Identification of an I-Ad restricted peptide on the 65-kilodalton heat shock protein of Mycobacterium avium

The 65 kilodalton heat shock protein (Hsp65) from mycobacterial species elicits immune responses and in some cases protective immunity. Here we have used a DNA sublibrary approach to identify antigenic fragments of Mycobacterium avium Hsp65 and a synthetic peptide approach to delineate CD4+ T cell determinants. A panel of Hsp65 reactive CD4+ T cell clones was established from lymph node cells obtained from BALB/c mice immunized with recombinant Hsp65. The clones were tested for proliferative reactivity against the products of the DNA sublibrary of the hsp65 gene. A T cell epitope, restricted by the I-Ad molecule, was identified within the C-terminal region of Hsp65 and the minimal epitope (amino acid residues 489-503) delineated using overlapping peptides spanning the C-terminal fragment. Additionally, the CD4+ T cell clone recognizing this epitope also responded to native Hsp65 present in M. avium lysates by both proliferation and cytokine production, indicating that the epitope was present and processed similarly both in the native and the recombinant forms of Hsp65. This sequence identified in BALB/c mice (Hsp65 489-503) is identical in other mycobacteria, notably M. tuberculosis, M. bovis and M. leprae, suggesting the epitope may have wider application in murine models of other mycobacterial infections.     

Administration of Mycobacterium leprae rHsp65 Aggravates Experimental Autoimmune Uveitis in Mice

The 60kDa heat shock protein family, Hsp60, constitutes an abundant and highly conserved class of molecules that are highly expressed in chronic-inflammatory and autoimmune processes. Experimental autoimmune uveitis [EAU] is a T cell mediated intraocular inflammatory disease that resembles human uveitis. Mycobacterial and homologous Hsp60 peptides induces uveitis in rats, however their participation in aggravating the disease is poorly known. We here evaluate the effects of the Mycobacterium leprae Hsp65 in the development/progression of EAU and the autoimmune response against the eye through the induction of the endogenous disequilibrium by enhancing the entropy of the immunobiological system with the addition of homologous Hsp. B10.RIII mice were immunized subcutaneously with interphotoreceptor retinoid-binding protein [IRBP], followed by intraperitoneally inoculation of M. leprae recombinant Hsp65 [rHsp65]. We evaluated the proliferative response, cytokine production and the percentage of CD4⁺IL-17⁺, CD4⁺IFN-γ⁺ and CD4⁺Foxp3⁺ cells ex vivo, by flow cytometry. Disease severity was determined by eye histological examination and serum levels of anti-IRBP and anti-Hsp60/65 measured by ELISA. EAU scores increased in the Hsp65 group and were associated with an expansion of CD4⁺IFN-γ⁺ and CD4⁺IL-17⁺ T cells, corroborating with higher levels of IFN-γ. Our data indicate that rHsp65 is one of the managers with a significant impact over the immune response during autoimmunity, skewing it to a pathogenic state, promoting both Th1 and Th17 commitment. It seems comprehensible that the specificity and primary function of Hsp60 molecules can be considered as a potential pathogenic factor acting as a whistleblower announcing chronic-inflammatory diseases progression.     

Detection and identification of mycobacteria by amplification of mycobacterial DNA

A 383bp segment of the gene coding for the 65kD mycobacterial antigens from Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium avium, Mycobacterium paratuberculosis, and Mycobacterium fortuitum was amplified using Taq polymerase and synthetic oligonucleotide primers and the amplified DNAs from four of these species were compared by nucleotide sequencing. Although the gene segments from these species showed considerable similarity, oligonucleotide probes which could distinguish M. tuberculosis/M. bovis, M. avium/M. paratuberculosis and M. fortuitum could be identified. Samples containing 10(6) human cells and serial dilutions of a suspension of intact mycobacteria were prepared, DNA was extracted, the segment of the mycobacterial DNA sequence amplified, and the amplified DNA hybridized with oligonucleotide probes. In two independent experiments, this procedure permitted the detection and identification of less than 100 mycobacteria in the original sample. These results suggest that this approach may prove useful in the early diagnosis of mycobacterial infection.     

The 65-kilodalton antigen of Mycobacterium tuberculosis.

