal skin testing (227). Themost frequently used preparation, and the one for which the response is best characterized, is Mitsuda lepromin. This is a suspension of whole, autoclaved leprosy bacilli (357) that is injected intradermally. Early studies used bacilli isolated directly from human lepromatous lesions, but armadillo-derived organisms have been used exclusively since the 1970s. In recent years, Mitsuda lepromin has been distributed for research applications by the World Health Organization. This skin test material is not approved by the Food and Drug Administration and is not recommended or provided for diagnostic use in the United States by the National Hansen's Disease Programs. Studies are under way to try to identify defined protein antigens that might be useful as diagnostic reagents (37, 94), but none of these has yet been determined to be satisfactorily sensitive or specific for this purpose.
Although the response to Mitsuda lepromin is not leprosy specific, a negative response is associated with lepromatous types of leprosy, i.e., with an inability to respond to M. leprae and to eliminate the bacilli. A positive lepromin test (at 4 weeks) is associated with the ability to develop a granulomatous response, involving antigen-presenting cells and CD4+ lymphocyte participation and, in leprosy patients, successful elimination of bacilli (121, 299).
Lepromin is probably the only widely studied skin test antigen that reflects the ability of an individual to generate a granulomatous response to mycobacterial antigens (as opposed to the 48- to 72-h delayed hypersensitivity response to tuberculin and other skin tests). For this reason, the possibility of genetic influences on lepromin responsiveness has been of interest to geneticists concerned with the inheritance of immunologic aspects of the granulomatous response (8, 30, 107).
In contrast to all other experience with mycobacterial infections in HIV-positive individuals, coinfection with M. leprae and HIV appears to have minimal effect upon the course of either leprosy or HIV/AIDS. This is best illustrated by studies following cohorts of infected patients (162; reviewed in reference 221).
The occurrence of leprosy reactions in HIV-positive patients with leprosy has been the focus of several reports (18, 31, 34, 50, 230, 298, 300), but since leprosy reactions affect a high percentage of all leprosy patients (see Leprosy Reactions, below), it is not clear that they are actually more frequent or more severe in HIV-positive individuals. Studies of cell phenotypes and cytokines in leprosy lesions in patients with and without HIV infection have found no significant differences between the two groups with respect to these immunologic parameters (136, 298, 329). It is possible that the very slow growth of M. leprae allows the host immune response to keep pace with this infection to a much greater degree than is the case with M. tuberculosis, M. avium, and the other mycobacterial pathogens in AIDS. Notably, however, infection with M. leprae may elicit antibodies cross-reactive with HIV screening assays (13, 15, 205, 372).
Treatment of HIV infection with highly active antiretroviral therapy has resulted in the emergence of previously unsuspected leprosy in a small number of reported cases (77, 230) and notably, many of these individuals have also developed type 1 reactions. This suggests that infection with M. leprae may be more common than has been documented among HIV-positive individuals in leprosy-endemic regions of the world, but that early leprosy lesions are overlooked when these patients are confronted by the other major, life-threatening complications of AIDS.
Interestingly, immunosuppression with
humanized monoclonal antibodies to tumor necrosis factor alpha
(TNF-
) has resulted in the rapid development of lepromatous
leprosy in at least two individuals who were being treated for severe
arthritis
(355a).These patients are presumed to have had minor, undetected leprosy
lesions prior to the administration of immunosuppressive treatment.
They responded promptly to antimicrobial treatment for M.
leprae with a multidrug regimen (see Chemotherapy,below), but did develop type 1 reactions during their recovery, as
noted above with AIDS patients receiving highly active antiretroviral
therapy. Similarly, a small number of patients who have been
immunosuppressed for renal or heart transplantation have developed
leprosy (266; Scollard,
unpublished observations), and these have also responded well to
antileprosy treatment.
Together, the evidence indicates that broad therapeutic immunosuppression does render individuals highly susceptible to infection with M. leprae, but that in HIV-positive individuals a degree of host response to M. leprae is maintained, comparable to that of non-HIV-infected individuals, even as HIV infection progresses and circulating CD4+ cell numbers decline. The experience with highly active antiretroviral therapy suggests that in leprosy-endemic areas, subclinical and early clinical infection with M. leprae may be more prevalent among HIV-positive individuals than has been generally recognized. In addition, it appears that the restoration of immune function after highly active antiretroviral therapy may be associated with the development of type 1 reactions.
