Mycobacterium Tuberculosis Culture Filtrate Research Articles

Isolation, Purification and Identification of CFP29 from Mycobacterium tuberculosis H37Rv culture filtrate proteins

Tuberculosis (TB) continues to be a serious worldwide health issue. Human tuberculosis (TB) is the commonest cause of mortality from one infectious agent, with eight million new cases and two million fatalities each year. In many animal models of TB, proteins isolated from the culture filtrate of Mycobacterium tuberculosis promote protective immunity. The extracellular proteins of Mycobacterium tuberculosis were isolated, purified and identifiedin the current study. M. tuberculosis H37Rv was culturedin Souton’s medium and the extracellular proteins were isolated employing an ion-exchange column chromatography before their purification and characterization using SDS- PAGE, western blotting and N- terminal sequencing. CFP29 was identified and purified in M. tuberculosis H37Rv culture filtrate. However, further characterization of this protein is needed to be utilized as an effective T-cell antigen produced from culture filtrates.

EmPAI-assisted strategy enhances screening and assessment of Mycobacteriumtuberculosis infection serological markers.

SummaryDiscovering new serological markers of Mycobacterium tuberculosis (MTB) infection and establishing a rapid and efficient detection technology is of great significance for the prevention and control of tuberculosis. In this study, we established an exponentially modified protein abundance index (emPAI) value‐assisted strategy to investigate and improve the screening efficiency of serological biomarkers of tuberculosis. First, we used LC‐MS/MS to analyse MTB culture filtrate proteins (MTB‐CFPs), and 632 MTB proteins were identified. Then, the characteristic values of MTB‐CFPs – including emPAI value, molecular weight (Mw), isoelectric point (pI), grand average of hydropathy (GRAVY), transmembrane domain (TMD) and functional groups were calculated. Next, we successfully prepared 10 MTB proteins with emPAI value > 1.0 and recombinantly expressed these proteins in Escherichia coli. At the same time, 3 MTB proteins with emPAI between 0.1 and 0.5 were randomly selected as the control groups, and the immunogenicity of the recombinant MTB proteins was detected using ELISA. The sensitivity and receiver operating characteristic (ROC) curves were calculated for each recombinant MTB protein. The results showed that the areas under the curve (AUC) value of Rv2031c, Rv0577, Rv0831c, Rv0934 and Rv3248c were all higher than those of Rv3875 (AUC, 0.6643). Further analysis of the relationship between emPAI value and antibody sensitivity, AUC value and antibody affinity in mice immunized with recombinant MTB protein showed that emPAI values were positively correlated with them, and R‐squared value ranged from 0.64 to 0.79. The only exception was ESAT‐6 (encoded by the Rv3875 gene), which AUC value was relatively low owing to its strong immunosuppressive properties. This study provides a rationale for the serological marker screening of emPAI‐assisted tuberculosis clinical test. The results also provide new technical support for the screening of candidate serological markers of infectious diseases in the future.

