Biomedical subjects

John T Belisle

Publications and source records attributed to John T Belisle.

At least 19 recordsLinked to original sources

PubMed2006

Use of protein microarrays to define the humoral immune response in leprosy patients and identification of disease-state-specific antigenic profiles.

Although the global prevalence of leprosy has decreased over the last few decades due to an effective multidrug regimen, large numbers of new cases are still being reported, raising questions as to the ability to identify patients likely to spread disease and the effects of chemotherapy on the overall incidence of leprosy. This can partially be attributed to the lack of diagnostic markers for different clinical states of the disease and the consequent implementation of differential, optimal drug therapeutic strategies. Accordingly, comparative bioinformatics and Mycobacterium leprae protein microarrays were applied to investigate whether leprosy patients with different clinical forms of the disease can be categorized based on differential humoral immune response patterns. Evaluation of sera from 20 clinically diagnosed leprosy patients using native protein and recombinant protein microarrays revealed unique disease-specific, humoral reactivity patterns. Statistical analysis of the serological patterns yielded distinct groups that correlated with phenolic glycolipid I reactivity and clinical diagnosis, thus demonstrating that leprosy patients, including those diagnosed with the paucibacillary, tuberculoid form of disease, can be classified based on humoral reactivity to a subset of M. leprae protein antigens produced in recombinant form.

Antibodies, Bacterial↗

PubMed2006

Phosphatidylinositol mannoside from Mycobacterium tuberculosis binds alpha5beta1 integrin (VLA-5) on CD4+ T cells and induces adhesion to fibronectin.

The pathological hallmark of the host response to Mycobacterium tuberculosis is the granuloma where T cells and macrophages interact with the extracellular matrix (ECM) to control the infection. Recruitment and retention of T cells within inflamed tissues depend on adhesion to the ECM. T cells use integrins to adhere to the ECM, and fibronectin (FN) is one of its major components. We have found that the major M. tuberculosis cell wall glycolipid, phosphatidylinositol mannoside (PIM), induces homotypic adhesion of human CD4+ T cells and T cell adhesion to immobilized FN. Treatment with EDTA and cytochalasin D prevented PIM-induced T cell adhesion. PIM-induced T cell adhesion to FN was blocked with mAbs against alpha5 integrin chain and with RGD-containing peptides. Alpha5beta1 (VLA-5) is one of two major FN receptors on T cells. PIM was found to bind directly to purified human VLA-5. Thus, PIM interacts directly with VLA-5 on CD4+ T lymphocytes, inducing activation of the integrin, and promoting adhesion to the ECM glycoprotein, FN. This is the first report of direct binding of a M. tuberculosis molecule to a receptor on human T cells resulting in a change in CD4+ T cell function.

CD4-Positive T-Lymphocytes↗

PubMed2006

Disease state differentiation and identification of tuberculosis biomarkers via native antigen array profiling.

A critical element of tuberculosis control is early and sensitive diagnosis of infection and disease. Our laboratories recently showed that different stages of disease were distinguishable via two-dimensional Western blot analyses of Mycobacterium tuberculosis culture filtrate proteins. However, this methodology is not suitable for high throughput testing. Advances in protein microarray technology provide a realistic mechanism to screen a large number of serum samples against thousands of proteins to identify biomarkers of disease states. Techniques were established for separation of native M. tuberculosis cytosol and culture filtrate proteins, resulting in 960 unique protein fractions that were used to generate protein microarrays. Evaluation of serological reactivity from 42 patients in three tuberculosis disease states and healthy purified protein derivative-positive individuals demonstrated that human immunodeficiency virus (HIV)-negative cavitary and noncavitary tuberculosis (TB) patients' sera recognized 126 and 59 fractions, respectively. Sera from HIV patients coinfected with TB recognized 20 fractions of which five overlapped with those recognized by non-HIV TB patients' sera and 15 were unique to the HIV+TB+ disease state. Identification of antigens within the reactive fractions yielded 11 products recognized by both cavitary and noncavitary TB patients' sera and four proteins (HspX, MPT64, PstS1, and TrxC) specific to cavitary TB patients. Moreover four novel B cell antigens (BfrB, LppZ, SodC, and TrxC) of human tuberculosis were identified.