An ancillary procedure, the slit-skin smear, can be used for the semiquantitative enumeration of acid-fast organisms in infected skin and is useful in follow-up of patients during and after treatment. This technique is reliable only when performed and interpreted by experienced technicians.
No serologic tests are available for the routine laboratory diagnosis of Hansen's disease, and no laboratories in the United States perform such assays routinely. Enzyme-linked immunosorbent assays and related immunoassays have been developed to detect antibodies to phenolic glycolipid 1 (PGL-1) of M. leprae (46, 97), and these have been used in epidemiological studies. Although they have some value in population follow-up studies, none of these assays has a satisfactory degree of sensitivity and specificity for diagnostic application. The greatest drawback to the serologic diagnosis of leprosy arises from the fact that patients in whom the diagnosis is most difficult, TT and BT patients with moderate to high-grade cellular immunity to M. leprae and only small numbers of organisms, do not reproducibly produce detectable, specific circulating antibodies.
Similarly, no skin test that enables the diagnosis of Hansen's disease has been developed. Intradermal injection of heat-killed M. leprae (the lepromin test, discussed above) reflects the ability of an individual to develop a granulomatous response to this organism, but it does not reflect infection by or even exposure to M. leprae. It has been used in epidemiological studies, but it has no diagnostic utility in individual cases, and it is not available in the United States.
M. leprae is not cultivable in vitro (see Basic Characteristics, below), and lack of growth on standard mycobacterial isolation media can be regarded as one laboratory criterion differentiating this organism from other mycobacterial pathogens. The major advance in the laboratory diagnosis of Hansen's disease in the last 15 years, however, has been the development of methods for the extraction, amplification, and identification of M. leprae DNA in clinical specimens using PCR and other molecular techniques. This is an invaluable addition to laboratory diagnosis and to studies of the basic microbiology of this uncultivable organism, although it is costly and has not yet been approved or become available as a routine clinical test. A detailed discussion of PCR evaluation of specimens for M. leprae DNA is presented below (see Molecular Identification by PCR, below).
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Annotation of M. leprae's genome and comparative genomic studies with other bacterial genomes have produced insight into the putative genes needed to direct the synthesis of this complex cell wall biopolymer (38). Most of the genes necessary to build the peptidoglycan-arabinogalactan-mycolate polymer appear to be present in the M. leprae genome and fit a reasonable strategy for its construction. A few exceptions are two genes involved in polyprenyl-phosphate synthesis (dxs-II and idi), a gene (fabH) involved in meromycolate synthesis, and a glycosyltransferase gene (pimB) involved in the biosynthesis of phosphatidylinositol, phosphatidylinositol mannosides, lipomannan, and lipoarabinomannan. It should be noted that much of this comparative work, while speculative, provides an important framework from which to investigate the authenticity of these putative pathways.
Growth. M. leprae has never been grown on artificial media but can be maintained in axenic cultures in what appears to be a stable metabolic state for a few weeks (414). As a result, propagation of M. leprae has been restricted to animal models, including the armadillo (415) and normal, athymic, and gene knockout mice (222). These systems have provided the basic resources for genetic, metabolic, and antigenic studies of the bacillus. Growth of M. leprae in mouse footpads also provides a tool for assessing the viability of a preparation of bacteria and testing the drug susceptibility of clinical isolates (364, 414). The viability of M. leprae harvested from several different sources is now known to vary greatly, and many standard laboratory practices, such as incubation at 37°C, rapidly reduce the viability of this organism (414). However, M. leprae stored at 33°C in 7H12 medium has been shown to remain viable for weeks.
Metabolism. The primary reasons for investigating the metabolic aspects of M. leprae have been to determine whether special media could be formulated to support in vitro growth of the bacilli and to learn more about metabolic pathways that could potentially be exploited for developing new antileprosy drugs. Early work provided a picture of a bacterium with some basic anabolic and catabolic pathways needed for survival in the host, but a thorough assessment of M. leprae's metabolic potential was still lacking. With the completed sequencing and annotation of M. leprae's genome, an improved understanding of M. leprae's metabolic capabilities now exists (43, 105, 432).
