M. leprae genome sequence.
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Base Sequence↗
Biomedical subjects
A Rambukkana
Publications and source records attributed to A Rambukkana.
Molecular basis for the peripheral nerve predilection of Mycobacterium leprae.
Mycobacterium leprae, the causative organism of leprosy, has a unique predilection for Schwann cells, the glial cells of the peripheral nervous system. M. leprae invasion of Schwann cells leads to the neurological damage that underlies the sensory motor loss and subsequent deformity and disability associated with this disease. Recent studies have begun to elucidate the early events of M. leprae infection of Schwann cells on a molecular level, and the host and bacterial factors that determine the neural predilection of this bacterium. These advances have now provided novel insights into the mechanisms of bacterial interactions with host cells.
Antigens, Bacterial↗
Role of the cell wall phenolic glycolipid-1 in the peripheral nerve predilection of Mycobacterium leprae.
The cell wall of pathogenic mycobacteria is abundant with complex glycolipids whose roles in disease pathogenesis are mostly unknown. Here, we provide evidence for the involvement of the specific trisaccharide unit of the phenolic glycolipid-1 (PGL-1) of Mycobacterium leprae in determining the bacterial predilection to the peripheral nerve. PGL-1 binds specifically to the native laminin-2 in the basal lamina of Schwann cell-axon units. This binding is mediated by the alpha(2LG1, alpha2LG4, and alpha2LG5 modules present in the naturally cleaved fragments of the peripheral nerve laminin alpha2 chain, and is inhibited by the synthetic terminal trisaccharide of PGL-1. PGL-1 is involved in the M. leprae invasion of Schwann cells through the basal lamina in a laminin-2-dependent pathway. The results indicate a novel role of a bacterial glycolipid in determining the nerve predilection of a human pathogen.
Animals↗
How does Mycobacterium leprae target the peripheral nervous system?
Mycobacterium leprae has the capacity to invade the peripheral nervous system and cause neuropathy. The molecular mechanisms responsible have remained unknown until recently. Identification of the endoneurial laminin-2 isoform and its receptor alpha-dystroglycan as neural targets of M. leprae has not only opened up a new area of scientific inquiry into the pathogenesis of neurological damage in leprosy, but has also revealed unexpected biological properties of these important host molecules.
Cytoskeletal Proteins↗
Molecular basis of the interaction of Mycobacterium leprae with peripheral nerve: implications for therapeutic strategies.
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Humans↗
A 21-kDa surface protein of Mycobacterium leprae binds peripheral nerve laminin-2 and mediates Schwann cell invasion.
Nerve damage is the hallmark of Mycobacterium leprae infection, which results from M. leprae invasion of the Schwann cell of the peripheral nervous system. We have recently shown that the laminin-2 isoform, specially the G domain of laminin alpha2 chain, on the Schwann cell-axon unit serves as an initial neural target for M. leprae. However, M. leprae surface molecules that mediate bacterial invasion of peripheral nerves are entirely unknown. By using human alpha2 laminins as a probe, a major 28-kDa protein in the M. leprae cell wall fraction that binds alpha2 laminins was identified. After N-terminal amino acid sequence analysis, PCR-based strategy was used to clone the gene that encodes this protein. Deduced amino acid sequence of this M. leprae laminin-binding protein predicts a 21-kDa molecule (ML-LBP21), which is smaller than the observed molecular size in SDS/PAGE. Immunofluorescence and immunoelectron microscopy on intact M. leprae with mAbs against recombinant (r) ML-LBP21 revealed that the protein is surface exposed. rML-LBP21 avidly bound to alpha2 laminins, the rG domain of the laminin-alpha2 chain, and the native peripheral nerve laminin-2. The role of ML-LBP21 in Schwann cell adhesion and invasion was investigated by using fluorescent polystyrene beads coated with rML-LBP21. Although beads coated with rML-LBP21 alone specifically adhered to and were ingested by primary Schwann cells, these functions were significantly enhanced when beads were preincubated with exogenous alpha2 laminins. Taken together, the present data suggest that ML-LBP21 may function as a critical surface adhesin that facilitates the entry of M. leprae into Schwann cells.