The immune response of the host to the antigens of Mycobacterium tuberculosis plays the key role in determining immunity from infection with as well as the pathogenicity of this organism. A 65-kilodalton (kDa) protein has been identified as one of the medically important antigens of M. tuberculosis. The gene encoding this antigen was isolated from a lambda gt11-M. tuberculosis recombinant DNA library using monoclonal antibodies directed against the 65-kDa antigen as the specific probes. The nucleotide sequence of this gene was determined, and a 540-amino-acid sequence was deduced. This sequence was shown to correspond to that of the 65-kDa antigen by constructing a plasmid in which this open reading frame was fused to the lacZ gene. The resulting fusion protein reacted specifically with the anti-65-kDa protein antibodies. A second long open reading frame was found downstream of the 65-kDa antigen gene which could encode a protein of 517 amino acids. This putative protein contained 29 tandemly arranged partial or complete matches to a pentapeptide sequence.     

T cells recognize an immunodominant epitope of heat shock protein 65 in Kawasaki disease

BACKGROUND: Kawasaki disease (KD) is an acute systemic vasculitis of infancy and early childhood that is characterized by endothelial cell damage associated with T-cell activation. Lymphocytes infiltrating damaged tissues might be responsible for the disease through secretion of cytokines, such as tumor necrosis factor (TNF)-alpha, that could cause fever, as well as endothelial tissue damage. Debate is growing about the nature of antigen responsible for T-cell activation in KD. Bacillus Calmette Guerin (BCG) and purified protein derivative (PPD) hyperresponsiveness was observed in KD patients and this phenomenon was hypothetically ascribed to cross-reactivity between mycobacterial Heat Shock Protein (HSP) 65 and human homologue HSP63. MATERIALS AND METHODS: CD4+ and CD8+ T-cell clones were obtained from peripheral blood of KD patients in acute phase, or control subjects. The clones were tested for reactivity toward HSP65 and derived peptides. Both proliferation and cytokine production were analyzed. RESULTS: A significant fraction of CD4 and CD8 T-cell clones from KD patients recognized an epitope from HSP65, spanning amino acids 65-85. T-cell clones cross-reacted with the corresponding 90-110 peptide sequence of human HSP-63. CONCLUSIONS: Cross-reactivity between specific epitopes of mycobacterial and human HSP could play a role in the development of the tissue-damage characteristic of KD.     

Immunogenicity of Mycobacterium ulcerans Hsp65 and protective efficacy of a Mycobacterium leprae Hsp65-based DNA vaccine against Buruli ulcer

Buruli ulcer, a disease caused by Mycobacterium ulcerans, is emerging as an increasingly important cause of morbidity throughout the world, for which surgery is the only efficient treatment to date. The aim of this work was to identify potential vaccine candidates in an experimental model of mouse infection. In BALB/c mice infected with M. ulcerans subcutaneously, Hsp65 appeared to be an immunodominant antigen eliciting both humoral and cellular responses. However, vaccination of mice with a DNA vector encoding Mycobacterium leprae Hsp65 only poorly limited the progression of M. ulcerans infection. In contrast, a substantial degree of protection was conferred by subcutaneous vaccination with BCG, suggesting that BCG antigens that are conserved in M. ulcerans, such as TB10.4, the 19 kDa antigen, PstS3 and Hsp70, may be interesting to consider as subunit vaccines in future prospects.     

Recombinant interleukin-4-treated macrophages, epithelioid cell surrogates, harbor and arrest Mycobacterium avium multiplication in vitro

