ESAT-6 is a secreted protein present in the short-term culture filtrate of Mycobacterium tuberculosis after growth on synthetic Sauton medium. has recently been demonstrated to induce strong T-cell responses mouse model memory immunity infection with M. tuberculosis. In Western blotting (immunoblotting), monoclonal antibody HYB76-8, reacting ESAT-6, gave 6-kDa region was observed filtrates from four eight substrains bovis BCG that produced high levels MPB64, while no band occurred any these...

Proteins secreted by Mycobacterium tuberculosis play an essential role in the pathogenesis of tuberculosis. The culture filtrates M. H37Rv made Sadamu Nagai (Japan), are considerably enriched for proteins compared to other filtrates. Complementary approaches were used identify these filtrates: (i) 2-DE combined with MALDI-TOF MS and (ii) LC coupled MS/MS. Peptides derived from a total 257 identified which 144 more than one peptide. Several members immunologically important early secretory...

Five actively secreted proteins (MPT32, MPT45, MPT51, MPT53, and MPT63) the MPT46 protein were purified to homogeneity from Mycobacterium tuberculosis culture fluid compared with previously by ourselves other investigators. Antisera obtained immunization of rabbits all newly isolated identified be immunogenic. Two-dimensional electrophoresis fluids each week for 2 10 weeks culturing M. revealed characteristic changes, permitting identification two distinct groups being mycobacterial cells or...

Fibronectin (FN)-binding antigens are prominent components of short-term culture supernatants Mycobacterium tuberculosis. In 3-day-old supernatants, a 30-kilodalton (kDa) protein was identified as the major FN-binding molecule. 21-day-old FN bound to double band 30 and 31 kDa, well group larger molecular mass (57 60 kDa). molecules in this size range, but not were also found sonicates. We showed that 31- 30-kDa bands correspond A B BCG85 complex, previously shown be abundant bovis BCG. Thus,...

Correct protein compartmentalization is a key step for molecular function and cell viability, this especially true membrane externalized proteins of bacteria. Recent proteomic reports Bacillus subtilis have shown that many with Sec-like signal peptides absence transmembrane helix domain are still observed in membrane-enriched fractions, but further evidence about peptide cleavage or soluble contamination needed. Here we report screening identified culture filtrate, fraction whole lysate...

Abstract Background Membrane- and membrane-associated proteins are important for the pathogenicity of bacteria. We have analysed content these in virulent Mycobacterium tuberculosis H37Rv using Triton X-114 detergent-phase separation extraction lipophilic proteins, followed by their identification with high resolution mass spectrometry. Results In total, 1417 different were identified. silico analysis identified revealed that 248 had at least one predicted trans-membrane region. Also, 64...

We have screened peripheral blood mononuclear cells (PBMC) from tuberculosis (TB) patients for proliferative reactivity and interferon-gamma (IFN-gamma) secretion against a panel of purified recombinant (r) natural (n) culture filtrate (rESAT-6, nMPT59, nMPT64 nMPB70) somatic-derived (rGroES, rPstS, rGroEL rDnaK) antigens Mycobacterium tuberculosis. The responses PBMC to these defined were compared with the corresponding results obtained complex antigens, such as whole-cell M. tuberculosis,...

MPB70 is a highly species specific protein which secreted from Mycobacterium bovis during culture. To investigate whether antibodies against can be used as an indicator of infection with M. bovis, enzyme-linked immunosorbent assay was developed, based on the use biotinylated G, to provide common for antibody formation in different species. During experimental cattle, characteristic pattern anti-MPB70 production observed initial flat plateau followed by marked rise 18 20 weeks after...

Reversible protein phosphorylation, regulated by kinases and phosphatases, mediates a switch between activity cellular pathways that contribute to large number of processes. The Mycobacterium tuberculosis genome encodes 11 Serine/Threonine (STPKs) which show close homology eukaryotic kinases. This study aimed elucidate the phosphoproteomic landscape clinical isolate M. tuberculosis. We performed high throughput mass spectrometric analysis proteins extracted from an early-logarithmic phase...