Adhesins, Bacterial↗
Role of alpha-dystroglycan as a Schwann cell receptor for Mycobacterium leprae.
alpha-Dystroglycan (alpha-DG) is a component of the dystroglycan complex, which is involved in early development and morphogenesis and in the pathogenesis of muscular dystrophies. Here, alpha-DG was shown to serve as a Schwann cell receptor for Mycobacterium leprae, the causative organism of leprosy. Mycobacterium leprae specifically bound to alpha-DG only in the presence of the G domain of the alpha2 chain of laminin-2. Native alpha-DG competitively inhibited the laminin-2-mediated M. leprae binding to primary Schwann cells. Thus, M. leprae may use linkage between the extracellular matrix and cytoskeleton through laminin-2 and alpha-DG for its interaction with Schwann cells.
Animals↗
Neural targeting of Mycobacterium leprae mediated by the G domain of the laminin-alpha2 chain.
We report that the molecular basis of the neural tropism of Mycobacterium leprae is attributable to the specific binding of M. leprae to the laminin-alpha2 (LN-alpha2) chain on Schwann cell-axon units. Using recombinant fragments of LN-alpha2 (rLN-alpha2), the M. leprae-binding site was localized to the G domain. rLN-alpha2G mediated M. leprae binding to cell lines and to sciatic nerves of dystrophic dy/dy mice lacking LN-alpha2, but expressing laminin receptors. Anti-beta4 integrin antibody attenuated rLN-alpha2G-mediated M. leprae adherence, suggesting that M. leprae interacts with cells by binding to beta4 integrin via an LN-alpha2G bridge. Our results indicate a novel role for the G domain of LN-2 in infection and reveal a model in which a host-derived bridging molecule determines nerve tropism of a pathogen.
Animals↗
Novel predictive assay for contact allergens using human skin explant cultures.
Contact allergens sensitize the immune system by the binding to and subsequent activation of Langerhans cells (LCs), the antigen-presenting cells of the skin. At present, new chemicals are usually tested for their contact allergenicity in animal models. To develop an animal-replacing predictive in vivo assay for the identification of potential contact allergens, we compared the effects of epicutaneous application of six known contact allergens, five known irritants and two dermatologically inactive chemicals on LCs in skin biopsy cultures from seven healthy donors. Immunohistochemical analysis of cryostat sections of all the biopsies treated with contact allergens showed 1) a large reduction in the number of LCs in epidermis, as evaluated by a decrease in human leukocyte antigens (HLA)-DR-expressing cells, and CD1a-expressing cells and 2) accumulation of the remaining LCs at the epidermal-dermal junction. In contrast, the irritants, inactive chemicals, and solvents did not induce these changes. Morphometrical analysis indicated that the contact allergen-induced reduction in the number of HLA-DR+ and CD1a+ LCs per millimeter of epidermis was significant and was dependent on the concentration of the contact allergens. Flow cytometric analysis of isolated epidermal cells confirmed the immunohistochemical findings. In combination, these results suggest that the culture of ex vivo human skin explants provides a promising model to predict potential allergenicity of newly produced chemical compounds and can therefore replace current animal models.
Allergens↗
Effects of contact allergens on human Langerhans cells in skin organ culture: migration, modulation of cell surface molecules, and early expression of interleukin-1 beta protein.