Prediction of o-glycosylation pathways in Mycobacterial immunogenic proteins

Aims and objectives: There is an emergence need to identifying Mycobacterial proteins of value to diagnostic assays and vaccine formulations. Glycosylation is an important post-translational modification of proteins and affects more than half of all the proteins in a cell. O-linked glycosylation refers to the event in which a carbohydrate is covalently linked to the hydroxyl group of serine or threonine. This modification influences a number of properties of proteins including proteolytic resistance, solubility, immunological properties and ligand binding. Host cells can bind to Mycobacterial cell wall carbohydrates via a class of surface localized or secreted proteins known as lectins, and these interactions strongly contribute to bacterial adhesion and uptake, and are also associated with the capability of Mtb to survive. This study represents the attempt to predict o-glycosylation pathways of immunogenic TB proteins as potential therapeutic strategies against TB. Methods: A Mycobacterium tuberculosis culture filtrate enriched with mannose-containing proteins that were purified by conA-Affinity chromatography. One and two dimensional gel electrophoresis carried out by standard methods. Gel staining procedures are based on the periodic Acid-Schiff reaction. The Protein spots of interest were excised from the gel and then analysis by MALDI Mass Spectrometry. GlycoPP software, Version 1.0, was used for glycosylation prediction and protein sequences. Results: Protein bands or spots were excised from the gel and analyzed by mass spectrometry. These proteins have O-glycosite residue and a glycan attached covalently and enzymatically at amide or hydroxyl group which includes Rv0475, Rv3367, Rv3699, Rv0251c, Rv2558 and Rv1284. Rv0475 has 17 Potential O-Linked Glycosylated Sites but 6 sites of Potential Glycosylated. Rv3367 has 67 Potential O-Linked Glycosylated Sites and 55 sites of Potential Glycosylated. Additionally, Rv3699 has 26 Potential O-Linked Glycosylated Sites and 10 sites Potential Glycosylated. Conclusions: This study may be shown the role of glycoprotein's in Mycobacterium pathogenesis, disease development, and its implications in drug development. All of these proteins have several putative antigenic epitopes and induces an immune response especially production of antibodies. In addition, Rv3367as a member of the Mycobacterium tuberculosis PE family and PGRS subfamily, gly-rich proteins have carbohydrate moieties and also are known to play a role in several pathological processes. Because of the importance of M.tuberculosis immunogenic antigens, the availability of the secretion and glycosylation of MTB proteins is a tool that will allow a deeper understanding of role these proteins play in the pathogenesis of disease and provide the basis for more sensitive and discriminative clinical diagnostic tests and improving to formulate new Mycobacterial vaccines.

Predictive value of TNF-\u03b1, IFN-\u03b3, and IL-10 for tuberculosis among recently exposed contacts in the United States and Canada

BackgroundWe examined cytokine immune response profiles among contacts to tuberculosis patients to identify immunologic and epidemiologic correlates of tuberculosis.MethodsWe prospectively enrolled 1272 contacts of culture-confirmed pulmonary tuberculosis patients at 9 United States and Canadian sites. Epidemiologic characteristics were recorded. Blood was collected and stimulated with Mycobacterium tuberculosis culture filtrate protein, and tumor necrosis factor (TNF-α), interferon gamma (IFN-γ), and interleukin 10 (IL-10) concentrations were determined using immunoassays.ResultsOf 1272 contacts, 41 (3.2%) were diagnosed with tuberculosis before or < 30 days after blood collection (co-prevalent tuberculosis) and 19 (1.5%) during subsequent four-year follow-up (incident tuberculosis). Compared with contacts without tuberculosis, those with co-prevalent tuberculosis had higher median baseline TNF-α and IFN-γ concentrations (in pg/mL, TNF-α 129 versus 71, P < .01; IFN-γ 231 versus 27, P < .001), and those who subsequently developed incident tuberculosis had higher median baseline TNF-α concentrations (in pg/mL, 257 vs. 71, P < .05). In multivariate analysis, contact age < 15 years, US/Canadian birth, and IFN or TNF concentrations > the median were associated with co-prevalent tuberculosis (P < .01 for each); female sex (P = .03) and smoking (P < .01) were associated with incident tuberculosis. In algorithms combining young age, positive skin test results, and elevated CFPS TNF-α, IFN-γ, and IL-10 responses, the positive predictive values for co-prevalent and incident tuberculosis were 40 and 25%, respectively.ConclusionsCytokine concentrations and epidemiologic factors at the time of contact investigation may predict co-prevalent and incident tuberculosis.

Integrative proteomic and glycoproteomic profiling of Mycobacterium tuberculosis culture filtrate.

