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

Sharma, Sumedha; Suri, Deepti; Aggarwal, Ashutosh N; Yadav, Rakesh; Sethi, Sunil; Laal, Suman; Verma, Indu
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.
PMID: 37198420
ISSN: 1678-4405
CID: 5508032

Association of Micronutrients with Tuberculosis Development in HIV Infected Patients

Banyal, Dinesh; Sharma, Sumedha; Ram, Anil Kumar; Kaur, Khushpreet; Jassal, Ravjit Singh; Attri, Savita; Sharma, Aman; Sharma, Kusum; Laal, Suman; Verma, Indu
Human immunodeficiency virus (HIV) infection associated with weakened immune system due to decreased CD4 T cell count favors development of tuberculosis. Effector immune responses are also associated with micronutrient status due to their prominent role in maintaining immune functions. Micronutrient deficiencies are quite common among HIV patients that further result into compromised immunity thus making the conditions even more favorable for mycobacteria to establish disease. So, current study was designed to assess association of different micronutrients with development of TB in HIV patients. Micronutrient levels were measured in asymptomatic HIV patients who were monitored for the development of TB during follow up period (incident TB) within one month to one year and also in symptomatic microbiologically confirmed HIV-TB patients. Among various micronutrients assessed, levels of ferritin were found to be significantly increased (p < 0.05) with significant decreased zinc (p < 0.05) and selenium (p < 0.05) levels in incident TB group as well as in HIV-TB subjects compared to asymptomatic HIV patients who did not develop TB in the follow up period. Importantly, increased levels of ferritin and decreased levels of selenium were significantly associated with development of tuberculosis in HIV patients.
PMCID:10205935
PMID: 37234181
ISSN: 0970-1915
CID: 5543932

Mycobacterium tuberculosis transcriptome in intraocular tuberculosis

Kaur, Kamaljit; Laal, Suman; Ryndak, Michelle B
PMID: 36762529
ISSN: 1473-5644
CID: 5420962

Evolution of antibodies to epitopes of PE_PGRS51 in the spectrum of active pulmonary tuberculosis

Sakamuri, Rama Murthy; Singh, Krishna Kumar; Ryndak, Michelle B; Laal, Suman
BACKGROUND:The PE_PGRS protein family isimplicated in virulence and immunopathogenesis of M. tuberculosis. Due to extensive intra-family homology, it has not been possible to correlate expression of specific members with stage of infection or disease progression. Here, we investigate the in vivo expression of PE_PGRS51, which besides the cross-reactive PE and PGRS domains, has 3 unique regions. METHODS:Expression in M. smegmatis confirmed PE_PGRS51 cell-wall localization. Patients at different stages of TB were classified across a spectrum akin to the Leprosy spectrum. Peptide arrays representing PE_PGRS51 were screened with sera from TB patients across the spectrum of active TB, with latent TB sera as controls. RESULTS:Antibodies directed only against conserved epitopes within the PE/PGRS domains were detectable at early stages of TB. As bacteriological burden and disease progresses, the epitope repertoire extends systematically to adjacent and overlapping peptides. Sera-reactivity to epitopes unique to PE_PGRS51 appear only in the most advanced TB patients, indicating PE_PGRS51 expression in vivo. DISCUSSION/CONCLUSIONS:The ability to classify active TB patients into a spectrum and delineate the in vivo expression of PE_PGRS proteins at different stages of disease has important implications for understanding their role in TB pathogenesis and their usefulness as diagnostic markers.
PMID: 31641771
ISSN: 1537-6613
CID: 4147392

Transcriptional signatures of Mycobacterium tuberculosis in mouse model of intraocular tuberculosis

Abhishek, Sudhanshu; Ryndak, Michelle Beth; Choudhary, Alpa; Sharma, Sumedha; Gupta, Amod; Gupta, Vishali; Singh, Nirbhai; Laal, Suman; Verma, Indu
BACKGROUND:Studies on human intraocular tuberculosis (IOTB) are extremely challenging. For understanding the pathogenesis of IOTB, it is important to investigate the mycobacterial transcriptional changes in ocular environment. METHODS:Mice were challenged intravenously with M. tuberculosis H37Rv and at 45 days post-infection, experimental IOTB was confirmed based on bacteriological and molecular assays. M. tuberculosis transcriptome was analyzed in the infected eyes using microarray technology. The identified M. tuberculosis signature genes were further validated and investigated in human IOTB samples using real-time polymerase chain reaction. RESULTS:Following intravenous challenge with M. tuberculosis, 45% (5/12) mice showed bacilli in the eyes with positivity for M. tuberculosis RNA in 100% (12/12), thus confirming the paucibacillary nature of IOTB similar to human IOTB. M. tuberculosis transcriptome in these infected eyes showed significant upregulation of 12 M. tuberculosis genes and five of these transcripts (Rv0962c, Rv0984, Rv2612c, Rv0974c and Rv0971c) were also identified in human clinically confirmed cases of IOTB. CONCLUSIONS:Differentially expressed mycobacterial genes identified in an intravenously challenged paucibacillary mouse IOTB model and presence of these transcripts in human IOTB samples highlights the possible role of these genes for survival of M. tuberculosis in the ocular environment thus contributing to pathogenesis of IOTB.
PMID: 31504463
ISSN: 2049-632x
CID: 4087842

