Characterisation and structural analysis of glyoxylate cycle enzymes of Teladorsagia circumcincta.

A 1332 bp full length cDNA encoding Teladorsagia circumcincta isocitrate lyase (TciICL) and a 1575 bp full length cDNA encoding T. circumcincta malate synthase (TciMS) were cloned, expressed in Escherichia coli and the recombinant proteins purified. The predicted TciICL protein of 444 amino acids was present as a single band of about 52 kDa on SDS-PAGE and the recombinant TciMS of 525 amino acids formed a single band about 62 kDa. Multiple alignments of the combined bifunctional TciICL-MS protein sequence with homologues from other nematodes showed that the greatest similarity (89-92 %) to the homologues of Ancylostoma ceylanicum, Haemonchus contortus and Haemonchus placei and 71-87 % similarity to the other nematode sequences. The 3-dimensional structures, binding and catalytic sites were determined for TciICL and TciMS and shown to be highly conserved. Substrate and metal ion binding sites were identified and were completely conserved in other homologues. TciICL was confirmed as a functional enzyme. At 30 °C, the optimum pH was pH 7.5, the Vmax was 275 ± 23 nmoles.min-1. mg-1 protein and the apparent Km for the substrate isocitrate was 0.7 ± 0.01µM (mean ± SEM, n = 3). Addition of 10 mM metal ions (except Mg2+) or 1 mM inhibitors reduced the recombinant TciICL activity by 60-90 %. Antibodies in both serum and saliva from field-immune, but not nematode-naïve, sheep recognised recombinant TciICL in ELISA, supporting similar antigenicity to that of the native enzyme.

The use of Mycobacterium tuberculosis HspX and GlcB proteins to identify latent tuberculosis in rheumatoid arthritis patients.

Rheumatoid arthritis (RA) is an autoimmune disease characterised by the destruction of articular cartilage and bone damage. The chronic treatment of RA patients causes a higher susceptibility to infectious diseases such as tuberculosis (TB); one-third of the world's population is latently infected (LTBI) with Mycobacterium tuberculosis (Mtb). The tuberculin skin test is used to identify individuals LTBI, but many studies have shown that this test is not suitable for RA patients. The goal of this work was to test the specific cellular immune responses to the Mtb malate synthase (GlcB) and heat shock protein X (HspX) antigens of RA patients and to correlate those responses with LTBI status. The T-helper (Th)1, Th17 and Treg-specific immune responses to the GlcB and HspX Mtb antigens were analysed in RA patients candidates for tumour necrosis factor-α blocker treatment. Our results demonstrated that LTBI RA patients had Th1-specific immune responses to GlcB and HspX. Patients were followed up over two years and 14.3% developed active TB. After the development of active TB, RA patients had increased numbers of Th17 and Treg cells, similar to TB patients. These results demonstrate that a GlcB and HspX antigen assay can be used as a diagnostic test to identify LTBI RA patients.

Antibodies against Mycobacterial proteins as biomarkers for HIV-associated smear-negative tuberculosis.

Serology data are limited for patients with sputum smear-negative HIV-associated active tuberculosis (TB). We evaluated the serum antibody responses against the mycobacterial proteins MPT51, MS, and echA1 and the 38-kDa protein via enzyme-linked immunosorbent assay (ELISA) in South African (S.A.) HIV-positive (HIV(+)) smear-negative TB patients (n = 56), U.S. HIV(+) controls with a positive tuberculin skin test (TST(+); n = 21), and S.A. HIV-negative (HIV(-)) (n = 18) and HIV(+) (n = 24) controls. TB patients had positive antibody reactivity against MPT51 (73%), echA1 (59%), MS (36%), and the 38-kDa protein (11%). Little reactivity against MPT51 and echA1 was observed in control groups at low risk for TB, i.e., S.A. HIV(-) (0% and 6%, respectively), and at moderate risk for TB development, i.e., U.S. HIV(+) TST(+) controls (14% and 10%, respectively). By contrast, more reactivity was detected in the S.A. HIV(+) control group at higher risk for TB (25% and 45%, respectively). Our data hold promise that antibody detection against MPT51 and echA1 might have adjunctive value in the detection of HIV(+) smear-negative TB and might reflect increasing Mycobacterium tuberculosis infection activity in asymptomatic HIV(+) individuals.