Despite being the subject of intensive research, tuberculosis, caused by Mycobacterium tuberculosis, remains at present the leading cause of death from an infectious agent. Secreted and cell wall proteins interact with the host and play important roles in pathogenicity. These proteins are explored as candidate diagnostic markers, potential drug targets or vaccine antigens, and more recently special attention is being given to the role of their post-translational modifications. With the purpose of contributing to the proteomic and glycoproteomic characterization of this important pathogen, we performed a shotgun analysis of culture filtrate proteins of M. tuberculosis based on a liquid nano-HPLC tandem mass spectrometry and a label-free spectral counting normalization approach for protein quantification. We identified 1314 M. tuberculosis proteins in culture filtrate and found that the most abundant proteins belong to the extracellular region or cell wall compartment, and that the functional categories with higher protein abundance factor were virulence, detoxification and adaptation, and cell wall and cell processes. We could identify a group of proteins consistently detected in previous studies, most of which were highly abundant proteins. In culture filtrate, 140 proteins were predicted to contain one of the three types of bacterial N-terminal signal peptides. Besides, various proteins belonging to the ESX secretion systems, and to the PE and PPE families, secreted by the type VII secretion system using nonclassical secretion signals, were also identified. O-glycosylation was identified in 46 proteins, many of them lipoproteins and cell wall associated proteins. Finally, we provide proteomic evidence for 33 novel O-glycosylated proteins, aiding to the glycoproteomic characterization of relevant antigenic membrane and exported proteins. These findings are expected to collaborate with the research on pathogen derived biomarkers, virulence factors and vaccine candidates, and to provide clues to the understanding of the pathogenesis and survival strategies adopted by M. tuberculosis.

Alanine dehydrogenases in mycobacteria.

Since NAD(H)-dependent L-alanine dehydrogenase (EC 1.1.4.1; Ald) was identified as one of the major antigens present in culture filtrates of Mycobacterium tuberculosis, many studies on the enzyme have been conducted. Ald catalyzes the reversible conversion of pyruvate to alanine with concomitant oxidation of NADH to NAD+ and has a homohexameric quaternary structure. Expression of the ald genes was observed to be strongly upregulated in M. tuberculosis and Mycobacterium smegmatis grown in the presence of alanine. Furthermore, expression of the ald genes in some mycobacteria was observed to increase under respiration-inhibitory conditions such as oxygen-limiting and nutrient-starvation conditions. Upregulation of ald expression by alanine or under respiration-inhibitory conditions is mediated by AldR, a member of the Lrp/AsnC family of transcriptional regulators. Mycobacterial Alds were demonstrated to be the enzymes required for utilization of alanine as a nitrogen source and to help mycobacteria survive under respiration-inhibitory conditions by maintaining cellular NADH/NAD+ homeostasis. Several inhibitors of Ald have been developed, and their application in combination with respiration-inhibitory antitubercular drugs such as Q203 and bedaquiline was recently suggested.

Mycobacterium tuberculosis Transfer RNA Induces IL-12p70 via Synergistic Activation of Pattern Recognition Receptors within a Cell Network.

Upon recognition of a microbial pathogen, the innate and adaptive immune systems are linked to generate a cell-mediated immune response against the foreign invader. The culture filtrate of Mycobacterium tuberculosis contains ligands, such as M. tuberculosis tRNA, that activate the innate immune response and secreted Ags recognized by T cells to drive adaptive immune responses. In this study, bioinformatics analysis of gene-expression profiles derived from human PBMCs treated with distinct microbial ligands identified a mycobacterial tRNA-induced innate immune network resulting in the robust production of IL-12p70, a cytokine required to instruct an adaptive Th1 response for host defense against intracellular bacteria. As validated by functional studies, this pathway contained a feed-forward loop, whereby the early production of IL-18, type I IFNs, and IL-12p70 primed NK cells to respond to IL-18 and produce IFN-γ, enhancing further production of IL-12p70. Mechanistically, tRNA activates TLR3 and TLR8, and this synergistic induction of IL-12p70 was recapitulated by the addition of a specific TLR8 agonist with a TLR3 ligand to PBMCs. These data indicate that M. tuberculosis tRNA activates a gene network involving the integration of multiple innate signals, including types I and II IFNs, as well as distinct cell types to induce IL-12p70.

Significance of immune response to Mycobacterium tuberculosis culture filtrate protein antigens in cerebrospinal fluid of tuberculous meningitis patients: A search for diagnostic marker

ABSTRACTMycobacterium tuberculosis (H37Ra) culture filtrate proteins (CFP) are explored as a diagnostic marker for tuberculous meningitis (TBM). Cerebrospinal fluid (CSF) samples from patients were categorized as confirmed (n = 47), suspected (n = 20), and non-TBM (n = 25) cases. Immune response by Western blot revealed TBM CSF samples are having heterogeneous response to CFP. CFP ELISA was 92% sensitive and 38.30% specific. ODs of confirmed TBM and non-TBM cases were significantly different (P < 0.0001) and also the suspected TBM and non-TBM cases (P = 0.0001). No significant difference noticed in TBM and suspected TBM (P = 0.90). Thus, CFP can be a better biomarker for the diagnosis of TBM.

