Evaluation of immunodominant peptides of in vivo expressed mycobacterial antigens in an ELISA-based diagnostic assay for pulmonary tuberculosis.

Non-sputum-based biomarker assay is urgently required as per WHO's target product pipeline for diagnosis of tuberculosis. Therefore, the current study was designed to evaluate the utility of previously identified proteins, encoded by in vivo expressed mycobacterial transcripts in pulmonary tuberculosis, as diagnostic targets for a serodiagnostic assay. A total of 300 subjects were recruited including smear+, smear- pulmonary tuberculosis (PTB) patients, sarcoidosis patients, lung cancer patients and healthy controls. Proteins encoded by eight in vivo expressed transcripts selected from previous study including those encoded by two topmost expressed and six RD transcripts (Rv0986, Rv0971, Rv1965, Rv1971, Rv2351c, Rv2657c, Rv2674, Rv3121) were analyzed for B-cell epitopes by peptide arrays/bioinformatics. Enzyme-linked immunosorbent assay was used to evaluate the antibody response against the selected peptides in sera from PTB and controls. Overall 12 peptides were selected for serodiagnosis. All the peptides were initially screened for their antibody response. The peptide with highest sensitivity and specificity was further assessed for its serodiagnostic ability in all the study subjects. The mean absorbance values for antibody response to selected peptide were significantly higher (p<0.001) in PTB patients as compared to healthy controls; however, the sensitivity for diagnosis of PTB was 31% for smear+ and 20% for smear- PTB patients. Thus, the peptides encoded by in vivo expressed transcripts elicited a significant antibody response, but are not suitable candidates for serodiagnosis of PTB.

Transcriptional profile of Mycobacterium tuberculosis replicating in type II alveolar epithelial cells.

Mycobacterium tuberculosis (M. tb) infection is initiated by the few bacilli inhaled into the alveolus. Studies in lungs of aerosol-infected mice provided evidence for extensive replication of M. tb in non-migrating, non-antigen-presenting cells in the alveoli during the first 2-3 weeks post-infection. Alveoli are lined by type II and type I alveolar epithelial cells (AEC) which outnumber alveolar macrophages by several hundred-fold. M. tb DNA and viable M. tb have been demonstrated in AEC and other non-macrophage cells of the kidney, liver, and spleen in autopsied tissues from latently-infected subjects from TB-endemic regions indicating systemic bacterial dissemination during primary infection. M. tb have also been demonstrated to replicate rapidly in A549 cells (type II AEC line) and acquire increased invasiveness for endothelial cells. Together, these results suggest that AEC could provide an important niche for bacterial expansion and development of a phenotype that promotes dissemination during primary infection. In the current studies, we have compared the transcriptional profile of M. tb replicating intracellularly in A549 cells to that of M. tb replicating in laboratory broth, by microarray analysis. Genes significantly upregulated during intracellular residence were consistent with an active, replicative, metabolic, and aerobic state, as were genes for tryptophan synthesis and for increased virulence (ESAT-6, and ESAT-6-like genes, esxH, esxJ, esxK, esxP, and esxW). In contrast, significant downregulation of the DevR (DosR) regulon and several hypoxia-induced genes was observed. Stress response genes were either not differentially expressed or were downregulated with the exception of the heat shock response and those induced by low pH. The intra-type II AEC M. tb transcriptome strongly suggests that AEC could provide a safe haven in which M. tb can expand dramatically and disseminate from the lung prior to the elicitation of adaptive immune responses.

Transcriptional profiling of Mycobacterium tuberculosis replicating ex vivo in blood from HIV- and HIV+ subjects.

Hematogenous dissemination of Mycobacterium tuberculosis (M. tb) occurs during both primary and reactivated tuberculosis (TB). Although hematogenous dissemination occurs in non-HIV TB patients, in ∼80% of these patients, TB manifests exclusively as pulmonary disease. In contrast, extrapulmonary, disseminated, and/or miliary TB is seen in 60-70% of HIV-infected TB patients, suggesting that hematogenous dissemination is likely more common in HIV+ patients. To understand M. tb adaptation to the blood environment during bacteremia, we have studied the transcriptome of M. tb replicating in human whole blood. To investigate if M. tb discriminates between the hematogenous environments of immunocompetent and immunodeficient individuals, we compared the M. tb transcriptional profiles during replication in blood from HIV- and HIV+ donors. Our results demonstrate that M. tb survives and replicates in blood from both HIV- and HIV+ donors and enhances its virulence/pathogenic potential in the hematogenous environment. The M. tb blood-specific transcriptome reflects suppression of dormancy, induction of cell-wall remodeling, alteration in mode of iron acquisition, potential evasion of immune surveillance, and enhanced expression of important virulence factors that drive active M. tb infection and dissemination. These changes are accentuated during bacterial replication in blood from HIV+ patients. Furthermore, the expression of ESAT-6, which participates in dissemination of M. tb from the lungs, is upregulated in M. tb growing in blood, especially during growth in blood from HIV+ patients. Preliminary experiments also demonstrate that ESAT-6 promotes HIV replication in U1 cells. These studies provide evidence, for the first time, that during bacteremia, M. tb can adapt to the blood environment by modifying its transcriptome in a manner indicative of an enhanced-virulence phenotype that favors active infection. Additionally, transcriptional modifications in HIV+ blood may further accentuate M. tb virulence and drive both M. tb and HIV infection.