Amino Acid Sequence↗

PubMed2006

Identification of cell cycle regulators in Mycobacterium tuberculosis by inhibition of septum formation and global transcriptional analysis.

In Mycobacterium tuberculosis the mechanism of septum formation and regulation of cell division remains undefined. In other bacterial species FtsZ polymerization and septum formation are influenced through protein interactions in addition to transcriptional regulation, and the combination of these provides tight regulation of this process. However, homologues of proteins known to affect FtsZ assembly have not been identified and substantiated in M. tuberculosis. This suggests that M. tuberculosis may possess unique processes for regulation of septum formation. To begin to address this poorly understood aspect of M. tuberculosis physiology, FtsZ inhibitors were used to block cell division and the effects on bacterial morphology and the transcriptional response were examined. Inhibition of septum formation prevented cell division and led to bacterial filamentation. Microarray-based transcriptional profiling allowed the evaluation of multiple metabolic processes in response to inhibition of septum formation and when coupled with bioinformatics provided a means to identify regulatory elements and other gene products that probably influence septum formation.

Bacterial Proteins↗

PubMed2006

Mycobacterium tuberculosis malate synthase is a laminin-binding adhesin.

Mycobacterium tuberculosis (M. tb) uses the glyoxalate bypass for intracellular survival in vivo. These studies provide evidence that the M. tb malate synthase (MS) has adapted to function as an adhesin which binds to laminin and fibronectin. This binding is achieved via the unique C-terminal region of the M. tb MS. The ability to function as an adhesin necessitates extracellular localization. We provide evidence that despite the absence of a Sec-translocation signal sequence the M. tb MS is secreted/excreted, and is anchored on the cell wall by an undefined mechanism. The MS of Mycobacterium smegmatis is cytoplasmic but the M. tb MS expressed in M. smegmatis localizes to the cell wall and enhances the adherence of the bacteria to lung epithelial A549 cells. Antibodies to the C-terminal laminin/fibronectin-binding domain interfere with the binding of the M. tb MS to laminin and fibronectin and reduce the adherence of M. tb to A549 cells. Coupled to the earlier evidence of in vivo expression of M. tb MS during active but not latent infection in humans, these studies show that a housekeeping enzyme of M. tb contributes to its armamentarium of virulence promoting factors.

Adhesins, Bacterial↗

PubMed2006

M. tuberculosis Rv2252 encodes a diacylglycerol kinase involved in the biosynthesis of phosphatidylinositol mannosides (PIMs).

Phosphorylated lipids play important roles in biological systems, not only as structural moieties but also as modulators of cellular function. Phospholipids of pathogenic bacteria are known to play roles both as membrane components and as factors that modulate the infectious process. Mycobacterium tuberculosis is, however, noteworthy in that it has an extremely diverse repertoire of biologically active phosphorylated lipids that, in the absence of a specialized protein translocation system, appear to constitute the main means of communication with the host. Many of these lipids are derived from phosphatidylinositol (PI) that is differentially processed to give rise to phosphatidylinositol mannosides (PIMs) or lipoarabinomannan. In preliminary studies on the lipid processing enzymes associated with the bacterial cell wall, a kinase activity was noted that gave rise to a novel lipid species released by the bacterium. It was determined that this kinase activity was encoded by the ORF Rv2252. Rv2252 demonstrates the capacity to phosphorylate various amphipathic lipids of host and bacterial origin, in particular a M. tuberculosis derived diacylglycerol. Targeted deletion of the rv2252 gene resulted in disruption of the production of certain higher order PIM species, suggesting a role for Rv2252 in the biosynthetic pathway of PI, a PIM precursor.

Amino Acid Sequence↗

PubMed2005

A randomised controlled trial of the effects of albendazole in pregnancy on maternal responses to mycobacterial antigens and infant responses to Bacille Calmette-Guérin (BCG) immunisation [ISRCTN32849447].