Epidermal Langerhans cells (LC) and cytokines play a critical role in the initiation phase of contact hypersensitivity reactions in the skin. Most of the studies of these aspects have been performed in animal models and relatively little is known about the human system. Short-term human skin organ cultures, in which LC preserved their characteristics and distribution within the epidermis, were used to examine the time course effects of contact allergens on human LC in situ and whether these effects are mediated by cytokines. Epicutaneous application of nontoxic concentrations of contact allergens 2,4-dinitrofluorobenzene, 2,4-dinitrochlorobenzene, and nickel sulphate, but not the irritants sodium dodecylsuphaye and croton oil or the tolerogen 2,4-dichloronitrobenzene, significantly reduced the total number of LC in the epidermis: remaining LC were localized along the epidermal-dermal junction, suggesting a migration of LC within and out of the epidermis. LC that are migrated to the epidermal-dermal junction showed a decreased expression of CD1a+ and MHC-II and an upregulation of ICAM-I. While these effects were observed after 24 hours, the expression of IL-1 beta protein was induced exclusively by LC as early as 4 hours after skin challenge with contact allergens alone. After 24 hours, contact allergens not only increased the expression of IL-1 beta but also induced the expression of IL-1 alpha, TNF-alpha, GM-CSF, and IL-6 proteins mainly by suprabasal keratinocytes. In an attempt to study the possible relation between allergen-induced epidermal cytokines and the migration and phenotypic changes of LC, skin explants were incubated with corresponding human recombinant (hr) cytokines. After 12 hours, hr IL-1 beta, but not other hr cytokines (IL-1 alpha, TNF-alpha, GM-CSF, and IL-6), induced the migration within and out of the epidermis and decreased the expression of CD1a+ and MHC-II on remaining epidermal LC similar to that caused by contact allergens. Pre-incubation of skin explants with neutralizing IL-1 beta antibodies, but not antibodies to IL-1 alpha, TNF-alpha, or GM-CSF, significantly prevented the allergen-induced migration of LC. This study showed that contact allergens preferentially induced the migration of LC within and out of the epidermis and modulated the expression of cell surface molecules on migrated LC as well as induced the early expression of LC-derived IL-1 beta. We also provide evidence that IL-1 beta is critically involved in contact allergen-induced changes on human epidermal LC and suggest that IL-1 beta plays a role in the initiation of contact hypersensitivity in human skin in vivo.
Allergens↗
In situ behavior of human Langerhans cells in skin organ culture.
BACKGROUND: Epidermal Langerhans cells (ELC) play a critical role in the initiation of cutaneous immune responses. ELC are characterized by the expression of major histocompatibility complex (MHC) class II Ag and a number of adhesion/costimulatory molecules. Evidence suggests that cytokines induced within the epidermis regulate the functions of ELC, including their phenotypic expression. In the human system, no information is available regarding the behavior of the ELC in situ: their changes in morphology, expression of functional molecules or migration within the microenvironment. In the present study, using an ex vivo human skin organ culture model, we addressed the above questions and also examined the phenotypic modulation of ELC in situ by cytokines. EXPERIMENTAL DESIGN: Skin explants were cultured either in a Trowell-type method or free in the medium. Skin explants were cultured with and without cytokines and were processed for light and electron microscopy and for immunohistochemical definition of ELC phenotypes. RESULTS: In the Trowell-type skin organ culture, morphologic integrity of ELC, CD1a molecule, and Birbeck granules could be preserved intact up to 3 to 4 days in culture. During the first 3 days of culture, the intensity of MHC-II (HLA-DR, DP, and DQ) and CD1a expression on ELC increased sharply, and the dendritic appearance of ELC became more prominent at Day 3. Adhesion molecules, ICAM-1, LFA-3, HECA-452, sLx, and B7/BB1 were also spontaneously acquired in varying amounts by CD1a+ ELC after 3 days in culture. Significant increase of CD1a and ICAM-1 expression on ELC was observed within 12 hours, when skin explants were cultured free in the medium with GM-CSF and TNF-alpha, respectively. Further, we demonstrated spontaneous migration of ELC within the epidermis and then to the dermis during the Trowell-type skin culture. We also showed the migration of ELC out of the human skin when skin explants were cultured directly in the medium. CONCLUSIONS: Human ELC showed significant phenotypic changes within the epidermis and acquired migratory capacity during the skin organ culture. ELC in skin organ culture appear to undergo a phenotypic maturation within the epidermis. ELC in situ rapidly respond to GM-CSF and TNF-alpha by increasing the expression of CD1a and ICAM-1 molecules, respectively. These results suggest the modulation of phenotypic characteristics of ELC and their migration in response to the changes of epidermal microenvironment and cytokines and implicate the potential use of skin organ culture model to elucidate the role of human ELC in the immunopathology of skin diseases.
Antigens, CD1↗
Antibodies to mycobacterial 65-kDa heat shock protein and other immunodominant antigens in patients with psoriasis.