The use of Mycobacterium tuberculosis HspX and GlcB proteins to identify latent tuberculosis in rheumatoid arthritis patients

Rheumatoid arthritis (RA) is an autoimmune disease characterised by the destruction of articular cartilage and bone damage. The chronic treatment of RA patients causes a higher susceptibility to infectious diseases such as tuberculosis (TB); one-third of the world’s population is latently infected (LTBI) with Mycobacterium tuberculosis (Mtb). The tuberculin skin test is used to identify individuals LTBI, but many studies have shown that this test is not suitable for RA patients. The goal of this work was to test the specific cellular immune responses to the Mtb malate synthase (GlcB) and heat shock protein X (HspX) antigens of RA patients and to correlate those responses with LTBI status. The T-helper (Th)1, Th17 and Treg-specific immune responses to the GlcB and HspX Mtb antigens were analysed in RA patients candidates for tumour necrosis factor-α blocker treatment. Our results demonstrated that LTBI RA patients had Th1-specific immune responses to GlcB and HspX. Patients were followed up over two years and 14.3% developed active TB. After the development of active TB, RA patients had increased numbers of Th17 and Treg cells, similar to TB patients. These results demonstrate that a GlcB and HspX antigen assay can be used as a diagnostic test to identify LTBI RA patients.

Detection of Mycobacterium tuberculosis GlcB or HspX Antigens or devR DNA impacts the rapid diagnosis of tuberculous meningitis in children.

BACKGROUND: Tuberculous meningitis (TBM) is the most common form of neurotuberculosis and the fifth most common form of extrapulmonary TB. Early diagnosis and prompt treatment are the cornerstones of effective disease management. The accurate diagnosis of TBM poses a challenge due to an extensive differential diagnosis, low bacterial load and paucity of cerebrospinal fluid (CSF) especially in children. METHODOLOGY/PRINCIPAL FINDINGS: We describe the utility of ELISA and qPCR for the detection of Mycobacterium tuberculosis (M. tb) proteins (GlcB, HspX, MPT51, Ag85B and PstS1) and DNA for the rapid diagnosis of TBM. CSF filtrates (n = 532) derived from children were classified as 'Definite' TBM (M. tb culture positive, n = 29), 'Probable and Possible' TBM (n = 165) and 'Not-TBM' including other cases of meningitis or neurological disorders (n = 338). ROC curves were generated from ELISA and qPCR data of 'Definite' TBM and Non-Tuberculous infectious meningitis (NTIM) samples and cut-off values were derived to provide ≥ 95% specificity. devR qPCR, GlcB, HspX and PstS1 ELISAs showed 100% (88;100) sensitivity and 96-97% specificity in 'Definite' TBM samples. The application of these cut-offs to 'Probable and Possible' TBM groups yielded excellent sensitivity (98%, 94;99) and specificity (98%, 96;99) for qPCR and for GlcB, HspX and MPT51 antigen ELISAs (sensitivity 92-95% and specificity 93-96%). A test combination of qPCR with GlcB and HspX ELISAs accurately detected all TBM samples at a specificity of ~90%. Logistic regression analysis indicated that these tests significantly added value to the currently used algorithms for TBM diagnosis. CONCLUSIONS: The detection of M. tb GlcB/HspX antigens/devR DNA in CSF is likely to improve the utility of existing algorithms for TBM diagnosis and also hasten the speed of diagnosis.

Comparative evaluation of profiles of antibodies to mycobacterial capsular polysaccharides in tuberculosis patients and controls stratified by HIV status.