Factors associated with tuberculosis infection, and with anti-mycobacterial immune responses, among five year olds BCG-immunised at birth in Entebbe, Uganda

BackgroundBCG is used widely as the sole licensed vaccine against tuberculosis, but it has variable efficacy and the reasons for this are still unclear. No reliable biomarkers to predict future protection against, or acquisition of, TB infection following immunisation have been identified. Lessons from BCG could be valuable in the development of effective tuberculosis vaccines. ObjectivesWithin the Entebbe Mother and Baby Study birth cohort in Uganda, infants received BCG at birth. We investigated factors associated with latent tuberculosis infection (LTBI) and with cytokine response to mycobacterial antigen at age five years. We also investigated whether cytokine responses at one year were associated with LTBI at five years of age. MethodsBlood samples from age one and five years were stimulated using crude culture filtrates of Mycobacterium tuberculosis in a six-day whole blood assay. IFN-γ, IL-5, IL-13 and IL-10 production was measured. LTBI at five years was determined using T-SPOT.TB® assay. Associations with LTBI at five years were assessed using multivariable logistic regression. Multiple linear regression with bootstrapping was used to determine factors associated with cytokine responses at age five years. ResultsLTBI prevalence was 9% at age five years. Only urban residence and history of TB contact/disease were positively associated with LTBI. BCG vaccine strain, LTBI, HIV infection, asymptomatic malaria, growth z-scores, childhood anthelminthic treatment and maternal BCG scar were associated with cytokine responses at age five. Cytokine responses at one year were not associated with acquisition of LTBI by five years of age. ConclusionAlthough multiple factors influenced anti-myocbacterial immune responses at age five, factors likely to be associated with exposure to infectious cases (history of household contact, and urban residence) dominated the risk of LTBI.

O-linked glycosylation sites profiling in Mycobacterium tuberculosis culture filtrate proteins

Mycobacterium tuberculosis (Mtb) causes tuberculosis, one of the leading causes of fatal infectious diseases worldwide. Cell-cell recognition between the pathogen Mtb and its host is mediated in part by glycosylated proteins. So far, glycoproteins in Mtb are understudied and for only very few glycoproteins glycosylation sites have been described, e.g., alanine and proline rich secreted protein apa, superoxide dismutase SODC, lipoprotein lpqH and MPB83/MPT83. In this study, glycosylated proteins in Mtb culture filtrate were investigated using liquid chromatography-mass spectrometry approaches and bioinformatic analyses. To validate the presence of glycoproteins, several strategies were pursued including collision induced dissociation, high energy collision dissociation and electron transfer dissociation techniques, and bioinformatics analyses involving a neutral loss search for glycosylated moieties. After extensive data curation, we report glycosylation sites for thirteen Mtb glycoproteins using a combination of mass spectrometry techniques on a dataset collected from culture filtrate proteins. This is the first glycoproteomics study identifying glycosylation sites on mycobacterial culture filtrate proteins (CFP) on a global scale. In this study, glycosylation sites in Mtb were characterized by collision-induced dissociation, electron-transfer dissociation and high energy collision dissociation techniques. The identification of glycosylation sites is important for our understanding of the physiology and pathophysiology of Mtb. Glycoproteins are often responsible for protein-protein interactions between host and pathogen and thus represent interesting targets for vaccine development. In addition, our strategy is not limited to Mtb, but could be extended to other organisms. This article is part of a Special Issue entitled: Trends in Microbial Proteomics.