Annotation of the genome identified genes showing that M. leprae has the capacity to generate energy by oxidizing glucose to pyruvate through the Embden-Meyerhof-Parnas pathway, supporting earlier biochemical observations. Acetyl-coenzyme A from glycolysis enters the Krebs cycle, producing energy in the form of ATP. In addition to glycolysis for energy production, genome analysis as well as biochemical studies in M. leprae and M. tuberculosis suggest that these organisms rely heavily upon lipid degradation and the glyoxylate shunt for energy production. In this regard, M. leprae contains a full complement of genes for B oxidation but, compared to M. tuberculosis, very few genes capable of lipolysis. Acetate has been lost to M. leprae as a carbon source since only pseudogenes are present for acetate kinase, phosphate acetyltransferase, and acetyl-coenzyme A synthase.
Overall, M. leprae has many fewer enzymes involved in degradative pathways for carbon and nitrogenous compounds than M. tuberculosis. This is reflected in the paucity of oxidoreductases, oxygenases, and short-chain alcohol dehydrogenases and their probable regulatory genes. In addition, other major problems associated with metabolism for M. leprae are that the bacilli have lost anaerobic and microaerophilic electron transfer systems and that the aerobic respiratory chain is severely curtailed, making it impossible for M. leprae to generate ATP from the oxidation of NADH. In contrast to the reduction in catabolic pathways, the anabolic capabilities of M. leprae appear relatively unharmed. For example, complete pathways are predicted for synthesis of purines, pyrimidines, most amino acids, nucleosides, nucleotides, and most vitamins and cofactors. The maintenance of these anabolic systems suggests that the intracellular niche that M. leprae has found for itself may not contain these compounds or transport systems.
Comparison of M. leprae's genome with that of its close relative M. tuberculosis (Table 1) suggests that M. leprae has undergone an extreme case of reductive evolution (reviewed in references 74 and 104). This is reflected by its smaller genome (3.3 Mb for M. leprae versus 4.4 Mb for M. tuberculosis) and a major reduction in G+C content (58% for M. leprae versus 66% for M. tuberculosis). M. leprae's annotated genome contains only 1,614 open reading frames potentially encoding functional proteins, compared to 3,993 open reading frames predicted in M. tuberculosis (Table 1).
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Downsizing of the genome has resulted in the elimination of several metabolic pathways, leaving a pathogen with very specific growth requirements, as discussed above (Metabolism). The largest functional groups of genes in M. leprae are those involved in gene regulation, metabolism and modification of fatty acids and polyketides, cell envelope synthesis, and transport of metabolites (74, 104, 105). Defense against toxic radicals is severely degenerative, as neither katG nor the narGHJI cluster is functional. In addition, several other genes involved with detoxification are pseudogenes or are missing from the genome. Redundancy as seen in the M. tuberculosis genome is often lost in M. leprae, as most paralogues seen in M. tuberculosis are pseudogenes in M. leprae. None of the additional 142 genes found only in M. leprae appear to be associated with metabolic pathways.
M. leprae appears to have a major deficiency in its ability to acquire iron from its environment. The entire mbt operon is deleted, rendering it unable to make either the membrane-associated or excreted form of mycobactin T. While genes known to be involved in iron acquisition are not obvious in its genome, there is little doubt that M. leprae utilizes iron. Genes are present for cytochrome c (ccsAB), a ferredoxin (fdxCD), biosynthesis of the heme group (hem genes), a hemogloblin-like oxygen carrier (glbO), the iron storage bacterioferritin bfrA, and ideR, the iron regulation protein dependent on intracellular iron (432). There are no intact polymorphic G+C-rich sequence- or major polymorphic tandem repeat-related repetitive sequences in the genome of M. leprae and only a limited number of proline-proline-glutamic acid and proline-glutamic acid proteins (72). Annotation of M. leprae's genome has thus provided insight into why the leprosy bacillus is an obligate intracellular parasite and has provided the basis for future experimentation to better understand its pathogenicity.
Transcriptome. While comparative genome analysis provides useful clues to identify deficits in general cellular metabolic potential and cellular composition, it offers only a starting point from which functional studies can proceed. Because of our inability to cultivate M. leprae axenically, extremely limited quantities of bacterial proteins can be purified for analysis. Transcriptional analysis of M. leprae genes provides a perspective that is complementary to protein analysis by identifying actively transcribed genes, thereby expanding our knowledge of genes expressed during infection that might otherwise be missed when examining M. leprae's proteome.