Despite the complexity of tuberculosis (TB) serology, antibodies (Abs) remain attractive biomarkers for TB. Recent evidence of a mycobacterial capsule that consists mainly of the polysaccharides arabinomannan (AM) and glucan provides new options for serologic targets. For this study, Ab responses to AM and glucan for 47 U.S. TB patients (33 HIV negative [HIV(-)], 14 HIV positive [HIV(+)]), 42 healthy controls, and 38 asymptomatic HIV(+) controls were evaluated by enzyme-linked immunosorbent assays (ELISAs). The results were compared with Ab responses to the mycobacterial glycolipid cell wall antigen lipoarabinomannan (LAM) and to the proteins malate synthase (MS) and MPT51. We found that the main immunoglobulin (Ig) isotype response to polysaccharides was IgG, predominantly of subclass IgG2. IgG responses to AM were significantly higher for HIV(-) and HIV(+) TB cases than for controls (P, <0.0001 and <0.01, respectively); significantly higher for HIV(-) than for HIV(+) TB cases (P, <0.01); and significantly higher in sputum smear-positive than smear-negative patients in both HIV(-) and HIV(+) cases (P, 0.01 and 0.02, respectively). In both TB groups, titers of Ab to glucan were significantly lower than titers of Ab to AM (P, <0.0001). IgG responses to AM and MS or to AM and MPT51 did not correlate with each other in HIV(-) TB patients, while they correlated significantly in HIV(+) TB patients (P, 0.01 and 0.05, respectively). We conclude that Ab responses to AM could contribute to the serodiagnosis of TB, especially for HIV(-) TB patients. This study also provides new and important insights into the differences in the profiles of Abs to mycobacterial antigens between HIV(-) and HIV(+) TB patients.

Correlation between serum and plasma antibody titers to mycobacterial antigens.

The ability to utilize serum or plasma samples interchangeably is useful for tuberculosis (TB) serology. We demonstrate a strong correlation between antibody titers to several mycobacterial antigens in serum versus plasma from HIV-infected and non-HIV-infected TB and non-TB patients (r = 0.99 to 0.89; P < 0.0001). Plasma and serum can be used interchangeably in the same antibody detection assays.

Antibodies against immunodominant antigens of Mycobacterium tuberculosis in subjects with suspected tuberculosis in the United States compared by HIV status.

The immunodominance of Mycobacterium tuberculosis proteins malate synthase (MS) and MPT51 has been demonstrated in case-control studies with patients from countries in which tuberculosis (TB) is endemic. The value of these antigens for the serodiagnosis of TB now is evaluated in a cross-sectional study of pulmonary TB suspects in the United States diagnosed to have TB, HIV-associated TB, or other respiratory diseases (ORD). Serum antibody reactivity to recombinant purified MS and MPT51 was determined by enzyme-linked immunosorbent assays (ELISAs) of samples from TB suspects and well-characterized control groups. TB suspects were diagnosed with TB (n = 87; 49% sputum microscopy negative, 20% HIV(+)) or ORD (n = 63; 58% HIV(+)). Antibody reactivity to MS and MPT51 was significantly higher in U.S. HIV(+)/TB samples than in HIV(-)/TB samples (P < 0.001), and it was significantly higher in both TB groups than in control groups with latent TB infection (P < 0.001). Antibody reactivity to both antigens was higher in U.S. HIV(+)/TB samples than in HIV(+)/ORD samples (P = 0.052 for MS, P = 0.001 for MPT51) but not significantly different between HIV(-)/TB and HIV(-)/ORD. Among U.S. HIV(+) TB suspects, a positive anti-MPT51 antibody response was strongly and significantly associated with TB (odds ratio, 11.0; 95% confidence interval, 2.3 to 51.2; P = 0.002). These findings have implications for the adjunctive use of TB serodiagnosis with these antigens in HIV(+) subjects.

The malate synthase of Paracoccidioides brasiliensis is a linked surface protein that behaves as an anchorless adhesin.

BACKGROUND: The pathogenic fungus Paracoccidioides brasiliensis is the agent of paracoccidioidomycosis (PCM). This is a pulmonary mycosis acquired by inhalation of fungal airborne propagules that can disseminate to several organs and tissues leading to a severe form of the disease. Adhesion and invasion to host cells are essential steps involved in the internalization and dissemination of pathogens. Inside the host, P. brasiliensis may use the glyoxylate cycle for intracellular survival. RESULTS: Here, we provide evidence that the malate synthase of P. brasiliensis (PbMLS) is located on the fungal cell surface, and is secreted. PbMLS was overexpressed in Escherichia coli, and polyclonal antibody was obtained against this protein. By using Confocal Laser Scanning Microscopy, PbMLS was detected in the cytoplasm and in the cell wall of the mother, but mainly of budding cells of the P. brasiliensis yeast phase. PbMLSr and its respective polyclonal antibody produced against this protein inhibited the interaction of P. brasiliensis with in vitro cultured epithelial cells A549. CONCLUSION: These observations indicated that cell wall-associated PbMLS could be mediating the binding of fungal cells to the host, thus contributing to the adhesion of fungus to host tissues and to the dissemination of infection, behaving as an anchorless adhesin.

