Identification Of Diagnostic Molecular Biomarkers Of Glycoconjugates For Mycobacterium Tuberculosis

Mycobacterium tuberculosis (M.tb) is the causative agent of most cases of Tuberculosis (TB) with high morbidity and mortality in many developing countries. World Health Organization (WHO) estimated that about1.3million individuals died from TB around the world in2012. China has the second largest number of active pulmonary tuberculosis patients in world. Recently the tuberculosis epidemic is increasing in developing countries due to co-infection with Human Immunodeficiency Virus (HIV) and emergence of multidrug-resistant tuberculosis (MDR-TB). Hence the early and definite diagnosis would benefit TB therapy and prognosis. Current laboratory methods for tuberculosis diagnosis had varied shortcomings, e.g. PPD test has a high false positive rate; the sputum culture takes long time; serological methods for the detection of M.tb antibodies exist, however, there is a "window of time" during which the antigens are present in the serum sample but antibodies have not been produced. The PCR assay has a sensitivity and specificity. However this technology needs expensive reagents and instruments and does not distinguish living bacteria and dead ones; Smear assay has a low positive rate and the T-Spot and gene chip are too expensive and complicated and T-Spot could not apply to detect T-cell deficiency patients. Therefore new diagnostic methods are needed for rapid and effective tuberculosis diagnosis, especially in the case of early tuberculosis infection and expulmonary tuberculosis infection. New effective methods for tuberculosis diagnosis and therapeutics are of great significance for tuberculosis prevention and control.We chose Beijing genotype strains of M.tb as the model system for diagnostic molecular marker screening, and selected Mannosylated lipoarabinomannan (ManLAM) as the main target marker molecule. Beijing genotype strains of M.tb were discovered in Beijing area in1995, with remarkable similar genetic characteristics of M.tb strain. It is widespread among TB patients in Asia, and it has stronger toxicity and higher transmissibility and resistance than H37Rv strain. Especially, the Beijing genotype strain is the dominant tuberculosis-causing pathogenic strain in China, so we chose Beijing genotype strains as the model system for current study. ManLAM is a glycolipid commonly found on the surface of toxic M.tb. Just like many toxic M.tb, ManLAM is also found on the surface of the Beijing genotype strains. When M.tb strain invades and interacts with host macrophages, ManLAM can be shed from toxic M.tb and released into the infection site and then into the blood circulation, which makes it as a perfect candidate diagnostic molecule. In this study, we screened ssDNA aptamers which specifically binding to ManLAM by systematic evolution of ligands by exponential enrichment (SELEX) and used the selected aptamer T9to detect ManLAM in human body liquid from clinical samples for use in TB diagnosis.Antibody against ManLAM is difficult to produce due to its weak immunogenicity. So we used SELEX to generate the ssDNA aptamer "antibody"which binding to the ManLAM of Beijing genotype strains of M.tb, named T9with high affinity (Kd=685±131nM) and specificity. Enzyme-linked oligonucleotide assay (ELONA) was applied to detect clinical sputum and serum samples using aptamer T9for diagnosis of active TB patients from healthy donors. The results of ELONA using aptamer T9was shown good correlation with ranks of detection by Auramine O staining for positive sputum samples (r2=0.8062). And the sensitivity and specificity of the ELONA using aptamer T9was92.65%and77.45%respectively for serology testing of active pulmonary TB patients. Aptamer T9could also be used to distinguish the extra-pulmonary TB from healthy donors by detection of serum samples, and the sensitivity and specificity of the aptamer T9was90.32%and94.12%respectively. Interestingly, in this study,6young babies and children (from6months to8years old) of confirmed tuberculosis were shown as negative results by T-Spot, but were positive by ELONA (T9), which suggested that aptamer T9ELONA might increase the detection rates among babies and children, comparison with T-Spot.Protein glycosylation is an important modification pathway after translation and participates in various life activities, such as proliferation, apoptosis, migration, immunoregulation and so on. Lectins can specifically recognize and bind to glycans with different structures. According to the characteristics of lectins, a lectin microarray system was set up and used to analysis the glycans alteration of host after M.tb infection. By the use of lectin microarray, we performed the following researches:(1) we detected the alterations of glycoproteins and glycans of macrophage membrane following macrophages infected with virulence H37Rv strain and non-virulence H37Ra strain respectively. We found that the membrane glycoproteins of macrophage specifically bound to lectin ABA were increased in the H37Rv infected macrophages, compared to H37Ra infected cells. These results were confirmed in the human peripheral blood monocyte cells (PBMCs) that membrane glycoproteins specifically bound to lectin ABA were increased in TB patients compared to healthy donors. We further found that lectin ABA could block the H37Rv attachment and invading to macrophage. The ABA binding glycoproteins from macrophage membrane were identified by mass spectrum (MS) and the CD44and Glectin-9were identified as the two candidate molecules;(2) We also analyzed the glycoproteins in serum samples from healthy donors and patients by lectin microarray and the results showed that the serum glycoproteins of TB patients preferred to binding three lectins, EBL, MAL-I and ABA. The differential expression levels of serum glycoproteins in TB patients were then identified by Western blot, and the differential glycoproteins with molecular weight between10kD-15kD were subjected to mass spectrum (MS) analysis. Platelet factor4was identified by MS as one of biomarkers for TB patients.In conclusion, firstly, ssDNA aptamer T9was successfully generated by use of SELEX, with high binding affinity and specificity to ManLAM from Beijing genotype of M.tb strain. ELONA with T9aptamer was established to detect the ManLAM antigens in the serum and sputum samples from TB patients and this method provide a new strategy for TB antigen diagnosis; secondly, we successfully established lectin microarray system to detect the glycoproteins alterations of macrophage membranes and serum samples in TB patients, and the glycoproteins CD44and galactin-9were identified by ABA specific binding from macrophage membrane, and the platelet factor4was identified from TB serum samples. These glycoproteins might be used as new clinical glycoprotein diagnosis markers, which are worthy of further clinical validation.