With less than 50% coding capacity and 1,133 pseudogenes, those genes that are expressed help define the minimal gene set necessary for in vivo survival of this mycobacterial pathogen as well as genes potentially required for infection and pathogenesis as seen in leprosy. To identify genes transcribed during infection, gene transcripts from M. leprae growing in athymic nude mice have been surveyed using reverse transcription-PCR and cross-species DNA microarray technologies with an M. tuberculosis microarray (440). Transcripts were detected for 221 open reading frames, which include genes involved in DNA replication, cell division, SecA-dependent protein secretion, energy production, intermediary metabolism, and iron transport and storage and genes associated with virulence (see supplemental Table S1 at http://www.leprosy-ila.org/Mycobacterium.html).
These results support the view that M. leprae actively catabolizes fatty acids for energy, produces a wide array of secretory proteins, utilizes the limited array of sigma factors available, produces several proteins involved in iron transport, storage, and regulation (in the absence of recognizable genes encoding iron scavengers), and transcribes several genes associated with virulence in M. tuberculosis. Transcript levels of nine of these potential virulence genes (aceA, esat6, fbpA, relA, sigA, sigE, soda, aphC, and mce1A) were compared in M. leprae derived from the lesions of multibacillary leprosy patients and from infected nude mouse footpad tissue using quantitative real-time reverse transcription-PCR. Gene transcript levels were comparable in the two isolates for all but one of the genes studied (esat6), suggesting that profiling of M. leprae genes from animal models such as the nude mouse should be continued. Identifying genes associated with growth and survival during infection should lead to a more comprehensive understanding of M. leprae's ability to cause disease.
Proteome. The functional complement of any genome is the proteome, which consists of the proteins expressed by a given organism under defined conditions. Understanding the basis of M. leprae's growth, virulence, and immunogenicity has always been the focus of proteomic discovery, with the goal of developing strategies for improved treatment and control of leprosy. Early work was driven by efforts to produce vaccines and diagnostic reagents. Prior to the publication of the full DNA sequence and annotation of the M. leprae genome in 2001 (74) protein analysis of M. leprae relied primarily on traditional subcellular fractionation of purified bacilli and genomic library screening using immunologic reagents. Of the two strategies, immunologic screening of genomic libraries proved more fruitful and expanded the number of purified and characterized proteins to over 40 (41, 131, 338, 407). Immunologic screening took advantage of the fact that serum and T cells from leprosy patients or healthy individuals previously sensitized to M. leprae were abundant, providing powerful probes for antigenic proteins of M. leprae expressed in Escherichia coli.
A major drawback to immunologic screening was that nonimmunogenic proteins were missed. Clark-Curtiss used an alternative genomic screening approach in which M. leprae genes were examined for expression in E. coli. This led to the discovery of a 46-kDa protein from M. leprae capable of complementing a citrate synthase mutant of E. coli (165) and the demonstration that M. leprae promoter activity was possible in E. coli (356). Unfortunately, while genetic complementation in E. coli had been very successful for identifying genes in other enteric bacteria, it appeared that problems associated with either M. leprae gene expression in E. coli or the relatively large evolutionary distance between the two bacteria limited further application of the approach.
Brennan and coworkers have combined subcellular fractionation of M. leprae with immunologic screening and chemical analysis of purified proteins using microsequencing and mass spectrometry to expand our understanding of the cellular location and function of many M. leprae proteins. Proteins have been identified from major cellular compartments, including cytosol, membrane, and cell wall, and a comprehensive list of these proteins and their characteristics was published earlier (248).
Much of this work has now been assimilated into a larger framework established from the completion of the M. leprae genome sequence. Annotation of the M. leprae genome indicates that approximately 1,600 open reading frames are scattered among a decaying genome, including approximately 1,100 pseudogenes, with gene remnants making up the remaining 23.5% of the genome. By comparative genomic analysis with M. tuberculosis and other known gene sequences, it appears that approximately 50% of M. leprae's open reading frames can be assigned putative functions. The other half of M. leprae's genes are considered to encode hypothetical proteins, with a small percentage designated unknown genes.
The power of this new portrait of M. leprae helps integrate what the bacterium can and cannot do. For example, bioinformatic analyses of metabolic pathways indicated that M. leprae has lost many metabolic pathways, together with their regulatory circuits, particularly those involved with catabolic potential (104). Aided by this metabolic picture, researchers can now investigate gene expression as it relates to various metabolic conditions either in limited culture with M. leprae or by studying gene function in cultivable mycobacteria into which specific mutations have been introduced. In this way it may be possible to determine the conditions that enhance or suppress M. leprae viability when it is held in axenic culture.