Our group has previously described that murine peritoneal macrophages treated in vitro for 7 days with recombinant interleukin-4 (rIL-4) acquire morphological and functional characteristics of epithelioid cells (ECs) found in granulomatous lesions. Although EC function has not been clarified so far, it has been suggested that these cells could present antigens and control multiplication of mycobacteria. These aspects have been addressed here using in vitro EC surrogates. Using immunocytochemistry and immunofluorescence methods, we have observed an increased expression of CD11b, CD54, CD86 and CD40 molecules on rIL-4-treated macrophages when compared to untreated ones. Cytokine-treated cells were less phagocytic for latex beads (P<0.03) and more pinocytic for dextran particles than untreated macrophages. T-cell lymphoproliferation assays using ovalbumin (OVA) and Mycobacterium avium as antigens showed that both cultured macrophages were equally efficient as antigen presenting cells (APCs). However, M. avium antigens were better presented in vivo by EC surrogates (P<0.01). Both macrophage cultures were similarly infected by M. avium. However, while the infection level was maintained in the cytokine-treated population, untreated macrophages showed a progressive increase in the number of bacilli/cell with time (P<0.01) and a reduction of about 65% in cell population. After 96 h of M. avium infection, untreated cells secreted higher amounts of tumor necrosis factor-alpha (P<0.005) while rIL-4-treated macrophages showed higher, although not significant, transforming growth factor-beta production. Also, EC surrogates produced less nitric oxide than control macrophages (P<0.05). Hence, EC surrogates restrain M. avium growth and act as APCs in vitro and in vivo.     

Experimental Preemptive Immunotherapy of Murine Cytomegalovirus Disease with CD8 T-Cell Lines Specific for ppM83 and pM84, the Two Homologs of Human Cytomegalovirus Tegument Protein ppUL83 (pp65)

CD8 T cells are the principal antiviral effectors controlling cytomegalovirus (CMV) infection. For human CMV, the virion tegument protein ppUL83 (pp65) has been identified as a source of immunodominant peptides and is regarded as a candidate for cytoimmunotherapy and vaccination. Two sequence homologs of ppUL83 are known for murine CMV, namely the virion protein ppM83 (pp105) expressed late in the viral replication cycle and the nonstructural protein pM84 (p65) expressed in the early phase. Here we show that ppM83, unlike ppUL83, is not delivered into the antigen presentation pathway after virus penetration before or in absence of viral gene expression, while other virion proteins of murine CMV are processed along this route. In cytokine secretion-based assays, ppM83 and pM84 appeared to barely contribute to the acute immune response and to immunological memory. Specifically, the frequencies of M83 and M84 peptide-specific CD8 T cells were low and undetectable, respectively. Nonetheless, in a murine model of cytoimmunotherapy of lethal CMV disease, M83 and M84 peptide-specific cytolytic T-cell lines proved to be highly efficient in resolving productive infection in multiple organs of cell transfer recipients. These findings demonstrate that proteins which fail to prime a quantitatively dominant immune response can nevertheless represent relevant antigens in the effector phase. We conclude that quantitative and qualitative immunodominance are not necessarily correlated. As a consequence of these findings, there is no longer a rationale for considering T-cell abundance as the key criterion for choosing specificities to be included in immunotherapy and immunoprophylaxis of CMV disease and of viral infections in general.     

T lymphocytes from healthy individuals with specificity to self-epitopes shared by the mycobacterial and human 65-kilodalton heat shock protein

The immune response to mycobacterial pathogens comprises a significant percentage of T cells with specificity for a 65-kDa heat shock protein (hsp) which is highly conserved in bacteria and man. PBMC were activated in vitro with killed Mycobacterium tuberculosis and afterward tested for CTL activity on autologous target cells primed with 1) killed M. tuberculosis, 2) intact recombinant 65-kDa hsp of Mycobacterium bovis/M. tuberculosis; or 3) tryptic fragments of the recombinant 65-kDa hsp. Strong CTL activity was observed on targets primed with killed M. tuberculosis or with tryptic fragments of the 65-kDa hsp, but not on those primed with the intact 65-kDa hsp. M. tuberculosis activated T cells from 2/13 donors tested exerted killer activity against unprimed targets. To assess whether T cell responses were directed against self-epitopes shared by the mycobacterial and human 65-kDa hsp, four peptides of at least 10 amino acids length were synthesized corresponding to fully or almost identical regions of these molecules. Peripheral blood T cells from 8/9 individuals tested, after activation with killed M. tuberculosis, expressed strong CTL activity toward autologous targets primed with one or more of these synthetic peptides. By using HLA-DR transfected murine L cells we found that the epitopes were recognized in the context of histocompatible HLA-DR (class II) molecules. We conclude that the demonstration of T cells with specificity to self-epitopes in vitro is not indicative for autoimmune disease. However, if at certain stages of infection such T cells are activated by crossreactive microbial epitopes they could cause autoimmune responses.     