Health care workers humoral immune response against GLcB, MPT51 and HSPX from Mycobacterium tuberculosis

Tuberculosis (TB) is one of the oldest human infectious diseases and one third of the world's population is latently infected. Brazil is an endemic area for TB. One of the most important challenges in TB control is the identification of latently infected individuals. Health Care Workers (HCW) are at high risk of being infected with Mycobacterium tuberculosis and even to become TB latently infected. The aim of this study was to increase knowledge about humoral immune response in TB latently infected individuals. HCW were classified according to their tuberculin skin test (TST), as positive or negative. The antibody response to GLcB, MPT51 and HSPX from Mycobacterium tuberculosis was evaluated. TST negative HCW constituted the majority of those who showed a humoral immune response. Antibody levels varied according to antigen characteristics, TST and BCG status. We suggest that possibly the presence of those antibodies could have a function in the protective immune response against Mycobacterium tuberculosis.

Health care workers humoral immune response against GLcB, MPT51 and HSPX from Mycobacterium tuberculosis.

Tuberculosis (TB) is one of the oldest human infectious diseases and one third of the world's population is latently infected. Brazil is an endemic area for TB. One of the most important challenges in TB control is the identification of latently infected individuals. Health Care Workers (HCW) are at high risk of being infected with Mycobacterium tuberculosis and even to become TB latently infected. The aim of this study was to increase knowledge about humoral immune response in TB latently infected individuals. HCW were classified according to their tuberculin skin test (TST), as positive or negative. The antibody response to GLcB, MPT51 and HSPX from Mycobacterium tuberculosis was evaluated. TST negative HCW constituted the majority of those who showed a humoral immune response. Antibody levels varied according to antigen characteristics, TST and BCG status. We suggest that possibly the presence of those antibodies could have a function in the protective immune response against Mycobacterium tuberculosis.

Biomarkers for clinical and incipient tuberculosis: performance in a TB-endemic country.

BACKGROUND: Simple biomarkers are required to identify TB in both HIV(-)TB(+) and HIV(+)TB(+) patients. Earlier studies have identified the M. tuberculosis Malate Synthase (MS) and MPT51 as immunodominant antigens in TB patients. One goal of these investigations was to evaluate the sensitivity and specificity of anti-MS and -MPT51 antibodies as biomarkers for TB in HIV(-)TB(+) and HIV(+)TB(+) patients from a TB-endemic setting. Earlier studies also demonstrated the presence of these biomarkers during incipient subclinical TB. If these biomarkers correlate with incipient TB, their prevalence should be higher in asymptomatic HIV(+) subjects who are at a high-risk for TB. The second goal was to compare the prevalence of these biomarkers in asymptomatic, CD4(+) T cell-matched HIV(+)TB(-) subjects from India who are at high-risk for TB with similar subjects from US who are at low-risk for TB. METHODS AND RESULTS: Anti-MS and -MPT51 antibodies were assessed in sera from 480 subjects including PPD(+) or PPD(-) healthy subjects, healthy community members, and HIV(-)TB(+) and HIV(+)TB(+) patients from India. Results demonstrate high sensitivity (approximately 80%) of detection of smear-positive HIV(-)TB(+) and HIV(+)TB(+) patients, and high specificity (>97%) with PPD(+) subjects and endemic controls. While approximately 45% of the asymptomatic HIV(+)TB(-) patients at high-risk for TB tested biomarker-positive, >97% of the HIV(+)TB(-) subjects at low risk for TB tested negative. Although the current studies are hampered by lack of knowledge of the outcome, these results provide strong support for the potential of these biomarkers to detect incipient, subclinical TB in HIV(+) subjects. CONCLUSIONS: These biomarkers provide high sensitivity and specificity for TB diagnosis in a TB endemic setting. Their performance is not compromised by concurrent HIV infection, site of TB and absence of pulmonary manifestations in HIV(+)TB(+) patients. Results also demonstrate the potential of these biomarkers for identifying incipient subclinical TB in HIV(+)TB(-) subjects at high-risk for TB.