BACKGROUND: Maternal schistosomiasis and filariasis have been shown to influence infant responses to neonatal bacille Calmette-Guérin (BCG) immunisation but the effects of maternal hookworm, and of de-worming in pregnancy, are unknown. METHODS: In Entebbe, Uganda, we conducted a randomised, double-blind, placebo-controlled trial of a single dose of 400 mg of albendazole in the second trimester of pregnancy. Neonates received BCG. Interferon-gamma (IFN-gamma) and interleukin (IL)-5 responses to a mycobacterial antigen (crude culture filtrate proteins (CFP) of Mycobacterium tuberculosis) were measured in a whole blood assay. We analysed results for binary variables using chi2 tests and logistic regression. We analysed continuous variables using Wilcoxon's tests. RESULTS: Maternal hookworm was associated with reduced maternal IFN-gamma responses to CFP (adjusted odds ratio for IFN-gamma > median response: 0.14 (95% confidence interval 0.02-0.83, p = 0.021). Conversely, maternal hookworm was associated with subsequent increased IFN-gamma responses in their one-year-old infants (adjusted OR 17.65 (1.20-258.66; p = 0.013)). Maternal albendazole tended to reduce these effects. CONCLUSION: Untreated hookworm infection in pregnancy was associated with reduced maternal IFN-gamma responses to mycobacterial antigens, but increased responses in their infants one year after BCG immunisation. The mechanisms of these effects, and their implications for protective immunity remain, to be determined.

Adult↗

PubMed2005

A major cell wall lipopeptide of Mycobacterium avium subspecies paratuberculosis.

Mycobacterium avium subspecies paratuberculosis (MAP), the causative agent of Johne disease in cattle and other ruminants, is proposed to be at least one of the causes of Crohn disease in humans. MAP and Mycobacterium avium subspecies avium, a closely related opportunistic environmental bacterium, share 95% of their genes and exhibit homologies of more than 99% between these genes. The identification of molecules specific for MAP is essential for understanding its pathogenicity and for development of useful diagnostic tools. The application of gas chromatography, mass spectrometry, and nuclear magnetic resonance led to the structural identification of a major cell wall lipopeptide of MAP, termed Para-LP-01, defined as C20 fatty acyl-D-Phe-N-Me-L-Val-L-Ile-L-Phe-L-Ala methyl ester. Variations of this lipopeptide with different fatty acyl moieties (C16 fatty acyl through C17, C18, C19, C21 to C22) were also identified. Besides the specificity of this lipopeptide for MAP, the presence of an N-Me-L-valine represents the first reported N-methylated amino acid within an immunogenic lipopeptide of mycobacteria. Sera from animals with Johne disease, but not sera from uninfected cattle, reacted with this lipopeptide, indicating potential biological importance.

Animals↗

PubMed2005

Export-mediated assembly of mycobacterial glycoproteins parallels eukaryotic pathways.

Protein O-mannosylation is an essential and evolutionarily conserved post-translational modification among eukaryotes. This form of protein modification is also described in Mycobacterium tuberculosis; however, the mechanism of mannoprotein assembly remains unclear. Evaluation of differentially translocated chimeric proteins and mass spectrometry to monitor glycosylation demonstrated that specific translocation processes were required for protein O-mannosylation in M. tuberculosis. Additionally, Rv1002c, a M. tuberculosis membrane protein homolog of eukaryotic protein mannosyltransferases, was shown to catalyze the initial step of protein mannosylation. Thus, the process of protein mannosylation is conserved between M. tuberculosis and eukaryotic organisms.

Cloning, Molecular↗

PubMed2005

The human CD1-restricted T cell repertoire is limited to cross-reactive antigens: implications for host responses against immunologically related pathogens.