Another important element of establishing the proteome of M. leprae impacts earlier work involving antigenic analysis of M. leprae with the purpose of identifying proteins useful for diagnostics and vaccines. For example, a group of M. leprae proteins that have gone understudied until recently is secreted proteins. Purified bacilli from armadillo, mouse, and human tissues by definition lack (or are significantly reduced in their concentration of) secreted proteins. Bioinformatic approaches have identified the basic genes necessary for a functional SecA-dependent secretory system in M. leprae (74). In addition, Williams and coworkers (440) have shown that all genes in the SecA-dependent pathway are transcribed during growth of M. leprae in nude mouse footpads and that some 25 proteins with potential for secretion are transcribed (see Transcriptome, above). Therefore, M. leprae has the potential to produce secreted proteins, most of which have not been studied for immunogenic potential. A full assessment of these proteins may give rise to important antigens useful for developing much-needed early diagnostic tests as well as therapeutic and prophylactic vaccines.
Finally, by virtue of its relatively small gene set and possibly smaller proteome, M. leprae has become an important model for conceptualizing the minimal gene set needed for obligate intracellular parasitism. It is unlikely that all of M. leprae's open reading frames are expressed during all phases of growth and parasitism. Teasing apart these intricate relationships should provide important insights into mechanisms of virulence, such as nerve infection, as well as identifying proteins expressed during early infection, which could give rise to better diagnostic reagents and potentially a vaccine to improve our chances of managing leprosy.
These assays have been based primarily on the amplification of M. leprae-specific sequences using PCR and identification of the M. leprae DNA fragment. This technique has been applied not only to skin biopsy samples but also to several different types of specimens, as indicated in Table 2. However, one should not infer from this that any tissue or specimen is suitable for PCR-based detection of M. leprae (see Table 3).
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Over the last two decades, a wide range of molecular tests have been applied to reveal genotypic variation in M. leprae. The results of initial studies suggested that the genome of M. leprae was highly conserved. Restriction fragment length polymorphism analysis of M. leprae isolates using a combination of restriction enzymes and probes and sequencing of the internal transcribed spacer region of the 16S-23S rRNA operon yielded no polymorphic DNA sequences (69, 92, 436). A polymorphic structure in the polA gene (119) and variation in a GACATC repeat in the rpoT gene (252) have been described, but the value of these elements for differentiating possible M. leprae strains appears to be limited.
In completing the sequence of the M. leprae genome, Cole et al. (74) identified several tandem repeats that could prove useful for discriminating M. leprae strains. Recently, Shin et al. reported evidence for diversity among M. leprae isolates obtained from several patient biopsy samples in the Philippines, based on the frequency of TTC repeats located downstream of a putative sugar transporter pseudogene (371). In addition to the TTC locus, in silico analysis of the genome sequence indicates that M. leprae has several other tandem repeat loci which may provide the genetic diversity necessary for creating a typing scheme capable of answering important questions related to the epidemiology of leprosy. Recent studies employing four different variable-number tandem repeat markers have successfully differentiated M. leprae strains used in the laboratory and successfully grouped identical samples and passage samples tested in blind panels (412).
Far less
diversity is seen with regard to single-nucleotide polymorphisms within
the genome. The M. leprae single-nucleotide polymorphism
frequency (
1 per 28 kb) is among the lowest seen for a human
pathogen, and only three informative single-nucleotide polymorphisms
have been identified. Among the 64 possible permutations of different
bases at each of these polymorphisms, only 4 are observed. The
variation in single-nucleotide polymorphism genotype is highly
correlated with the geographic origin of the strain, and analysis of
strains from different continents has been useful in predicting the
evolution and global spread of leprosy. The disease appears to have
originated in eastern Africa or the Near East and spread with
successive human immigrations
(272). Europeans and
North Africans appear to have introduced leprosy into West Africa and
the Americas within the last 500
years.