The protective immune response to heat shock protein 60 of Histoplasma capsulatum is mediated by a subset of V beta 8.1/8.2+ T cells

Immunization with recombinant heat shock protein 60 (rHsp60) from Histoplasma capsulatum or a region of the protein designated fragment 3 (F3) confers protection from a subsequent challenge in mice. To determine the T cell repertoire involved in the response to Hsp60, T cell clones from C57BL/6 mice immunized with rHsp60 were generated and examined for Vbeta usage by flow cytometry and RT-PCR. Vbeta8.1/8.2(+) T cells were preferentially expanded; other clones bore Vbeta4, -6, or -11. When Vbeta8.1/8.2(+) cells were depleted in mice, Vbeta4(+) T cell clones were almost exclusively isolated. Measurement of cytokine production demonstrated that nine of 16 Vbeta8.1/8.2(+) clones were Th1, while only three of 13 non-Vbeta8.1/8.2(+) clones were Th1. In mice immunized with rHsp60, depletion of Vbeta8.1/8.2(+), but not Vbeta6(+) plus Vbeta7(+), T cells completely abolished the protective efficacy of Hsp60 to lethal and sublethal challenges. Examination of the TCR revealed that a subset of Vbeta8.1/2(+) clones that produced IFN-gamma and were reactive to F3 shared a common CDR3 sequence, DGGQG. Transfer of these T cell clones into TCR alpha/beta(-/-) or IFN-gamma(-/-) mice significantly improved survival, while transfer of other Vbeta8.1/8.2(+) clones that were F3 reactive but were Th2 or clones that were not reactive to F3 but were Th1 did not confer protection. These data indicate that a distinct subset of Vbeta8.1/8.2(+) T cells is crucial for the generation of a protective response to rHsp60.     

Macrophage's proinflammatory response to a mycobacterial infection is dependent on sphingosine kinase-mediated activation of phosphatidylinositol phospholipase C, protein kinase C, ERK1/2, and phosphatidylinositol 3-kinase

Previous studies have shown that the ability of Mycobacterium tuberculosis to block a Ca(2+) flux is an important step in its capacity to halt phagosome maturation. This affect on Ca(2+) release results from M. tuberculosis inhibition of sphingosine kinase (SPK) activity. However, these studies did not address the potential role of SPK and Ca(2+) in other aspects of macrophage activation including production of proinflammatory mediators. We previously showed that nonpathogenic Mycobacterium smegmatis and to a lesser extent pathogenic Mycobacterium avium, activate Ca(2+)-dependent calmodulin/calmodulin kinase and MAPK pathways in murine macrophages leading to TNF-alpha production. However, whether SPK functions in promoting MAPK activation upon mycobacterial infection was not defined in these studies. In the present work we found that SPK is required for ERK1/2 activation in murine macrophages infected with either M. avium or M. smegmatis. Phosphoinositide-specific phospholipase C (PI-PLC) and conventional protein kinase C (cPKC) were also important for ERK1/2 activation. Moreover, there was increased activation of cPKC and PI3K in macrophages infected with M. smegmatis compared with M. avium. This cPKC and PI3K activation was dependent on SPK and PI-PLC. Finally, in macrophages infected with M. smegmatis compared with M. avium, we observed enhanced secretion of TNF-alpha, IL-6, RANTES, and G-CSF and found production of these inflammatory mediators to be dependent on SPK, PI-PLC, cPKC, and PI3K. These studies are the first to show that the macrophage proinflammatory response following a mycobacterial infection is regulated by SPK/PI-PLC/PKC activation of ERK1/2 and PI3K pathways.     

Isolation and diagnostic potential of ISMav2, a novel insertion sequence-like element from Mycobacterium avium subspecies paratuberculosis

A novel Mycobacterium avium ssp. paratuberculosis (M. paratuberculosis) specific insertion sequence has been identified by representational difference analysis and designated as ISMav2. ISMav2 has no similarity to known mycobacterial IS elements but shows more than 50% identity to a non-composite transposon of Streptomyces coelicolor at the DNA and protein level. ISMav2 is present in at least three copies on the genome as assessed by Southern blot analysis and its potential value as a diagnostic tool was confirmed by PCR analyses on 79 M. paratuberculosis field isolates, nine M. avium ssp. avium isolates, and the reference strains of nine other mycobacterial species.     