An association of microbial agents and autoimmunity has been suggested for the pathogenesis of psoriasis. Mycobacteria are common environmental microbes and their antigens, especially the highly conserved mycobacterial 65-kDa heat shock protein (hps65), have been implicated in the pathogenesis of autoimmune arthritis and other idiopathic diseases. In this context, we investigated a possible mycobacterium-induced humoral immune response in psoriasis. Sera from 17 patients with chronic plaque-type psoriasis were studied by immunoblotting using the whole sonicate of Mycobacterium tuberculosis and purified recombinant mycobacterial hsp65. Immunoblot analysis demonstrated that 58% of the psoriasis patients compared to patients with acne and DLE, and normal controls showed strong antibody activity to 65-kDa and 48/45 doublet antigens from M. tuberculosis sonicate, whereas 47% of the patients showed antibody activity to mycobacterial hsp65. Only 10-20% of the patients had an antibody response to 16-kDa and 80-kDa antigens. Similar antibody activity to 65 kDa and 48/45 kDa was also found consistently with eight different sonicated mycobacterial species by immunoblotting, indicating that these seroreactive antigens are crossreactive and are present in common environmental mycobacteria. Antibody activities to both mycobacterial 65-kDa and hsp65 showed a positive correlation (r = 0.76) with the psoriasis disease activity, whereas antibodies to 48/45-kDa doublet antigens showed a weak correlation (r = 0.54). By enzyme-linked immunosorbent assay (ELISA), 47% of the psoriasis patients showed significantly elevated antibody titers to hsp65 (p < 0.003) as compared to control groups, and the antibody response by ELISA also showed a significant positive correlation (r = 0.76) with disease activity. Anti-mycobacterial antibody activity may be related to severity of disease and may be useful in monitoring disease activity in psoriasis.
Adult↗
Identification of a novel 27-kDa protein from Mycobacterium tuberculosis culture fluid by a monoclonal antibody specific for the Mycobacterium tuberculosis complex.
Mycobacterium tuberculosis antigens inducing species-specific immune responses are likely to be particularly important for serodiagnosis or for skin testing of tuberculosis. In the present study, we describe the characterization of two novel monoclonal antibodies (MoAbs) A3h4 (IgG2a) and B5g1 (IgM) that are directed to M. tuberculosis 27-kDa and 25-kDa proteins respectively. Specificity analysis by immunoblotting using 20 different species of mycobacterial sonicates revealed that MoAb A3h4 was specific for M. tuberculosis complex alone while MoAb B5g1 showed a limited cross-reactivity. Direct comparison with previously characterized MoAbs revealed that these MoAbs A3h4 and B5g1 defined new antigenic determinants of M. tuberculosis. By using M. tuberculosis complex-specific MoAb A3h4 we have identified a distinct 27-kDa protein in the M. tuberculosis H37Rv culture fluid. Since this MoAb did not bind to the previously characterized MPT44, MPT59, MPT45, MPT51 and MPT64 proteins as well as the 23-kDa superoxide dismutase (SOD) protein of M. tuberculosis, we conclude that MoAb A3h4 recognizes a novel protein in the M. tuberculosis H37Rv culture fluid. Studies of the subcellular distribution of these MoAb-reactive proteins indicate that the MoAb A3h4-reactive 27-kDa protein is present not only in the culture fluid but also in the cytosol and the cell wall of M. tuberculosis. By contrast, B5g1-reactive protein is mainly a cytosolic protein. When these MoAbs were tested in a previously established ELISA with intact mycobacteria derived from early cultures, only MoAb A3h4 showed the positive reactivity to mycobacteria belonging to the M. tuberculosis complex. In addition, during the present comparative studies of MoAbs we have also found that the previously described MoAb F116-5, which is known to recognize the mycobacterial 23-kDa SOD protein [17], cross-reacted with the MPT44, MPT59, MPT45 and MPT51 secreted proteins but not with MPT64 and MPB70. These findings indicate that the family of four secreted proteins of M. tuberculosis share a common epitope with M. tuberculosis SOD protein.
