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-α 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.
Trends in increased tuberculosis infection and a fatality rate of ϳ23% 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.
Initially, the occurrence of bona fide glycosylated proteins in eubacteria was difficult to accept. Now, as a result of the application of critical analytical techniques, the presence of such molecules in such eubacteria as Neisseria meningitidis (43), Flavobacterium meningosepticum (36, 37), Streptococcus sanguis (11), Bacillus alvei (32), Clostridium spp. (15, 31), Bacteroides cellulosolvens (15), Thermoanaerobacter thermohydrosulfuricus (3), and Mycobacterium tuberculosis (10) is recognized. In the case of M. tuberculosis, full definition of glycosylation sites and the nature and extent of glycosylation are lacking. Initial evidence for the presence of glycoproteins in M. tuberculosis was based on the observation of discrete concanavalin A-binding products upon polyacrylamide gel electrophoresis (PAGE) and electroblotting of protein preparations (13,14). However, since these patterns occurred in the midst of considerable quantities of mannose (Man)-containing lipoglycans and phospholipids (10), chemical proof of amino acid glycosylation was considered necessary.The 45-kDa culture filtrate protein of M. tuberculosis is one case in point. Recent antibody reactivity studies and N-terminal amino acid sequencing conducted by both Espitia et al. (12,13) and Dobos et al. (10) demonstrated that this 45-kDa protein is the same as MPT 32, a culture filtrate protein originally isolated by Nagai and colleagues (34). Also, more recently, the DNA sequence of a gene designated apa encoding a 45/47-kDa M. tuberculosis protein was elucidated by Laqueyrerie et al. (27), and the deduced amino acid sequence of this gene yielded 100% homology with the N-terminal sequence of the 45-kDa
The occurrence of glycosylated proteins in Mycobacterium tuberculosis has been widely reported. However, unequivocal proof for the presence of true glycosylated amino acids within these proteins has not been demonstrated, and such evidence is essential because of the predominance of soluble lipoglycans and glycolipids in all mycobacterial extracts. We have confirmed the presence of several putative glycoproteins in subcellular fractions of M. tuberculosis by reaction with the lectin concanavalin A. One such product, with a molecular mass of 45 kDa, was purified from the culture filtrate. Compositional analysis demonstrated that the protein was rich in proline and that mannose, galactose, glucose, and arabinose together represented about 4% of the total mass. The 45-kDa glycoprotein was subjected to proteolytic digestion with either the Asp-N or the Glu-C endopeptidase or subtilisin, peptides were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and glycopeptides were identified by reaction with concanavalin A. Peptides were further separated, and when they were analyzed by liquid chromatography-electrospray mass spectrometry for neutral losses of hexoses (162 mass units), four peptides were identified, indicating that these were glycosylated with hexose residues. One peptide, with an average molecular mass of 1,516 atomic mass units (AMU), exhibited a loss of two hexose units. The N-terminal sequence of the 1,516-AMU glycopeptide was determined to be DPEPAPPVP, which was identical to the sequence of the amino terminus of the mature protein, DPEPAP PVPXTA. Furthermore, analysis of the glycopeptide by secondary ion mass spectrometry demonstrated that the complete sequence of the glycopeptide was DPEPAPPVPTTA. From this, it was determined that the 10th amino acid, threonine, was O-glycosidically linked to a disaccharide composed of two hexose residues, probably mannose. This report establishes that true, O-glycosylated proteins exist in mycobacteria.
Background The development of a fast and accurate, non-sputum-based point-of-care triage test for tuberculosis (TB) would have a major impact on combating the TB burden worldwide. A new fingerstick blood test has been developed by Cepheid (the Xpert-MTB-Host Response (HR)-Prototype), which generates a ‘TB score’ based on mRNA expression of 3 genes. Here we describe the first prospective findings of the MTB-HR prototype. Methods Fingerstick blood from adults presenting with symptoms compatible with TB in South Africa, The Gambia, Uganda and Vietnam was analysed using the Cepheid GeneXpert MTB-HR prototype. Accuracy of the Xpert MTB-HR cartridge was determined in relation to GeneXpert Ultra results and a composite microbiological score (GeneXpert Ultra and liquid culture) with patients classified as having TB or other respiratory diseases (ORD). Results When data from all sites (n=75 TB, 120 ORD) were analysed, the TB score discriminated between TB and ORD with an AUC of 0·94 (CI, 0·91-0·97), sensitivity of 87% (CI, 77-93%) and specificity of 94% (88-97%). When sensitivity was set at 90% for a triage test, specificity was 86% (CI, 75-97%). These results were not influenced by HIV status or geographical location. When evaluated against a composite microbiological score (n=80 TB, 111 ORD), the TB score was able to discriminate between TB and ORD with an AUC of 0·88 (CI, 0·83-0·94), 80% sensitivity (CI, 76-85%) and 94% specificity (CI, 91-96%). Conclusions Our interim data indicate the Cepheid MTB-HR cartridge reaches the minimal target product profile for a point of care triage test for TB using fingerstick blood, regardless of geographic area or HIV infection status.
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