Mycobacterium tuberculosis Primary Infection and Dissemination: A Critical Role for Alveolar Epithelial Cells

Ryndak, Michelle B; Laal, Suman
Globally, tuberculosis (TB) has reemerged as a major cause of morbidity and mortality, despite the use of the Mycobacterium bovis BCG vaccine and intensive attempts to improve upon BCG or develop new vaccines. Two lacunae in our understanding of the Mycobacterium tuberculosis (M. tb)-host pathogenesis have mitigated the vaccine efforts; the bacterial-host interaction that enables successful establishment of primary infection and the correlates of protection against TB. The vast majority of vaccine efforts are based on the premise that cell-mediated immunity (CMI) is the predominating mode of protection against TB. However, studies in animal models and in humans demonstrate that post-infection, a period of several weeks precedes the initiation of CMI during which the few inhaled bacteria replicate dramatically and disseminate systemically. The "Trojan Horse" mechanism, wherein M. tb is phagocytosed and transported across the alveolar barrier by infected alveolar macrophages has been long postulated as the sole, primary M. tb:host interaction. In the current review, we present evidence from our studies of transcriptional profiles of M. tb in sputum as it emerges from infectious patients where the bacteria are in a quiescent state, to its adaptations in alveolar epithelial cells where the bacteria transform to a highly replicative and invasive phenotype, to its maintenance of the invasive phenotype in whole blood to the downregulation of invasiveness upon infection of epithelial cells at an extrapulmonary site. Evidence for this alternative mode of infection and dissemination during primary infection is supported by in vivo, in vitro cell-based, and transcriptional studies from multiple investigators in recent years. The proposed alternative mechanism of primary infection and dissemination across the alveolar barrier parallels our understanding of infection and dissemination of other Gram-positive pathogens across their relevant mucosal barriers in that barrier-specific adhesins, toxins, and enzymes synergize to facilitate systemic establishment of infection prior to the emergence of CMI. Further exploration of this M. tb:non-phagocytic cell interaction can provide alternative approaches to vaccine design to prevent infection with M. tb and not only decrease clinical disease but also decrease the overwhelming reservoir of latent TB infection.
PMCID:6712944
PMID: 31497538
ISSN: 2235-2988
CID: 4087482

Mycobacterium tuberculosis(M.tb)antibody and antigen biomarkers for rapid diagnosis of intra-ocular tuberculosis [Meeting Abstract]

Kaur, Kamaljit; Ryndak, Michelle B.; Agarwal, Aniruddha; Verma, Indu; Gupta, Vishali; Laal, Suman
ISI:000488628102011
ISSN: 0146-0404
CID: 4154202

Transcriptional Profile of Mycobacterium tuberculosis in an in vitro Model of Intraocular Tuberculosis