Antibodies, Monoclonal↗
The mycobacterial secreted antigen 85 complex possesses epitopes that are differentially expressed in human leprosy lesions and Mycobacterium leprae-infected armadillo tissues.
The granulomatous skin lesions in leprosy are thought to be initiated by the immune response to certain antigens of the causative agent, Mycobacterium leprae. The antigen 85 complex is one of the major targets in the immune response to M. leprae infection. In the present study, a panel of previously characterized monoclonal antibodies (MAbs) (3A8, Rb2, A4g4, A2h11, Pe12, and A3c12) reacting with different epitopes of the 85 complex proteins of Mycobacterium tuberculosis and M. leprae was employed in a comparative immunohistological analysis to demonstrate the in situ expression of 85 complex antigenic epitopes in leprosy lesions across the clinical spectrum and in M. leprae-infected armadillo liver tissues. These MAbs showed a heterogeneous staining pattern in a given leprosy lesion. In highly bacilliferous borderline and lepromatous leprosy lesions, MAbs Rb2, A4g4, A2h11, and Pe12 stained clear rod-shaped M. leprae bacilli within macrophages, and the degree of staining correlated with the bacillary index of the lesion. On the other hand, MAbs 3A8 and A3c12 staining was mostly seen as a diffuse staining pattern within interstitial spaces and on the membranes of the infiltrated cells but not the bacilli. In paucibacillary borderline and tuberculoid leprosy lesions, only 3A8, Rb2, and A3c12 showed distinct staining in association with infiltrates in the granuloma. None of these MAbs showed any detectable reaction with control nonleprosy skin lesions, while MAb A3c12 positively stained the granulomas of both leprosy and control specimens. In situ reactivity of these MAbs with M. leprae-infected armadillo liver tissues also showed a heterogeneous staining pattern. Interestingly, a clear difference in expression of these epitopes was observed between armadillo tissues and human leprosy lesions. By immunogold ultracytochemistry, we further showed the differential localization of these MAb-reactive epitopes on the cell surface, in the cytosol, and at the vicinity of M. leprae within Kupffer cells of armadillo liver tissues. Our results indicate that these antigenic epitopes of the antigen 85 complex are differentially expressed in leprosy lesions and infected armadillo tissues and that they could be target determinants in the immunopathological responses during M. leprae infection.
Animals↗
Association of the mycobacterial 30-kDa region proteins with the cutaneous infiltrates of leprosy lesions. Evidence for the involvement of the major mycobacterial secreted proteins in the local immune response of leprosy.
The granulomatous skin lesions of human leprosy are known to be due to the cutaneous immune reaction to various mycobacterial antigens. In the present study, by immunohistochemical analysis using a previously characterized monoclonal antibody (MoAb) 3A8 we have demonstrated a selective expression of the 3A8 epitope of mycobacterial 30-kDa proteins, the major secreted proteins of mycobacteria, in various forms of leprosy lesions across the clinical spectrum. The localization of MoAb 3A8 staining is confined to the areas of cellular infiltrates of the lesions. In tuberculoid lesions the intense 3A8 staining was seen mostly in association with the membrane of the dermal cellular infiltrates whereas in highly bacilliferous lepromatous lesions the staining seems to be diffused with granular appearance but not in the form of bacteria. In patients with reversal reaction the staining was specifically extended to cells infiltrating the epidermis. MoAb 3A8 did not show any reactivity with inflammatory skin lesions of patients other than those with leprosy. Since the 3A8 epitope of 30-kDa proteins has been shown to be present in all cellular compartments of the mycobacteria and in the actively secreted BCG 85 antigen complex, MoAb 3A8 reactive protein(s) in leprosy lesions may be derived either from degraded somatic mycobacterial products or from antigens actively secreted by live bacilli. The latter could be true in the cases of untreated lepromatous lesions with high bacterial load since live M. leprae has also been considered to secrete corresponding 30-kDa proteins similar to other closely related mycobacteria. By double immunoenzyme staining we clearly demonstrate the expression of 3A8 epitope on CD68+ macrophages in the granulomas of tuberculoid leprosy, whereas in highly bacilliferous lepromatous lesions most of the double staining was seen in a diffuse pattern within the interstitial space of the cellular infiltrate as well as in the cytoplasm of CD68+ macrophages. In lesions from reversal reaction the 3A8 epitope is more strongly expressed on CDla+ dendritic Langerhans cells (LC) both in the epidermis and in the dermis as compared with other types of leprosy. This provides evidence for the involvement of LC in handling of mycobacterial antigenic epitopes in leprosy lesions. Further, immunoenzyme double staining revealed that the expression of this mycobacterial 3A8 epitope on antigen presenting cells such as CD68+ macrophages and CDla+ LC is present in juxtaposition with CD3+ T cells including the alpha beta and gamma delta receptor-bearing T cells in the granuloma.(ABSTRACT TRUNCATED AT 400 WORDS)
Antibodies, Monoclonal↗
Identification and characterization of epitopes shared between the mycobacterial 65-kilodalton heat shock protein and the actively secreted antigen 85 complex: their in situ expression on the cell wall surface of Mycobacterium leprae.