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One obstacle to the development of an animal model for leprosy has been the poor quality of the M. leprae inoculum, which usually consisted of fresh or frozen homogenates of nodules and lesions from untreated human lepromas. An important research emphasis in the National Hansen's Disease Program laboratories over the past few years has been the production, characterization, and provision of viable Mycobacterium leprae for our own researchers and qualified investigators around the world. A large (>200 mice) colony of M. leprae-infected athymic nu/nu mice is maintained for this purpose. A protocol of rigorously programmed passage of M. leprae, with radiorespirometry (the oxidation of 14C-labeled palmitic acid) as a measure of viability, has enabled the harvest, on a routine (weekly) schedule, of 4 to 6 billion bacilli that are 80 to 90% viable, a resource unprecedented in almost 130 years of leprosy research. With these organisms we have confirmed the preference of M. leprae for cooler temperatures (4°C for storage, 26°C to 33°C for metabolic activity) and the rapidly deleterious effects of incubation at 37°C or a single freezing-thawing cycle (414).
Whereas
radiorespirometry measures the metabolic activity of a suspension of
M. leprae, and this has been shown to be correlated with
growth in the mouse footpad, a novel, two-color fluorescence viability
staining assay (Molecular Probes BacLight bacterial viability kit) has
recently been adapted to provide a rapid (1-h), reliable,
quantitative, direct-count viability assay that measures the cell wall
integrity of individual bacilli
(229). This confirms
previous findings regarding biophysical optima for maintaining M.
leprae.
The second impediment in the early attempts to develop a leprosy model in animals was failure to recognize the prolonged growth cycle of M. leprae and not acknowledging its preference for cooler body sites. A new era was entered with description of the mouse footpad model by Shepard in 1960 (363). Passage of M. leprae infection was achieved, drug evaluation and rudimentary immunology studies became feasible, and the basis for subsequent exploration of M. leprae infection in various immunocompromised, transgenic, and knockout murine models was established.
The importance of the T lymphocyte in host resistance was revealed in experimental M. leprae infection of neonatally thymectomized or congenitally athymic mice and rats (75, 208, 315). In immunocompetent mice, an inoculum of a few thousand bacilli grows locally and plateaus at approximately 1 million organisms per footpad. There is virtually no disease in these footpads, and the histopathological changes are minor, consisting of small granulomas containing a few lymphocytes and very few bacilli. Furthermore, there is essentially no dissemination of infection. In athymic nu/nu mice, however, local footpad multiplication of M. leprae appears to be unimpeded, reaching 1010 or more bacilli per footpad.
Histopathologically,
the infected footpad tissue becomes an enormous foreign body-type
macrophage (M
) granuloma, or leproma, and the cells are
engorged with bacilli
(61) (Fig.
4). Unlike the course in an immunologically
intact mouse, some dissemination does occur, and if observed for a long
enough interval, evidence of growth in the opposite hind footpad or
forefeet is seen. Thus, development of this mouse model was a second
major milestone in leprosy research for, in addition to its
immunological significance, the athymic nu/nu mouse footpad
allowed the routine culture of large numbers of highly viable M.
leprae for experimental use
(208,
229,
414) (see Basic
Characteristics, above). More recently, other immunosuppressed strains
of mice and rats have been reported to allow enhanced growth of M.
leprae, including severe combined immunodeficiency mice, which
lack both T and B cells
(22).
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An invaluable means for studying immunological parameters of host defense is via gene transfer technology in the murine system. Through a variety of mechanisms a specific gene can be rendered either totally or conditionally inactive (244). The resulting knockout models allow investigation at the level of the direct effects due to the loss of the functional gene as well as the compensatory mechanisms operational in its absence. Numerous targeted gene KO strains are now commercially available, including those unable to produce specific cytokines and chemokines, their receptors, immune modulators, or cell surface markers. In addition, new generations of mice, including tissue-specific KO, conditional KO, multiple KO, and knockin mutants, are continually being developed.
We have examined several strains of KO mice in our
studies on M. leprae growth and granuloma development in
experimental leprosy. Perhaps the best characterized is the inducible
nitric oxide synthase (iNOS) KO
(NOS2–/–)
strain. M isolated from
NOS2–/– mice
are incapable of producing reactive nitrogen products and they cannot
inhibit M. leprae metabolic activity in vitro, although they
are fully competent in producing reactive oxygen products
(3). When inoculated into
NOS2–/– mouse
footpads, M. leprae initially grew to slightly higher levels
than seen in wild-type controls, but thereafter the course of infection
was similar. The granulomas formed in wild-type mice in response to
M. leprae infection consisted of only small, focal collections
of mononuclear cells; in contrast, the granulomas formed in
NOS2–/– mice
contained large, dense, organized collections of epithelioid cells and
lymphocytes which infiltrated the perineurium and destroyed muscle
bundles (6). In addition,
Th-1-type cytokine expression was significantly augmented in
NOS2–/– mice,
and granulomas in liver tissue demonstrated a pattern similar to that
seen in human leprosy lesions
(268) with
CD4+ T cells distributed throughout the lesion,
surrounded by CD8+ T cells (D. A. Hagge et al.,
submitted for publication). Overall, the M.