Abhishek, Sudhanshu; Saikia, Uma Nahar; Gupta, Amod; Bansal, Reema; Gupta, Vishali; Singh, Nirbhai; Laal, Suman; Verma, Indu
Background: Intraocular tuberculosis (IOTB), an extrapulmonary manifestation of tuberculosis of the eye, has unique and varied clinical presentations with poorly understood pathogenesis. As it is a significant cause of inflammation and visual morbidity, particularly in TB endemic countries, it is essential to study the pathogenesis of IOTB. Clinical and histopathologic studies suggest the presence of Mycobacterium tuberculosis in retinal pigment epithelium (RPE) cells. Methods: A human retinal pigment epithelium (ARPE-19) cell line was infected with a virulent strain of M. tuberculosis (H37Rv). Electron microscopy and colony forming units (CFU) assay were performed to monitor the M. tuberculosis adherence, invasion, and intracellular replication, whereas confocal microscopy was done to study its intracellular fate in the RPE cells. To understand the pathogenesis, the transcriptional profile of M. tuberculosis in ARPE-19 cells was studied by whole genome microarray. Three upregulated M. tuberculosis transcripts were also examined in human IOTB vitreous samples. Results: Scanning electron micrographs of the infected ARPE-19 cells indicated adherence of bacilli, which were further observed to be internalized as monitored by transmission electron microscopy. The CFU assay showed that 22.7 and 8.4% of the initial inoculum of bacilli adhered and invaded the ARPE-19 cells, respectively, with an increase in fold CFU from 1 dpi (0.84) to 5dpi (6.58). The intracellular bacilli were co-localized with lysosomal-associated membrane protein-1 (LAMP-1) and LAMP-2 in ARPE-19 cells. The transcriptome study of intracellular bacilli showed that most of the upregulated transcripts correspond to the genes encoding the proteins involved in the processes such as adherence (e.g., Rv1759c and Rv1026), invasion (e.g., Rv1971 and Rv0169), virulence (e.g., Rv2844 and Rv0775), and intracellular survival (e.g., Rv1884c and Rv2450c) as well as regulators of various metabolic pathways. Two of the upregulated transcripts (Rv1971, Rv1230c) were also present in the vitreous samples of the IOTB patients. Conclusions:M. tuberculosis is phagocytosed by RPE cells and utilizes these cells for intracellular multiplication with the involvement of late endosomal/lysosomal compartments and alters its transcriptional profile plausibly for its intracellular adaptation and survival. The findings of the present study could be important to understanding the molecular pathogenesis of IOTB with a potential role in the development of diagnostics and therapeutics for IOTB.
PMID: 30333960
ISSN: 2235-2988
CID: 3368592

Transcriptome analysis of mycobacteria in sputum samples of pulmonary tuberculosis patients

Sharma, Sumedha; Ryndak, Michelle B; Aggarwal, Ashutosh N; Yadav, Rakesh; Sethi, Sunil; Masih, Shet; Laal, Suman; Verma, Indu
Pulmonary tuberculosis, the disease caused by Mycobacterium tuberculosis, still retains a top rank among the deadliest communicable diseases. Sputum expectorated during the disease continues to be a primary diagnostic specimen and also serves as a reservoir of bacteria. The expression pattern of mycobacteria in sputum will lead to an insight into bacterial adaptation at the most highly transmissible stage of infection and can also help in identifying newer diagnostic as well as drug targets. Thus, in the present study, a whole genome microarray of Mycobacterium tuberculosis was used to elucidate the transcriptional profile of mycobacteria in the sputum samples of smear positive pulmonary tuberculosis patients. Overall, the mycobacteria in sputum appeared to be in a low energy and low replicative state as compared to in vitro grown log phase M. tb with downregulation of genes involved in ATP synthesis, aerobic respiration and translational machinery. Simultaneously, downregulation was also seen in the genes involved in secretion machinery of mycobacteria along with the downregulation of genes involved in the synthesis of phthiocerol dimycocerosate and phenol glycolipids. In contrast, the majority of the genes which showed an upregulation in sputum mycobacteria were of unknown function. Further identification of these genes may provide new insights into the mycobacterial behavior during this phase of infection and may help in deciphering candidates for development of better diagnostic and drug candidates.
PMCID:5345810
PMID: 28282458
ISSN: 1932-6203
CID: 2477492

Understanding dissemination of Mycobacterium tuberculosis from the lungs during primary infection

Ryndak, Michelle B; Chandra, Dinesh; Laal, Suman
Understanding how inhaled M. tuberculosis achieves dramatic replication and crosses the alveolar barrier to establish systemic latent infection before adaptive immunity is elicited in humans, is limited by the small infecting inoculum carried in aerosol droplets (1-5 m diameter) and the inability to identify time of infection. M. tuberculosis is believed to disseminate via infected macrophages. However, like other invasive bacterial pathogens, M. tuberculosis could also cross the barrier directly using adhesins and toxins. An in vitro alveolar-barrier mimicking gas-exchange regions of the alveolus was devised comprising monolayers of human alveolar epithelial and endothelial cells cultured on opposing sides of a basement membrane. Migration of dissemination-competent strains of M. tuberculosis, and dissemination-attenuated M. tuberculosis and M. bovis mutant strains lacking adhesin/toxin ESAT-6 and adhesin HBHA were tested for macrophage-free migration across the barrier. Strains that disseminate similarly in vivo migrated similarly across the in vitro alveolar-barrier. Strains lacking ESAT-6 expression/secretion were attenuated, and absence of both ESAT-6 and HBHA increased attenuation of bacterial migration across the barrier. Thus, as reported for other bacteria, M. tuberculosis utilizes adhesins and toxins for macrophage-independent crossing of the alveolar-barrier. This in vitro model will allow identification and characterization of molecules/mechanisms employed by M. tuberculosis to establish systemic LTBI during primary infection.
PMID: 26905324
ISSN: 1473-5644
CID: 2045772