Both mycobacterial hsp65 and the actively secreted antigen 85 complex of 30-kDa region proteins are considered to be major immune targets in mycobacterial diseases. In this study, by using a novel series of monoclonal antibodies (MAbs) directed to these antigens, we identified and partially characterized three unique epitopes (Rb2, Pe12, and A2h11) that are shared between mycobacterial hsp65 and the individual components of the antigen 85 complex. Dot blot assays with native purified proteins revealed that all three MAbs are strongly bound to hsp65 and antigens 85A (MPT44) and 85B (MPT59), while a weak reaction or no reaction was found with antigen 85C (MPT45). Immunoblotting showed that MAb Rb2 reacted strongly with both hsp65 and the antigen 85 complex proteins, whereas MAbs Pe12 and A2h11 reacted strongly with the former but weakly with the latter. Moreover, these MAbs did not react with other closely related MPT51 and MPT64 secreted proteins. Further characterization of these epitopes was performed by using recombinant fusion and truncated proteins of Mycobacterium bovis BCG hsp65 (MbaA) and the M. leprae 30- and 31-kDa antigen 85 complex fusion proteins. In hsp65, Rb2-Pe12- and A2h11-reactive epitopes were found to reside in the C-terminal region of amino acid residues 479 to 540 and 303 to 424, respectively. In the M. leprae 30- and 31-kDa antigen 85 complex, all three epitopes were located in an N-terminal region of amino acid residues 55 to 266, one of the known fibronectin-binding sites of the M. leprae antigen 85 complex. Comparison of these MAb-reactive amino acid sequence regions between mycobacterial hsp65 and the components of the antigen 85 complex revealed that these regions show certain amino acid sequence identities. Furthermore, by immunoperoxidase and immunogold ultracytochemistry, we demonstrated that Rb2-, Pe12-, and A2h11-reactive epitopes are expressed both on the cell wall surface and in the cytosol of M. leprae bacilli within the lesions of lepromatous leprosy patients and in M. leprae-infected armadillo liver tissue.
Amino Acid Sequence↗
Heterogeneity of monoclonal antibody-reactive epitopes on mycobacterial 30-kilodalton-region proteins and the secreted antigen 85 complex and demonstration of antigen 85B on the Mycobacterium leprae cell wall surface.