LipC (Rv0220) is an immunogenic cell surface esterase of Mycobacterium tuberculosis.

We have reported previously the identification of novel proteins of Mycobacterium tuberculosis by the immunoscreening of an expression library of M. tuberculosis genomic DNA with sera obtained from M. tuberculosis-infected rabbits at 5 weeks postinfection. In this study, we report the further characterization of one of these antigens, LipC (Rv0220). LipC is annotated as a member of the Lip family based on the presence of the consensus motif "GXSXG" characteristic of esterases. Although predicted to be a cytoplasmic enzyme, we provide evidence that LipC is a cell surface protein that is present in both the cell wall and the capsule of M. tuberculosis. Consistent with this localization, LipC elicits strong humoral immune responses in both HIV-negative (HIV-) and HIV-positive (HIV+) tuberculosis (TB) patients. The absence of anti-LipC antibodies in sera from purified protein derivative-positive (PPD+) healthy subjects confirms its expression only during active M. tuberculosis infection. Epitope mapping of LipC identified 6 immunodominant epitopes, 5 of which map to the exposed surface of the modeled LipC protein. The recombinant LipC (rLipC) protein also elicits proinflammatory cytokine and chemokine responses from macrophages and pulmonary epithelial cells. rLipC can hydrolyze short-chain esters with the carbon chain containing 2 to 10 carbon atoms. Together, these studies demonstrate that LipC is a novel cell surface-associated esterase of M. tuberculosis that is highly immunogenic and elicits both antibodies and cytokines/chemokines.

Potential role for ESAT6 in dissemination of M. tuberculosis via human lung epithelial cells.

ESAT6 has recently been demonstrated to cause haemolysis and macrophage lysis. Our studies demonstrate that ESAT6 causes cytolysis of type 1 and type 2 pneumocytes. Both types of pneumocytes express membrane laminin, and ESAT6 exhibits dose-dependent binding to both cell types and to purified human laminin. While minimal ESAT6 was detected on the surface of Mycobacterium tuberculosis grown in vitro, exogenously provided ESAT6 specifically associated with the bacterial cell surface, and the bacterium-associated ESAT6 retained its cytolytic ability. esat6 transcripts were upregulated approximately 4- to approximately 13-fold in bacteria replicating in type 1 cells, and approximately 3- to approximately 5 fold in type 2 cells. In vivo, laminin is primarily concentrated at the basolateral surface of pneumocytes where they rest on the basement membrane, which is composed primarily of laminin and collagen. The upregulation of esat6 transcripts in bacteria replicating in pneumocytes, the specific association of ESAT6 with the bacterial surface, the binding of ESAT6 to laminin and the lysis of pneumocytes by free and bacterium-associated ESAT6 together suggest a scenario wherein Mycobacterium tuberculosis replicating in pneumocytes may utilize surface ESAT6 to anchor onto the basolateral laminin-expressing surface of the pneumocytes, and damage the cells and the basement membrane to directly disseminate through the alveolar wall.

Performance of purified antigens for serodiagnosis of pulmonary tuberculosis: a meta-analysis.