The repertoires of CD1- and MHC-restricted T cells are complementary, permitting the immune recognition of both lipid and peptide Ags, respectively. To compare the breadth of the CD1-restricted and MHC-restricted T cell repertoires, we evaluated T cell responses against lipid and peptide Ags of mycobacteria in leprosy, comparing tuberculoid patients, who are able to restrict the pathogen, and lepromatous patients, who have disseminated infection. The striking finding was that in lepromatous leprosy, T cells did not efficiently recognize lipid Ags from the leprosy pathogen, Mycobacterium leprae, or the related species, Mycobacterium tuberculosis, yet were able to efficiently recognize peptide Ags from M. tuberculosis, but not M. leprae. To identify a mechanism for T cell unresponsiveness against mycobacterial lipid Ags in lepromatous patients, we used T cell clones to probe the species specificity of the Ags recognized. We found that the majority of M. leprae-reactive CD1-restricted T cell clones (92%) were cross-reactive for multiple mycobacterial species, whereas the majority of M. leprae-reactive MHC-restricted T cells were species specific (66%), with a limited number of T cell clones cross-reactive (34%) with M. tuberculosis. In comparison with the MHC class II-restricted T cell repertoire, the CD1-restricted T cell repertoire is limited to recognition of cross-reactive Ags, imparting a distinct role in the host response to immunologically related pathogens.

Adult↗

PubMed2005

Antigens of Mycobacterium tuberculosis recognized by antibodies during incipient, subclinical tuberculosis.

Serum samples obtained from human immunodeficiency virus (HIV)-infected tuberculosis (TB) patients months prior to clinical TB were used to delineate the profile of Mycobacterium tuberculosis culture filtrate proteins recognized during subclinical TB. A subset of approximately 12 antigens was recognized by antibodies in these serum samples. Antibodies to two of these antigens (81 [88]-kDa malate synthase [GlcB] and MPT51) were present in serum samples obtained during incipient subclinical TB in 19 (approximately 90%) of the 21 HIV-infected TB patients tested. These antigens will be useful for devising diagnostic tests that can identify HIV-positive individuals who are at a high risk for developing clinical TB.

Antibodies, Bacterial↗

PubMed2005

Demonstration of components of antigen 85 complex in cerebrospinal fluid of tuberculous meningitis patients.

Tuberculous meningitis (TBM) is the most common form of chronic infection of the central nervous system. Despite the magnitude of the problem, the general diagnostic outlook is discouraging. Specifically, there is no generally accepted early confirmative diagnosis protocol available for TBM. Various Mycobacterium tuberculosis antigens are now recognized as potential markers for diagnosis of TBM. However, their presence remains questionable, and many of these antigens are reported in the blood but not in the cerebrospinal fluid (CSF). This study identifies a specific protein marker in CSF which will be useful in early diagnosis of TBM. We have demonstrated the presence of a 30-kDa protein band in CSF of 100% (n = 5) of confirmed and 90% (n = 138) of suspected TBM patients out of 153 TBM patients. The 30-kDa band was excised from the gel, destained extensively, and digested with trypsin. The resulting peptides were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Partially purified proteins from CSF samples of TBM were analyzed by two-dimensional polyacrylamide gel electrophoresis and Western blotting. Immunoblotting and enzyme-linked immunosorbent assay (ELISA) were performed to confirm the presence of proteins in the 30-kDa protein band. The antigen 85 (Ag 85) complex was detected in CSF of TBM patients by indirect ELISA using antibodies against Ag 85 complex. The results of this study showed the 30-kDa protein band contained MTB proteins Rv3804c (Ag85A) and Rv1886c (Ag 85B), both members of the Ag85 complex. This was also confirmed by using immunotechniques such as indirect ELISA and the dot immunobinding assay. Detection of Ag85 complex was observed in CSF of 89% (71 out of 80) of suspected TBM patients that were 30-kDa protein positive. The observed 30-kDa protein in the CSF is comprised of the MTB Ag85 complex. This protein was earlier reported to be present in the blood of patients with extra-central nervous system tuberculosis. Therefore, this finding suggests that this protein can be used as a molecular marker for any type of tuberculous infection. It also provides a more sensitive immunoassay option for the early and confirmatory diagnosis of TBM.

Acyltransferases↗

PubMed2005

Peripheral blood and pleural fluid mononuclear cell responses to low-molecular-mass secretory polypeptides of Mycobacterium tuberculosis in human models of immunity to tuberculosis.