Proteins of the antigen 85 complex in the 30-kDa region secreted by live mycobacteria are important in the immune response against mycobacterial infections and may play an important biological role in the host-parasite interaction. In the present study, we have characterized epitopes of the 30-kDa-region proteins and the antigen 85 complex by using a panel of 13 monoclonal antibodies (MAbs) reacting with these antigens, 6 of which have not been described before. By using five previously characterized related secreted proteins of Mycobacterium tuberculosis, MPT44 (85A), MPT59 (85B), MPT45 (85C), MPT51 (27 kDa), and MPT64 (26 kDa), we have identified at least 10 different MAb-reactive epitopes on the proteins of the antigen 85 complex. A heterogeneous distribution of epitopes was observed within the components of the antigen 85 complex. Two distinct epitopes specific for antigen 85B and two other epitopes restricted to the 85A and 85B components were recognized. Two of them were shared with a previously unidentified 27-kDa protein present in M. tuberculosis culture fluid from which all MPT proteins were derived. The rest of the MAb-reactive epitopes were found to be present mostly in antigens 85A and 85B and to a lesser extent in antigen 85C. None of these MAbs recognized component 85C alone nor did they bind to the related MPT51 and MPT64 proteins. Interestingly, most of the MAbs reacted with purified native proteins of the antigen 85 complex but not to them in their denatured forms. In contrast, reactivity of the MAbs with the cytosol fraction of M. tuberculosis in immunoblotting revealed that they bound to a closely related cytosolic 30-kDa protein(s) even when they were denatured. Heterogeneity of these MAb-reactive epitopes of the antigen 85 complex was further evident as they were found to be distributed in various patterns among 19 different mycobacterial species. By using fusion proteins of the Mycobacterium leprae 30/31-kDa antigen 85 complex, we have localized at least six different epitopes within amino acid residues 55 to 266 of the M. leprae antigen 85 complex. Finally, by immunohistochemical analysis, we have demonstrated the in situ expression of one of the novel MAb-reactive epitopes specific for antigen 85B on the cell wall surface of M. leprae within macrophages in lepromatous leprosy lesions and thus provide direct evidence for the presence of the B component of the antigen 85 complex on the surface of intact M. leprae.
Animals↗
Mycobacterial 65,000 MW heat-shock protein shares a carboxy-terminal epitope with human epidermal cytokeratin 1/2.
Molecular mimicry between mycobacterial heat-shock protein (hsp) 65 and host tissue antigens have been implicated in the autoimmune pathogenesis of certain idiopathic diseases. Here, we demonstrated that two of our previously characterized monoclonal antibodies (mAb), Ne5 and Nd4 that were directed to a carboxy-terminal epitope on the mycobacterial hsp 65, specifically cross-reacted with suprabasal cytokeratin of the normal human skin. These mAb also showed similar keratin staining of hair follicle epithelia and produced no reaction with other dermal components. Both mAb strongly stained the cytoplasm of the majority of freshly isolated epidermal keratinocytes from the normal human skin. None of these mAb showed staining with human HeLa cells and with human skin fibroblasts. Immunoblotting using total keratin extract prepared from isolated epidermal keratinocytes revealed that mAb Ne5 and Nd4 specifically reacted with a molecular size of 65,000-67,000 MW keratin protein(s) and such reactivity was not observed from cytoskeletal proteins extracted from HeLa cells and skin fibroblasts. Comparison of immunoblotting reactivity with conventional anti-cytokeratin mAb further revealed that mAb Ne5/Nd4 recognized a 65,000-67,000 MW molecular-sized protein corresponding to cytokeratin 1/2 from the same keratinocyte extract as anti-cytokeratin mAb. Preincubation of mAb Ne5/Nd4 with the purified mycobacterial hsp 65 abolished this keratin cross-reactivity in both immunohistochemistry and immunoblotting. Moreover, these mAb showed no keratin staining in lesional psoriatic skin and also reacted weakly with cultured epidermal keratinocytes. Since mAb Ne5/Nd4 specifically recognized a 67,000-65,000 MW molecular-sized protein(s) derived from epidermal keratinocytes and the known characteristics of epidermal cytokeratin 1/2 appeared to be consistent with present results, we concluded that Ne5/Nd4 cross-reactive protein(s) in the human epidermis is suprabasal cytokeratin 1/2. Comparison of the previously mapped Ne5/Nd4 epitope region of amino acid residues 525-540 of the mycobacterial hsp 65 with the entire sequence of human 65,000 MW keratin revealed that a stretch of nine amino acids of the Ne5/Nd4 epitope sequence resembled certain regions of the carboxy-terminus of the human 65,000 MW keratin. This similarity of the mycobacterial hsp 65 probably contributes to the cytokeratin cross-reactive epitope. Our results presented here demonstrate direct evidence of immunological cross-reactivity between mycobacterial hsp 65 and human epidermal cytokeratin 1/2. We speculate that Ne5/Nd4 cross-reactive epitope of epidermal cytokeratins might be an important target for skin diseases.
Amino Acid Sequence↗