Expression profiles of low molecular mass heat shock proteins by Mycobacterium avium and Mycobacterium intracellulare

Closely related non-tuberculous mycobacterial species, Mycobacterium avium and Mycobacterium intracellulare, were compared for the profiles of their production of low molecular mass heat shock proteins at 45 degrees C, by performing polyacrylamide gel electrophoresis analysis of bacterial cell lysate proteins. All of the M. intracellulare but not M. avium strains potently increased the production of the 18-kDa heat shock protein, when cultured at 45 degrees C. Half of the M. intracellulare strains with high sensitivity to 45 degrees C produced not only the 18-kDa heat shock protein but also the 16-kDa heat shock protein at 45 degrees C. These findings indicate that M. avium and M. intracellulare differentially respond to 45 degrees C heat shock in terms of the production of low molecular mass heat shock proteins.     

fecB, a gene potentially involved in iron transport in Mycobacterium avium, is not induced within macrophages

FecB is a protein involved in the transport of iron from ferric citrate in Escherichia coli and is present in the Mycobacterium tuberculosis genome sequence. Since the ability to retrieve iron from the host is crucial and may be related to virulence, we characterized the gene fecB from Mycobacterium avium, strain 101. An E. coli-mycobacterial shuttle plasmid with a fecB-promoter green fluorescence protein (gfp)-fusion was transformed into M. avium strain 104 to study the fecB-regulation. In vitro, the fecB expression in M. avium weakly correlated with the amount of iron present in the medium but the expression was maximal when there was no iron in the culture medium. In macrophages, M. avium fec B was not induced during the early phase of infection, suggesting that the iron concentration in the mycobacterial phagosome is not sufficiently low to stimulate the expression of fecB in M. avium.     

Characterization of the fibronectin-attachment protein of Mycobacterium avium reveals a fibronectin-binding motif conserved among mycobacteria

Mycobacterium avium is an intracellular pathogen and a major opportunistic infectious agent observed in patients with acquired immune deficiency syndrome (AIDS). Evidence suggests that the initial portal of infection by M. avium is often the gastrointestinal tract. However, the mechanism by which the M. avium crosses the epithelial barrier is unclear. A possible mechanism is suggested by the ability of M. avium to bind fibronectin, an extracellular matrix protein that is a virulence factor for several extracellular pathogenic bacteria which bind to mucosal surfaces. To further characterize fibronectin binding by M. avium , we have cloned the M. avium fibronectin-attachment protein (FAP). The M. avium FAP (FAP-A) has an unusually large number of Pro and Ala residues (40% overall) and is 50% identical to FAP of both Myco-bacterium leprae and Mycobacterium tuberculosis . Using recombinant FAP-A and FAP-A peptides, we show that two non-continuous regions in FAP-A bind fibronectin. Peptides from these regions and homologous sequences from M. leprae FAP inhibit fibronectin binding by both M. avium and Mycobacterium bovis Bacillus Calmette–Guerin (BCG). These regions have no homology to eukaryotic fibronectin-binding proteins and are only distantly related to fibronectin-binding peptides of Gram-positive bacteria. Nevertheless, these fibronectin-binding regions are highly conserved among the mycobacterial FAPs, suggesting an essential function for this interaction in mycobacteria infection of their metazoan hosts.     

结核杆菌Hsp65与人IL-2融合蛋白在耻垢杆菌表达

目的：构建能够分泌表达结核分枝杆菌热休克蛋白65（Hsp65）与人IL-2融合蛋白的重组耻垢分枝杆菌（recombinant Mycobacterium Smegmatis,rMs）。方法：用EcoRV和HindIII双酶切含Hsp65．IL-2融合基因的pPRO-hsp65-IL-2载体,回收目的基因片断Hsp65-IL-2,并将其亚克隆入同样双酶切的大肠埃希菌-分枝杆菌穿梭分泌表达载体pDE22中。重组质粒pDE22-hsp65-IL-2酶切鉴定正确后,电穿孔转化MS感受态,潮霉素抗性压力筛选阳性rMs。Westem—blot鉴定rMs培养上清蛋白中目的蛋白的表达。结果：重组pDE22-hsp65-IL-2质粒酶切后可获得约2000bp片段,与预期大小一致。Western-blot结果表明,rMs培养上清蛋白中有特异性反应条带,大小为78kD,与Hsp65-IL-2融合蛋白大小相一致。结论：成功构建了大肠埃希菌．分枝杆菌穿梭分泌表达载体pDE22-hsp65-IL-2,为该rMs的免疫学特性及抗结核分枝杆菌感染的保护效果研究奠定了基础。     