Abstract Injection of plasmid DNA encoding antigens into rodents followed by electroporation improved the immune response when compared with injection without (Widera et al. J Immunol 2000;164:4635–40; Zucchelli Virol 2000;74:11598–607; Kadowaki al . Vaccine 2000;18:2779–88). The present study describes extension this technology to farm animals, injecting mycobacterial (MPB70, Ag85B and Hsp65) muscles goats cattle using two different types electrodes, both allowing delivery at site...

A series of mycobacterial antigens were quantified by immunoelectrophoresis, enzyme-linked immunosorbent assays, or SDS-PAGE with immunoblotting using antisera against purified antigens. The showed a characteristic distribution profile. Some had marked quantitative dominance in the culture fluid while others sonicates whole washed bacilli. majority tested could thus be located and grouped as either secreted cytoplasmic terms localization index (LI) which is described. 5-week-old...

Summary Mycobacterium bovis Bacille Calmette–Guérin (BCG) strains are genetically and phenotypically heterogeneous. Expression of the antigenic proteins MPB70 MPB83 is known to vary considerably across BCG strains; however, reason for this phenotypic difference has remained unknown. By immunoblot, we separated into high‐ low‐producing strains. quantitative reverse transcription polymerase chain reaction (RT‐PCR), determined that antigen‐encoding genes, mpb70 mpb83 , follows same strain...

Recombinant phage clones, TB1 and TB2, were selected from a Mycobacterium tuberculosis lambda gt11 DNA expression library by screening with polyclonal antiserum raised against the antigen 85 complex of bovis BCG. Analysis recombinant inserts expressed fusion proteins showed that two new genes had been isolated. The product clone TB2 was identified as member 30/31-kDa complex. Restriction enzyme analysis this gene differs previously cloned members complex, detailed serological indicating it...

The aim of this study was to evaluate the diagnostic potential immunohistochemistry using an antibody secreted mycobacterial antigen MPT64, in abdominal and lymph node tuberculosis.We used formalin-fixed histologically diagnosed tuberculosis (n = 33) cervical tuberculous lymphadenitis 120) biopsies. These were investigated a combination Ziehl-Neelsen method, culture, with specific for Mycobacterium complex organisms. Abdominal biopsies from non-mycobacterial diseases 50) similarly tested as...

Abstract Background The potential causes for variation in virulence between distinct M. tuberculosis strains are still not fully known. However, differences protein expression probably an important factor. In this study we used a label-free quantitative proteomic approach to estimate abundance two closely related strains; the virulent H37Rv strain and its attenuated counterpart H37Ra. Results We were able identify more than 1700 proteins from both strains. As expected, majority of identified...

Abstract Culture filtrates of Mycobacterium tuberculosis H37Rv highly enriched with secreted proteins were used to identify antigens recognized by a serum pool from patients. Two different approaches separate the culture filtrate protein mixture: (i) fractionated according their hydrophobicity using an HPLC‐C18 chromatography column followed separation based on molecular mass SDS‐PAGE and subsequent immunoblotting or (ii) separated two‐dimensional gel electrophoresis, isoelectric point mass....

ABSTRACT RipSeq (iSentio, Bergen, Norway) is a web-based application for the analysis of mixed DNA chromatograms. It opens possibility to analyze chromatograms obtained by direct 16S rRNA gene sequencing from polybacterial human clinical samples. In this study, we used investigate 264 samples wide range suspected bacterial infections. The sequence-based identification was compared with results routine culture-based identification. A total 151 were positive first PCR, producing 85 pure and 66...

ABSTRACT Broad-range amplification and sequencing of the bacterial 16S rRNA gene directly from clinical specimens are offered as a diagnostic service in many laboratories. One major pitfall is primer cross-reactivity with human DNA which will result mixed chromatograms. Mixed chromatograms complicate subsequent sequence analysis impede identification. In SYBR green real-time PCR assays, it can also affect crossing threshold values consequently status specimen positive or negative. We...