Syndecan-1 and Heparanase

Syndecan-1 (SDC1) and its endo-beta-D-glucuronidase heparanase (HPA) are implicated in the maintenance of intestinal barrier function, but their detailed functions in Crohn's disease (CD) are not fully investigated. The aim of this study was to determine alteration patterns of SDC1 and HPA and their potential roles in evaluating disease activity and differentiating CD from intestinal tuberculosis (ITB). Tissue and serum specimens were obtained from 89 patients, including 15 patients with functional bowel disorders, 18 active patients with ITB, and 56 patients with CD (remission = 19, active = 37). Basic clinical data were collected and routine blood tests were analyzed. SDC1 and HPA were measured by immunohistochemistry, enzyme-linked immunosorbent assay, reverse transcriptase polymerase chain reaction, and western blot. Colonic epithelial cells were incubated with recombinant HPA, tumor necrosis factor alpha (TNF-α), and mycobacterium tuberculosis culture filtrate protein to detect the alterations of SDC1 and HPA. In the CD group, SDC1 was significantly decreased in mucosa and increased in serum, whereas HPA level in both were elevated. Such alterations were associated with clinicopathological features representing disease activity and injury severity and were not available in functional bowel disorder and ITB groups. Recombinant HPA incubation increased soluble SDC1 in culture supernatants (P = 2 × 10(-4)), and low-dose TNF-α effectively enhanced HPA's activity (P = 3 × 10(-6)). Exogenous TNF-α destroyed cellular SDC1 and raised HPA expressions dose dependently, whereas mycobacterium tuberculosis culture filtrate protein showed no effects. Unique alterations of SDC1 and HPA are shown in both patients with CD and in vitro model. The results indicate SDC1 and HPA are potential markers for CD in evaluating its disease activity and differentiating it from ITB.

Bacterial proteins with cleaved or uncleaved signal peptides of the general secretory pathway

Correct protein compartmentalization is a key step for molecular function and cell viability, and this is especially true for membrane and externalized proteins of bacteria. Recent proteomic reports of Bacillus subtilis have shown that many proteins with Sec-like signal peptides and absence of a transmembrane helix domain are still observed in membrane-enriched fractions, but further evidence about signal peptide cleavage or soluble protein contamination is still needed. Here we report a proteomic screening of identified peptides in culture filtrate, membrane fraction and whole cell lysate of Mycobacterium tuberculosis. We were able to detect peptide sequencing evidence that shows that the predicted signal peptide was kept uncleaved for several types of proteins such as mammalian cell entry (Mce) proteins and PE or PE-PGRS proteins. Label-free quantitation of all proteins identified in each fraction showed that the majority of these proteins with uncleaved signal peptides are, indeed, enriched in the Triton X-114 lipid phase. Some of these proteins are likely to be located in the inner membrane while others may be outer membrane proteins.

Changes in cell-mediated immune response after lung resection surgery for MDR-TB patients

The immune responses of multidrug-resistant tuberculosis (MDR-TB) patients undergoing lung resection surgery were investigated in order to understand the mechanism of strong immune suppression in MDR-TB. We examined changes in cell-mediated immune response (CMI) of a total of sixteen MDR-TB patients, three of them extensively drug-resistant tuberculosis (XDR-TB) patients, after the removal of the heavily diseased lung section. The IFN-γ response to Mycobacterium tuberculosis culture filtrate proteins (Mtb-CFP), one of the most important CMI to defend TB, showed a statistically significant elevation in 2-4 months after operation when compared to the preoperative CMI in patients who were converted into AFB negative and cured in two years' follow-up, suggesting that the recovery of CMI may be one of the key factors in the successful treatment of MDR-TB. Interestingly, IL-10 response to Mtb-CFP was also elevated in 2-4 months after surgery in cured patients although both proliferative response and PBMC composition were not significantly changed. Infection with first- or second-line drugs resistant Mtb reduces the efficiency of chemotherapeutic treatment of MDR-TB to about 50%. Thus, this study suggests that chemotherapeutic treatment of MDR-TB may be more effective when combined with accompanying therapy that increases CMI, includes lung resection surgery.

Mycobacterium tuberculosis Glycoproteomics Based on ConA-Lectin Affinity Capture of Mannosylated Proteins

A Mycobacterium tuberculosis culture filtrate enriched with mannose-containing proteins was resolved by 2-DE gel. After ConA ligand blotting, 41 proteins were identified by mass spectrometry as putative glycoproteins with 34 of them new probably mannosylated proteins. These results contribute to the construction of the ConA affinity glycoprotein database of M. tuberculosis, and provide useful information for understanding the biological role of glycoproteins in mycobacteria.