Humoral immune responses of tuberculosis patients in Brazil indicate recognition of Mycobacterium tuberculosis MPT-51 and GlcB.

The humoral responses to recombinant MPT-51 and GlcB was determined by using an enzyme-linked immunosorbent assay. Levels of immunoglobulin M (IgM) against MPT-51 and IgG against GlcB were higher among tuberculosis (TB) patients than among control individuals. When the MPT-51 and GlcB assays were combined, 90.8% specificity and 75.5% sensitivity were observed. MPT-51 and GlcB were recognized in the humoral responses of Brazilian TB patients.

Antibody markers of incident tuberculosis among HIV-infected adults in the USA: a historical prospective study.

OBJECTIVE: To assess whether serum levels of antibodies against Mycobacterium tuberculosis antigens increase before diagnosis of active tuberculosis (TB). DESIGN: Serial serum samples were obtained from 30 human immunodeficiency virus (HIV) co-infected individuals who developed active TB during a multicenter prospective study on pulmonary complications of HIV/AIDS conducted among >1300 subjects in the USA in the 1980s. Multiple serum samples from 47 matched control individuals who did not develop TB in the same study were also tested. Immunoglobulin G (IgG) antibodies to 10 M. tuberculosis proteins were detected by enzyme-linked immunosorbent assay (ELISA), and data were analyzed by descriptive and inferential statistical techniques to assess patterns, trends and differences in antibody levels relative to time from TB diagnosis. RESULTS: Antibodies to five antigens (ESAT-6, 38 kDa Ag, 16 kDa Ag, malate synthase and MTSA-10/CFP-10), but not to five other antigens (Rv2626c, ferredoxin A, glutamine synthetase, alanine dehydrogenase and Ag85) increased before diagnosis of TB relative to control levels. The earliest increase in the TB group was detected for MTSA-10/CFP-10 (24-30 months pre-diagnosis). CONCLUSIONS: Levels of serum antibodies to particular proteins of M. tuberculosis increase before microbiological and clinical symptoms of active TB. The use of antibody biomarkers for prognostic purposes should therefore be feasible.

Humoral response to HspX and GlcB to previous and recent infection by Mycobacterium tuberculosis.

BACKGROUND: Tuberculosis (TB) remains a major world health problem. Around 2 billions of people are infected by Mycobacterium tuberculosis, the causal agent of this disease. This fact accounts for a third of the total world population and it is expected that 9 million people will become infected each year. Only approximately 10% of the infected people will develop disease. However, health care workers (HCW) are continually exposed to the bacilli at endemic sites presenting increased chance of becoming sick. The objective of this work was to identify LTBI (latent tuberculosis infection) among all asymptomatic HCW of a Brazilian Central Hospital, in a three year follow up, and evaluate the humoral response among HCW with previous and recent LTBI to recombinant HspX and GlcB from M. tuberculosis. METHODS: Four hundred and thirty seven HCW were screened and classified into three different groups according to tuberculin skin test (TST) status: uninfected, previous LTBI and recent LTBI. ELISA test were performed to determine the humoral immune response to HspX and GlcB. RESULTS: The levels of IgG and IgM against the HspX and GlcB antigens were the same among HCW with recent and previous LTBI, as well as among non infected HCW. However, the IgM levels to HspX was significantly higher among HCW with recent LTBI (OD = 1.52 +/- 0.40) than among the uninfected (OD = 1.09 +/- 0.50) or subjects with previous LTBI (OD = 0.96 +/- 0.51) (p < 0.001). CONCLUSION: IgG and IgM humoral responses to GlcB antigens were similar amongst all studied groups; nevertheless IgM levels against HspX were higher among the recent LTBI/HCW.

Mycobacterium tuberculosis malate synthase- and MPT51-based serodiagnostic assay as an adjunct to rapid identification of pulmonary tuberculosis.