Serological antibody detection tests for tuberculosis may offer the potential to improve diagnosis. Recent meta-analyses have shown that commercially available tests have variable accuracies and a limited clinical role. We reviewed the immunodiagnostic potential of antigens evaluated in research laboratories (in-house) for the serodiagnosis of pulmonary tuberculosis and conducted a meta-analysis to evaluate the performance of comparable antigens. Selection criteria included the participation of at least 25 pulmonary tuberculosis patients and the use of purified antigens. Studies evaluating 38 kDa, MPT51, malate synthase, culture filtrate protein 10, TbF6, antigen 85B, alpha-crystallin, 2,3-diacyltrehalose, 2,3,6-triacyltrehalose, 2,3,6,6'-tetraacyltrehalose 2'-sulfate, cord factor, and TbF6 plus DPEP (multiple antigen) were included in the meta-analysis. The results demonstrated that (i) in sputum smear-positive patients, sensitivities significantly >or=50% were provided for recombinant malate synthase (73%; 95% confidence interval [CI], 58 to 85) and TbF6 plus DPEP (75%; 95% CI, 50 to 91); (ii) protein antigens achieved high specificities; (iii) among the lipid antigens, cord factor had the best overall performance (sensitivity, 69% [95% CI, 28 to 94]; specificity, 91% [95% CI, 78 to 97]); (iv) compared with the sensitivities achieved with single antigens (median sensitivity, 53%; range, 2% to 100%), multiple antigens yielded higher sensitivities (median sensitivity, 76%; range, 16% to 96%); (v) in human immunodeficiency virus (HIV)-infected patients who are sputum smear positive, antibodies to several single and multiple antigens were detected; and (vi) data on seroreactivity to antigens in sputum smear-negative or pediatric patients were insufficient. Potential candidate antigens for an antibody detection test for pulmonary tuberculosis in HIV-infected and -uninfected patients have been identified, although no antigen achieves sufficient sensitivity to replace sputum smear microscopy. Combinations of select antigens provide higher sensitivities than single antigens. The use of a case-control design with healthy controls for the majority of studies was a limitation of the review. Efforts are needed to improve the methodological quality of tuberculosis diagnostic studies.

Peptide-based antibody detection for tuberculosis diagnosis.

Tuberculosis (TB) is a major cause of morbidity and mortality, especially in developing countries. Despite significant limitations, microscopy remains the cornerstone of the global TB control strategy. As the TB epidemic escalates, new diagnostic methods that are accurate and also economical and simple to manufacture and deploy are urgently needed. Although several promising antigens have been identified and evaluated in recent years, the reproducible production of high-quality recombinant mycobacterial proteins with minimal batch-to-batch variation is difficult, laborious, and expensive. To determine the feasibility of devising a synthetic peptide-based diagnostic test for TB, we have delineated the immunodominant epitopes of three candidate antigens, Ag85B, BfrB, and TrxC, that were previously identified to be immunogenic in TB patients. The results demonstrate that combinations of carefully selected synthetic peptides derived from highly immunogenic proteins can be the basis for devising an immunodiagnostic test for TB.

Disease state differentiation and identification of tuberculosis biomarkers via native antigen array profiling.

A critical element of tuberculosis control is early and sensitive diagnosis of infection and disease. Our laboratories recently showed that different stages of disease were distinguishable via two-dimensional Western blot analyses of Mycobacterium tuberculosis culture filtrate proteins. However, this methodology is not suitable for high throughput testing. Advances in protein microarray technology provide a realistic mechanism to screen a large number of serum samples against thousands of proteins to identify biomarkers of disease states. Techniques were established for separation of native M. tuberculosis cytosol and culture filtrate proteins, resulting in 960 unique protein fractions that were used to generate protein microarrays. Evaluation of serological reactivity from 42 patients in three tuberculosis disease states and healthy purified protein derivative-positive individuals demonstrated that human immunodeficiency virus (HIV)-negative cavitary and noncavitary tuberculosis (TB) patients' sera recognized 126 and 59 fractions, respectively. Sera from HIV patients coinfected with TB recognized 20 fractions of which five overlapped with those recognized by non-HIV TB patients' sera and 15 were unique to the HIV+TB+ disease state. Identification of antigens within the reactive fractions yielded 11 products recognized by both cavitary and noncavitary TB patients' sera and four proteins (HspX, MPT64, PstS1, and TrxC) specific to cavitary TB patients. Moreover four novel B cell antigens (BfrB, LppZ, SodC, and TrxC) of human tuberculosis were identified.

Mycobacterium tuberculosis malate synthase is a laminin-binding adhesin.

Mycobacterium tuberculosis (M. tb) uses the glyoxalate bypass for intracellular survival in vivo. These studies provide evidence that the M. tb malate synthase (MS) has adapted to function as an adhesin which binds to laminin and fibronectin. This binding is achieved via the unique C-terminal region of the M. tb MS. The ability to function as an adhesin necessitates extracellular localization. We provide evidence that despite the absence of a Sec-translocation signal sequence the M. tb MS is secreted/excreted, and is anchored on the cell wall by an undefined mechanism. The MS of Mycobacterium smegmatis is cytoplasmic but the M. tb MS expressed in M. smegmatis localizes to the cell wall and enhances the adherence of the bacteria to lung epithelial A549 cells. Antibodies to the C-terminal laminin/fibronectin-binding domain interfere with the binding of the M. tb MS to laminin and fibronectin and reduce the adherence of M. tb to A549 cells. Coupled to the earlier evidence of in vivo expression of M. tb MS during active but not latent infection in humans, these studies show that a housekeeping enzyme of M. tb contributes to its armamentarium of virulence promoting factors.