Improved diagnosis of pulmonary tuberculosis by detection of antibodies against multiple Mycobacterium tuberculosis antigens

Two secreted antigens (38 and 30 kDa) and 1 cytosolic antigen (16 kDa) were purified in our laboratory from Mycobacterium tuberculosis culture filtrate and cytosol using chromatographic/electrophoretic methods. One recombinant antigen (27 kDa, MPT51) expressed in Escherichia coli was also isolated. All the 4 antigens were tested individually for detection of serum IgG, IgA, and IgM (a total of 476 sera from 5 groups) by indirect enzyme-linked immunosorbent assay. Keeping the well-reported 38 kDa as the main candidate, the usefulness of the other antigens, which may add to the test positivity in cases not diagnosed by 38 kDa, was analyzed. The individual antigens ranged in their sensitivity from 57% to 67% (IgG). Addition of other antigen results, with that of 38 kDa, offered a sensitivity of 91% in smear- and culture-positive tuberculosis (TB), 78% in smear-negative culture-confirmed TB, and 97% specificity in normal healthy subjects. IgG antibody to multiple antigens (38, 30, and 16 kDa) may be a sensitive, specific, rapid, and cost-effective test to rule-in clinical suspicion of pulmonary TB.

A LOW MOLECULAR WEIGHT ES-20 PROTEIN RELEASED IN VIVO AND IN VITRO WITH DIAGNOSTIC POTENTIAL IN LYMPH NODE TUBERCULOSIS

Purpose: To determine role of antigens released in vivo and in vitro in immunodiagnosis of tuberculosis (TB). Methods:In vivo released circulating tuberculosis antigen (CTA) was obtained from TB sera by ammonium sulphate precipitation and in vitro released excretory-secretory (ES) antigens from Mycobacterium tuberculosis culture filtrate. CTA and ES antigens were fractionated by SDS-PAGE and electro-eluted gel fractions were analysed for antigen by ELISA. Results: Low molecular weight proteins CTA-9 and ES-9 showed high titre of antigen activity. To explore the diagnostic potential of low molecular weight ES antigen, M. tuberculosis ES antigen was further fractionated by gel filtration chromatography followed by purification on anion exchange column using fast protein liquid chromatography and a highly seroreactive ESG-5D (ES-20) antigen was obtained. Competitive inhibition showed that CTA-9 and ES-9 antigens inhibit the binding of ES-20 antigen to its antibody. Seroanalysis showed sensitivity of 83 and 80% for ES-20 antigen and antibody detection, respectively, in pulmonary TB and 90% in lymph node TB. Conclusions: Seroreactivity studies using M. tuberculosis ES-20 antigen showed usefulness in detection of TB; in particular, lymph node TB.

A low molecular weight ES-20 protein released &lt;i&gt; in vivo&lt;/i&gt; and &lt;i&gt; in vitro&lt;/i&gt; with diagnostic potential in lymph node tuberculosis

To determine role of antigens released in vivo and in vitro in immunodiagnosis of tuberculosis (TB). In vivo released circulating tuberculosis antigen (CTA) was obtained from TB sera by ammonium sulphate precipitation and in vitro released excretory-secretory (ES) antigens from Mycobacterium tuberculosis culture filtrate. CTA and ES antigens were fractionated by SDS-PAGE and electro-eluted gel fractions were analysed for antigen by ELISA. Low molecular weight proteins CTA-9 and ES-9 showed high titre of antigen activity. To explore the diagnostic potential of low molecular weight ES antigen, M. tuberculosis ES antigen was further fractionated by gel filtration chromatography followed by purification on anion exchange column using fast protein liquid chromatography and a highly seroreactive ESG-5D (ES-20) antigen was obtained. Competitive inhibition showed that CTA-9 and ES-9 antigens inhibit the binding of ES-20 antigen to its antibody. Seroanalysis showed sensitivity of 83 and 80% for ES-20 antigen and antibody detection, respectively, in pulmonary TB and 90% in lymph node TB. Seroreactivity studies using M. tuberculosis ES-20 antigen showed usefulness in detection of TB; in particular, lymph node TB.