The 81-kDa malate synthase (MS; Rv 1837c) and the 27-kDa MPT51 (Rv 3803c) of Mycobacterium tuberculosis are immunodominant antigens recognized by serum antibodies from approximately 80% of human immunodeficiency virus-negative smear-positive tuberculosis patients from India. We now provide evidence that the use of the MS/MPT51-based serodiagnostic assay can serve as an adjunct to sputum microscopy in the rapid diagnosis of pulmonary tuberculosis.

Rapid purification of malate synthase from cotyledons of Brassica napus L.

A rapid and efficient method for the purification of malate synthase, an enzyme uniquely confined to glyoxysomes, from cotyledons of Brassica napus L. has been developed. The two step purification procedure is based on the consequent utilization of the tendency of malate synthase to form high molecular weight aggregates. Malate synthase was purified 75-fold to apparent homogeneity with a specific activity of 180 nkat/mg protein. The estimated molecular weight of malate synthase subunits was 63 kDa. Polyclonal antibodies raised against malate synthase in rabbits detect on Western blots only one single polypeptide with an identical molecular weight.

Glyoxysomal malate dehydrogenase and malate synthase from soybean cotyledons (Glycine max L.): enzyme association, antibody production and cDNA cloning.

In order to investigate a possible association between soybean malate synthase (MS; L-malate glyoxylate-lyase, CoA-acetylating, EC 4.1.3.2) and glyoxysomal malate dehydrogenase (gMDH; (S)-malate: NAD+ oxidoreductase, EC 1.1.1.37), two consecutive enzymes in the glyoxylate cycle, their elution profiles were analyzed on Superdex 200 HR fast protein liquid chromatography columns equilibrated in low- and high-ionic-strength buffers. Starting with soluble proteins extracted from the cotyledons of 5-d-old soybean seedlings and a 45% ammonium sulfate precipitation, MS and gMDH coeluted on Superdex 200 HR (low-ionic-strength buffer) as a complex with an approximate relative molecular mass (Mr) of 670,000. Dissociation was achieved in the presence of 50 mM KCl and 5 mM MgCl2, with the elution of MS as an octamer of M(r) 510,000 and of gMDH as a dimer of M(r) 73,000. Polyclonal antibodies raised to the native copurified enzymes recognized both denatured MS and gMDH on immunoblots, and their native forms after gel filtration. When these antibodies were used to screen a lambda ZAP II expression library containing cDNA from 3-d-old soybean cotyledons, they identified seven clones encoding gMDH, whereas ten clones encoding MS were identified using an antibody to SDS-PAGE-purified MS. Of these cDNA clones a 1.8 kb clone for MS and a 1.3-kb clone for gMDH were fully sequenced. While 88% identity was found between mature soybean gMDH and watermelon gMDH, the N-terminal transit peptides showed only 37% identity. Despite this low identity, the soybean gMDH transit peptide conserves the consensus R(X6)HL motif also found in plant and mammalian thiolases.

Purification and immunochemical characterization of malate synthase from Euglena gracilis.

Malate synthase (EC 4.1.3.2) from dark-grown Euglena gracilis was purified to homogeneity by the criterion of polyacrylamide-gel electrophoresis. The enzyme was released from acetate-grown cells by treatment with ultrasonic waves and purified from broken-cell suspensions by high-speed centrifugation and (NH4)2SO4 fractionation, followed by gel-filtration on Sepharose 6B. The final enzyme preparation was purified 190-fold compared with the crude extract. The mol.wt. of the enzyme was about 350000 as determined by gel filtration on Sepharose 6B. Treatment with sodium dodecyl sulphate and urea dissociated the enzyme into subunits of mol.wt. 175000. The pH optimum for the enzyme was 8.0 and the Km values for glyoxylate and acetyl-CoA were 50 and 80 micron respectively. Antibodies raised to the purified enzyme were shown to be monospecific by radiochemical immunoassay. Euglena anti-(malate synthase) tested on Ouchterlony double-diffusion gels gave a sharp precipitation band against acetate-grown Escherichia coli, but no immunological correspondence was observed with acetate-grown Chlorella fusca, Zea mays (maize) scutella or purified malate synthase from Ricinus communis.