Biomedical subjects

Karen M Dobos

Publications and source records attributed to Karen M Dobos.

8 recordsLinked to original sources

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↗

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

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

Continued proteomic analysis of Mycobacterium leprae subcellular fractions.

Recently the sequence of the Mycobacterium leprae chromosome, the only known obligate intracellular mycobacterium, was completed. It has a dramatic reduction in functional genes, with a coding capacity of only 49.5%, the lowest one so far observed among bacterial genomes. The leprosy bacillus seems to preserve a minimal set of genes that allows its survival in the host. The identification of genes that are actually expressed by the bacterium is of high significance in the context of mycobacterial pathogenesis. In this current study, a proteomic approach was undertaken to identify the proteins present in the soluble/cytosol and membrane subcellular fractions obtained from armadillo derived M. leprae. Proteins from each fraction were separated by two-dimensional gel electrophoresis (2-DE) and identified by mass spectrometry. A total of 147 protein spots were identified from 2-DE patterns and shown to comprise products of 44 different genes, twenty eight of them corresponding to new proteins. Additionally, two highly basic proteins (with pI >10.0) were isolated by heparin affinity chromatography and identified by N-terminal sequencing. This study constitutes the first application of proteomics to a host-derived Mycobacterium.

Bacterial Proteins↗

PubMed2004

A limited antigen-specific cellular response is sufficient for the early control of Mycobacterium tuberculosis in the lung but is insufficient for long-term survival.

Mice that were transgenic for a T-cell receptor (TCR) specific for ovalbumin peptide(323-339) (DO11.10) were able to survive an infection with Mycobacterium tuberculosis for approximately 80 days. This limited early control of infection was associated with gamma interferon production, inducible nitric oxide synthase expression within the lung, and an influx of clonotypic lymphocytes. The control of M. tuberculosis was lost in DO11.10 mice bred in a rag mutant background, demonstrating that the immune responsiveness was recombinase dependent and likely to be associated with the expression of an alternative alpha TCR by DO11.10 mice. A characterization of the antigen specificity in DO11.10 TCR transgenic mice demonstrated that the specificity was limited and dominated by the 26-kDa (Rv1411c) lipoprotein of M. tuberculosis. This study identifies this lipoprotein as an important and potent inducer of protective T cells within the lungs of mice infected with M. tuberculosis and therefore as a possible target for vaccination.

Animals↗

PubMed2004

Proteomic approaches to antigen discovery.

Proteomics has been widely applied to develop two-dimensional polyacrylamide gel electrophoresis maps and databases, evaluate gene expression profiles under different environmental conditions, assess global changes associated with specific mutations, and define drug targets of bacterial pathogens. When coupled to immunological assays, proteomics may also be used to identify B-cell and T-cell antigens within complex protein mixtures. This chapter describes the proteomic approaches developed by our laboratories to accelerate the antigen discovery program for Mycobacterium tuberculosis. As presented or with minor modifications, these techniques may be universally applied to other bacterial pathogens or used to identify bacterial proteins possessing other immunological properties.

Animals↗

PubMed2004

Buruli ulcer and schistosomiasis: no association found.

Helminth infections elicit an immune response potentially enhancing susceptibility to mycobacterial diseases. Schistosomiasis and infection with Mycobacterium ulcerans show a remarkable similarity in epidemiologic characteristics in Ghana. In 2000, a case-control study was conducted in three districts in Ghana endemic for M. ulcerans. One hundred six patients with confirmed M. ulcerans disease and 106 matched community controls were included. Schistosome infection of these patients and controls was measured by an enzyme-linked immunosorbent assay that detected circulating anodic antigen in serum. Fifty percent of the participants tested positive for schistosomiasis. There was no difference in detection rates among patients and matched controls. Similarly, there were no differences in worm burden between patients and controls. These results do not support the hypothesis that susceptibility to M. ulcerans disease is driven by a co-infection with schistosomes.

Adolescent↗

PubMed2003

Comprehensive proteomic profiling of the membrane constituents of a Mycobacterium tuberculosis strain.

Mycobacterium tuberculosis is an infectious microorganism that causes human tuberculosis. The cell membranes of pathogens are known to be rich in possible diagnostic and therapeutic protein targets. To compliment the M. tuberculosis genome, we have profiled the membrane protein fraction of the M. tuberculosis H37Rv strain using an analytical platform that couples one-dimensional SDS gels to a microcapillary liquid chromatography-nanospray-tandem mass spectrometer. As a result, 739 proteins have been identified by two or more distinct peptide sequences and have been characterized. Interestingly, approximately 450 proteins represent novel identifications, 79 of which are membrane proteins and more than 100 of which are membrane-associated proteins. The physicochemical properties of the identified proteins were studied in detail, and then biological functions were obtained by sorting them according to Sanger Institute gene function category. Many membrane proteins were found to be involved in the cell envelope, and those proteins with energy metabolic functions were also identified in this study.

Amino Acid Sequence↗