Identification of novel bacterial plasminogen‐binding proteins in the human pathogen Mycobacterium tuberculosis

Binding and activation of human plasminogen (Plg) to generate the proteolytic enzyme plasmin (Plm) have been associated with the invasive potential of certain bacteria. In this work, proteomic analysis together with ligand blotting assays identified several major Plg-binding spots in Mycobacterium tuberculosis soluble extracts (SEs) and culture filtrate proteins. The identity of 15 different proteins was deduced by N-terminal and/or MS and corresponded to DnaK, GroES, GlnA1, Ag85 complex, Mpt51, Mpt64, PrcB, MetK, SahH, Lpd, Icl, Fba, and EF-Tu. Binding of Plg to recombinant M. tuberculosis DnaK, GlnA1, and Ag85B was further confirmed by ELISA and ligand blotting assays. The binding was inhibited by epsilon-aminocaproic acid, indicating that the interaction involved lysine residues. Plg bound to recombinant mycobacterial proteins was activated to Plm by tissue-type Plg activator. In contrast with recombinant proteins, M. tuberculosis SE enhanced several times the Plg activation mediated by the activator. Interestingly, GlnA1 was able to bind the extracellular matrix (ECM) protein fibronectin. Together these results show that M. tuberculosis posses several Plg receptors suggesting that bound Plg to bacteria surface, can be activated to Plm, endowing bacteria with the ability to break down ECM and basal membranes proteins contributing to tissue injury in tuberculosis.

A Single Dose of Vitamin D Enhances Immunity to Mycobacteria

Vitamin D was used to treat tuberculosis (TB) in the preantibiotic era. Prospective studies to evaluate the effect of vitamin D supplementation on antimycobacterial immunity have not previously been performed. To determine the effect of vitamin D supplementation on antimycobacterial immunity and vitamin D status. A double-blind randomized controlled trial was conducted in 192 healthy adult TB contacts in London, United Kingdom. Participants were randomized to receive a single oral dose of 2.5 mg vitamin D or placebo and followed up at 6 weeks. The primary outcome measure was assessed with a functional whole blood assay (BCG-lux assay), which measures the ability of whole blood to restrict luminescence, and thus growth, of recombinant reporter mycobacteria in vitro; the readout is expressed as a luminescence ratio (luminescence postinfection/baseline luminescence). IFN-gamma responses to the Mycobacterium tuberculosis antigens early secretory antigenic target-6 and culture filtrate protein 10 were determined with a second whole blood assay. Vitamin D supplementation significantly enhanced the ability of participants' whole blood to restrict BCG-lux luminescence in vitro compared with placebo (mean luminescence ratio at follow-up, 0.57, vs. 0.71, respectively; 95% confidence interval for difference, 0.01-0.25; p=0.03) but did not affect antigen-stimulated IFN-gamma secretion. A single oral dose of 2.5 mg vitamin D significantly enhanced the ability of participants' whole blood to restrict BCG-lux luminescence in vitro without affecting antigen-stimulated IFN-gamma responses. Clinical trials should be performed to determine whether vitamin D supplementation prevents reactivation of latent TB infection. Clinical trial registered with www.clinicaltrials.gov (NCT 00157066).

Clinical value of specific detection of immune complex-bound antibodies in pulmonary tuberculosis

Two actively secreted (38 and 30 kDa) and 1 cytosolic (16 kDa) antigens were purified from Mycobacterium tuberculosis culture filtrate and cytosol, respectively, using a combination of chromatographic and electrophoretic methods. One recombinant antigen (27 kDa) overexpressed in Escherichia coli was also isolated. The diagnostic test characteristics of circulating immune complex (CIC)-bound antibodies to purified protein antigens, singly and in combination, were evaluated in patients with pulmonary tuberculosis. The individual antigens ranged in their sensitivity from 73% to 88%, while considering the IgG response. Addition of IgA results improved the sensitivity. The combination of IgG results for 38, 30, and 16 kDa offered >95% sensitivity and specificity for the smear- and culture-positive tuberculosis, as well as for the smear-negative, culture-positive group. CIC-bound antibodies promise to be a better diagnostic tool than serum antibodies.