TGF-β-mediated sustained ERK1/2 activity promotes the inhibition of intracellular growth of Mycobacterium avium in epithelioid cells surrogates

Transforming growth factor beta (TGF-β) has been implicated in the pathogenesis of several diseases including infection with intracellular pathogens such as the Mycobacterium avium complex. Infection of macrophages with M. avium induces TGF-β production and neutralization of this cytokine has been associated with decreased intracellular bacterial growth. We have previously demonstrated that epithelioid cell surrogates (ECs) derived from primary murine peritoneal macrophages through a process of differentiation induced by IL-4 overlap several features of epithelioid cells found in granulomas. In contrast to undifferentiated macrophages, ECs produce larger amounts of TGF-β and inhibit the intracellular growth of M. avium. Here we asked whether the levels of TGF-β produced by ECs are sufficient to induce a self-sustaining autocrine TGF-β signaling controlling mycobacterial replication in infected-cells. We showed that while exogenous addition of increased concentration of TGF-β to infected-macrophages counteracted M. avium replication, pharmacological blockage of TGF-β receptor kinase activity with SB-431542 augmented bacterial load in infected-ECs. Moreover, the levels of TGF-β produced by ECs correlated with high and sustained levels of ERK1/2 activity. Inhibition of ERK1/2 activity with U0126 increased M. avium replication in infected-cells, suggesting that modulation of intracellular bacterial growth is dependent on the activation of ERK1/2. Interestingly, blockage of TGF-β receptor kinase activity with SB-431542 in infected-ECs inhibited ERK1/2 activity, enhanced intracellular M. avium burden and these effects were followed by a severe decrease in TGF-β production. In summary, our findings indicate that the amplitude of TGF-β signaling coordinates the strength and duration of ERK1/2 activity that is determinant for the control of intracellular mycobacterial growth.     

Development of monoclonal antibodies reacting against mycobacterial 65 kDa heat shock protein by using recombinant truncated products.

A mycobacterial 65 kDa molecule is a member of the GroEL heat shock protein family. We developed mAbs reacting against recombinant 65 kDa protein by using a gene (pTB12) which encodes this protein. Three mAbs (B20, B97 and B167) reacted selectively with 65 kDa proteins of Mycobacterium tuberculosis, BCG and Mycobacterium leprae, although B20 and B167 may weakly react with a 15 kDa molecule of mammalian cells. One (B108) was obviously cross-reactive between mycobacterial 65 kDa and the mammalian intracytoplasmic protein. We also developed deletion mutants of pTB12. The localization of these mAb-defined epitopes was determined by using truncated proteins of the Mycobacterium tuberculosis 65 kDa molecule produced in E. coli. Immunohistochemical analysis showed that B20, B97 and B167 mAbs could detect this antigen in experimental granulomas induced by injection of BCG in the subcutaneous tissue of rats. These mAbs should be useful for analyzing the immunobiologic roles of mycobacterial 65 kDa molecules.     

Etiology of Crohn's disease: the role of Mycobacterium avium paratuberculosis

Crohn's disease is a chronic inflammatory bowel disease characterized by transmural inflammation and granuloma formation. Several theories regarding the etiology of Crohn's disease have been proposed, one of which is infection with Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis), which causes a similar disease in animals, and is present in the human food chain. Considerable evidence supports the presence of M. paratuberculosis in the intestinal tissues of many patients with Crohn's disease including culture, detection of homologous mycobacterial DNA, detection of the mycobacterial insertion sequence IS900 by both PCR and in situ hybridization in tissues, and a serologic immune response to recombinant M. paratuberculosis antigens. Despite this evidence, and our personal belief that M. paratuberculosis is a cause of Crohn's disease, widespread acceptance of this hypothesis will require evidence that specific anti-mycobacterial chemotherapy will cure the disease.