A total of 104 polypeptides were purified from the low-molecular-mass secretory proteome of Mycobacterium tuberculosis H(37)Rv using a combination of anion exchange column chromatography and high resolution preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by electroelution. The goal of this study was to identify polypeptides from a low-molecular-mass secretory proteome recognized by human subjects infected with M. tuberculosis and to ascertain the differences in specificity of antigen recognition by the peripheral blood mononuclear cells (PBMCs) and pleural fluid mononuclear cells (PFMCs) of these individuals. The study identified CFP-8 (Rv0496), CFP-11 (Rv2433c), CFP-14.5 (Rv2445c), and CFP-31 (Rv0831c) as novel T-cell antigens apart from previously characterized ESAT-6, TB10.4, CFP10, GroES, MTSP14, MTSP17, CFP21, MPT64, Ag85A, and Ag85B on the basis of recognition by PBMCs of tuberculosis contacts and treated tuberculosis patients. Further, polypeptides prominently recognized by PFMCs of tuberculous pleurisy patients were the same as those recognized by PBMCs of healthy contacts and treated tuberculosis patients. The results of our study indicate the homogeneity of antigenic target recognition by lymphocytes at the site of infection and at the periphery in the human subjects studied and the need to evaluate these antigenic targets as components of future antituberculous vaccines.

Adult↗

PubMed2005

Mycobacterial lipid II is composed of a complex mixture of modified muramyl and peptide moieties linked to decaprenyl phosphate.

Structural analysis of compounds identified as lipid I and II from Mycobacterium smegmatis demonstrated that the lipid moiety is decaprenyl phosphate; thus, M. smegmatis is the first bacterium reported to utilize a prenyl phosphate other than undecaprenyl phosphate as the lipid carrier involved in peptidoglycan synthesis. In addition, mass spectrometry showed that the muropeptides from lipid I are predominantly N-acetylmuramyl-L-alanine-D-glutamate-meso-diaminopimelic acid-D-alanyl-D-alanine, whereas those isolated from lipid II form an unexpectedly complex mixture in which the muramyl residue and the pentapeptide are modified singly and in combination. The muramyl residue is present as N-acetylmuramic acid, N-glycolylmuramic acid, and muramic acid. The carboxylic functions of the peptide side-chains of lipid II showed three types of modification, with the dominant one being amidation. The preferred site for amidation is the free carboxyl group of the meso-diaminopimelic acid residue. Diamidated species were also observed. The carboxylic function of the terminal D-alanine of some molecules is methylated, as are all three carboxylic acid functions of other molecules. This study represents the first structural analysis of mycobacterial lipid I and II and the first report of extensive modifications of these molecules. The observation that lipid I was unmodified strongly suggests that the lipid II intermediates of M. smegmatis are substrates for a variety of enzymes that introduce modifications to the sugar and amino acid residues prior to the synthesis of peptidoglycan.

Cell Wall↗

PubMed2004

Mycobacterium tuberculosis functional network analysis by global subcellular protein profiling.

Trends in increased tuberculosis infection and a fatality rate of approximately 23% have necessitated the search for alternative biomarkers using newly developed postgenomic approaches. Here we provide a systematic analysis of Mycobacterium tuberculosis (Mtb) by directly profiling its gene products. This analysis combines high-throughput proteomics and computational approaches to elucidate the globally expressed complements of the three subcellular compartments (the cell wall, membrane, and cytosol) of Mtb. We report the identifications of 1044 proteins and their corresponding localizations in these compartments. Genome-based computational and metabolic pathways analyses were performed and integrated with proteomics data to reconstruct response networks. From the reconstructed response networks for fatty acid degradation and lipid biosynthesis pathways in Mtb, we identified proteins whose involvements in these pathways were not previously suspected. Furthermore, the subcellular localizations of these expressed proteins provide interesting insights into the compartmentalization of these pathways, which appear to traverse from cell wall to cytoplasm. Results of this large-scale subcellular proteome profile of Mtb have confirmed and validated the computational network hypothesis that functionally related proteins work together in larger organizational structures.

Automation↗

PubMed2004

Utilization of a ts-sacB selection system for the generation of a Mycobacterium avium serovar-8 specific glycopeptidolipid allelic exchange mutant.