leprae-infected
NOS2–/– mouse
model exhibits findings similar to those of BT leprosy in humans.
Interestingly, in contrast to
NOS2–/– mice,
macrophages from mice that are superoxide deficient due to a defective
gp91 subunit of the phagocyte oxidase
(phox91–/–)
(93) efficiently kill
M. leprae in vitro, and infected
phox91–/–
mice develop a footpad induration similar to that of wild-type mice
(Adams, Scollard, and Krahenbuhl, unpublished).
Gamma interferon
(IFN-
) KO (IFN-–/–) mice
also exhibited enlarged footpads and enhanced cellular infiltration
upon infection with M. leprae
(7). The footpad, however,
contained epithelioid M and scattered lymphocytes that were
not assembled into organized granulomas. Growth of M. leprae
was enhanced approximately 1 log over that in wild-type mice and a
Th-2-type cytokine profile was generated. Overall, the general features
exhibited by the IFN-–/– model were
similar to those of BB-BL leprosy.
Studies in other KO mouse
models are currently under way. Growth of M. leprae in mice
deficient in interleukin-10 (IL-10) was similar to that seen in
immunocompetent mice, whereas bacillary growth was augmented in mice
deficient in IL-12, TNF-, tumor necrosis factor receptor,
lymphotoxin , CD4, and CD8 (Adams, unpublished data). It is
important to note, however, that M. leprae growth in these KO
footpad models, including the
IFN-–/– model, did not reach the
enormous levels seen in the T-cell-deficient mouse models. Thus, even
though these cytokines and cell types are important for the expression
of cell-mediated immunity, compensatory mechanisms are able to limit
bacillary growth. Immunoregulation in these mice is being studied
further by additional "conditional" approaches, such as
treatment with competitive inhibitors to create a second KO or
restoration of the KO in infected mice. These immunoregulatory
mechanisms may be crucial in the manifestation of the unstable
borderline areas of the leprosy
spectrum.
The nine-banded armadillo (Dasypus novemcintus) is the only immunologically intact animal that regularly develops fully disseminated M. leprae infections. Intravenous inoculation with 108 to 109 bacilli regularly results in 10,000-fold increases in the number of M. leprae over a span of about 18 months, and armadillos have been the hosts of choice for in vivo propagation of M. leprae for more than 30 years. With high burdens of bacilli in their reticuloendothelial tissues, armadillos can yield gram quantities of M. leprae (185, 413).
Approximately 65% of all armadillos experimentally inoculated with M. leprae will develop a fully disseminated infection. Infected armadillos exhibit few discernible clinical signs. Histopathological examination of infected animals typically reveals heavy infiltration of M. leprae-laden macrophages throughout the liver, spleen, and lymph nodes, as well as notable involvement of the lips, tongue, nose, nasal mucosa, skin, bone marrow, eyes, lungs, peripheral nervous system, gonads, and other tissues. Like humans, armadillos can upgrade and downgrade their response to M. leprae over the course of infection, but 90% of the animals that exhibit signs of systemic dissemination will eventually succumb to their leprosy (181). Intravenous inoculation promotes the most rapid and severe infections. However, respiratory instillation and percutaneous and intraperitoneal inoculation are also known to be effective (413).
Free-ranging armadillos are exposed to a number of atypical mycobacterial species in the environment and may develop nonspecific antibody responses cross-reactive with antigens shared by M. leprae (413). Armadillo immunoglobulin M (IgM) is highly cross-reactive with human IgM, and armadillo IgG reacts well with protein A or G.