BACKGROUND: Mycobacterium avium are ubiquitous environmental organisms and a cause of disseminated infection in patients with end-stage AIDS. The glycopeptidolipids (GPL) of M. avium are proposed to participate in the pathogenesis of this organism, however, establishment of a clear role for GPL in disease production has been limited by the inability to genetically manipulate M. avium. METHODS: To be able to study the role of the GPL in M. avium pathogenesis, a ts-sacB selection system, not previously used in M. avium, was employed as a means to achieve homologous recombination for the rhamnosyltransferase (rtfA) gene of a pathogenic serovar 8 strain of M. avium to prevent addition of serovar-specific sugars to rhamnose of the fatty acyl-peptide backbone of GPL. The genotype of the resultant rtfA mutant was confirmed by polymerase chain reaction and southern hybridization. Disruption in the proximal sugar of the haptenic oligosaccharide resulted in the loss of serovar specific GPL with no change in the pattern of non-serovar specific GPL moieties as shown by thin layer chromatography and gas chromatography/mass spectrometry. Complementation of wild type (wt) rtfA in trans through an integrative plasmid restored serovar-8 specific GPL expression identical to wt serovar 8 parent strain. RESULTS: In this study, we affirm our results that rtfA encodes an enzyme responsible for the transfer of Rha to 6d-Tal and provide evidence of a second allelic exchange mutagenesis system suitable for M. avium. CONCLUSION: We report the second allelic exchange system for M. avium utilizing ts-sacB as double-negative and xylE as positive counter-selection markers, respectively. This system of allelic exchange would be especially useful for M. avium strains that demonstrate significant isoniazid (INH) resistance despite transformation with katG. Through the construction of mutants in GPL or other mycobacterial components, their roles in M. avium pathogenesis, biosynthesis, or drug resistance can be studied in a consistent manner.

Journal Article↗

PubMed2004

Mycobacterium tuberculosis LprG (Rv1411c): a novel TLR-2 ligand that inhibits human macrophage class II MHC antigen processing.

MHC class II (MHC-II)-restricted CD4(+) T cells are essential for control of Mycobacterium tuberculosis infection. This report describes the identification and purification of LprG (Rv1411c) as an inhibitor of primary human macrophage MHC-II Ag processing. LprG is a 24-kDa lipoprotein found in the M. tuberculosis cell wall. Prolonged exposure (>16 h) of human macrophages to LprG resulted in marked inhibition of MHC-II Ag processing. Inhibition of MHC-II Ag processing was dependent on TLR-2. Short-term exposure (<6 h) to LprG stimulated TLR-2-dependent TNF-alpha production. Thus, LprG can exploit TLR-2 signaling to inhibit MHC-II Ag processing in human macrophages. Inhibition of MHC-II Ag processing by mycobacterial lipoproteins may allow M. tuberculosis, within infected macrophages, to avoid recognition by CD4(+) T cells.

Amino Acid Sequence↗

PubMed2004

Mycobacterium tuberculosis antigen 85A and 85C structures confirm binding orientation and conserved substrate specificity.

The maintenance of the highly hydrophobic cell wall is central to the survival of Mycobacterium tuberculosis within its host environment. The antigen 85 proteins (85A, 85B, and 85C) of M. tuberculosis help maintain the integrity of the cell wall 1) by catalyzing the transfer of mycolic acids to the cell wall arabinogalactan and 2) through the synthesis of trehalose dimycolate (cord factor). Additionally, these secreted proteins allow for rapid invasion of alveolar macrophages via direct interactions between the host immune system and the invading bacillus. Here we describe two crystal structures: the structure of antigen 85C co-crystallized with octylthioglucoside as substrate, resolved to 2.0 A, and the crystal structure of antigen 85A, which was solved at a resolution of 2.7 A. The structure of 85C with the substrate analog identifies residues directly involved in substrate binding. Elucidation of the antigen 85A structure, the last of the three antigen 85 homologs to be solved, shows that the active sites of the three antigen 85 proteins are virtually identical, indicating that these share the same substrate. However, in contrast to the high level of conservation within the substrate-binding site and the active site, surface residues disparate from the active site are quite variable, indicating that three antigen 85 enzymes are needed to evade the host immune system.

Acyltransferases↗