The granulomatous response of armadillos to M. leprae is histopathologically identical to that seen in humans. Armadillo-derived lepromin (lepromin-A) can be used effectively to index the cell-mediated response of individual animals and to classify their type of leprosy according to the Ridley-Jopling scale. The reactions of individual animals can range from polar lepromatous to polar tuberculoid. Lepromatous armadillos develop the very large burdens of bacilli needed to obtain M. leprae from their tissues with high purity and are selected most commonly for propagation purposes (182-186), However, both lepromin-positive and lepromin-negative armadillos can resist challenge with M. leprae, and armadillos are useful models for studying both susceptibility and the variable resistance to leprosy that may result from treatment or vaccination (185, 187-189).
In 1973 Kirchheimer showed that 80% of the armadillos sensitized with heat-killed M. leprae could resist infectious challenge (213). Subsequent vaccination studies with BCG demonstrated effective protection of armadillos against M. leprae. The armadillo is the only animal model that can demonstrate effective protection against M. leprae with BCG, equivalent to rates observed for BCG in several human vaccination trials. Studies with these animals could potentially benefit our efforts to generate more efficacious antituberculosis and antileprosy vaccines (29, 188, 214, 215). Unfortunately, the number of armadillos and duration of time required (up to 1,140 days) for effective challenge studies with armadillos currently limit the utility of armadillos as vaccine models.
In addition to the armadillo's value as a source of organisms and an immunological model, the infection of peripheral nerves in the armadillo constitutes a unique model of lepromatous nerve involvement in humans (350, 351) (reviewed in Mechanisms of Nerve Injury, below).
Recently, the Human Genome Consortium completed the sequencing of the armadillo genome as part of a comparative genomics initiative. Armadillos are the most common modern representatives of Xenathra, a family of mammals that diverged from the rodent-primate tree in the Cretaceous period. The availability of extensive sequence information on the armadillo (http://www.ncbi.nlm.nih.gov/BLAST) is likely to rapidly expand the availability of new immunological probes and reagents for use with armadillos and will advance their use as models for resistance, vaccination, and nerve injury.
Wild nine-banded armadillos in the south central United States are highly susceptible natural hosts of Mycobacterium leprae.Surveys conducted over the last 30 years on more than 5,000 animals confirm that the infection is present among armadillos in Arkansas, Louisiana, Mississippi, and Texas. Little evidence for M. leprae infection is found among armadillos elsewhere in the U.S. range, and only a few reports relate finding the infection among animals in Central or South America. However, the issue has received only scant attention in other countries. Armadillos only recently expanded their range into the United States, and leprosy was present in Texas and Louisiana prior to the arrival of armadillos. The ecological relationship between humans and armadillos with M. leprae in this region remains unclear. However, infected armadillos constitute a large reservoir of M. leprae, and they may be a source of infection for some humans in this country, and perhaps in other locations across the animal's range (415).
The impact of armadillo leprosy on humans is difficult to measure. A number of anecdotal reports have associated handling armadillos with individuals' developing leprosy, and case-control studies have yielded conflicting results (45, 239). Among Louisiana residents developing leprosy, no association with armadillo contact was found (110), but among Mexican-born patients who presented in Los Angeles and who had lived in areas where they could have been exposed to armadillos, an increased likelihood for lepromatous-type leprosy was reported (408). Leprosy remains rare in the United States, and the degree of risk attributable to armadillos is quite low. Nonetheless, armadillos are a large natural reservoir and can be an effective vehicle for exposure to large numbers of M. leprae (112). However, understanding the actual impact of armadillos on human infection will likely require the evolution of better molecular techniques that can track transmission.
| HOST RESPONSE TO M. LEPRAE |
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The idea that at least two different genes might control the human immune response to leprosy was proposed in the 1970s (89) and supported by subsequent investigations (1, 427). A convincing body of evidence now exists to indicate that different genes do influence the human immune response to M. leprae, operating at two levels (Fig. 5). The first level, overall susceptibility/resistance to the infection, is a manifestation of innate resistance mediated by cells of the monocyte lineage. If innate resistance is insufficient and infection becomes established, genetic influence is expressed at the second level, i.e., influencing the degree of specific cellular immunity and delayed hypersensitivity generated by the infected individual. Such acquired immunity is mediated primarily through the function of T lymphocytes, in cooperation with antigen-presenting cells (see Adaptive Immunity, below). The association of genes involved in both innate and acquired immunity to leprosy has recently been reviewed by Marquet and Schurr (249), and genetic defects in different components of the type 1 cytokine pathway that affect human resistance to mycobacteria have been reviewed by van de Vosse and colleagues (420).
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