Mycobacterial excretory secretory-31 protein with serine protease and lipase activities: An immunogen and drug target against tuberculosis infection

Abstract

Objective/background: Tuberculosis (TB) has been declared as a global emergency by the World Health Organization in 1993 and still remains one of the world's biggest threats. Worldwide, 9.6 million people have been estimated to have fallen ill with TB in 2014: 5.4 million men, 3.2 million women, and 1.0 million children. To reduce this burden, detection and treatment gaps must be addressed and new tools developed (Global TB report 2015). Methods: Seroreactivity of the purified excretory secretory (ES) antigens ES-31, ES-43, ES-41, and ES-6 have been assessed in pulmonary TB (fresh, relapse, chronic, and latent), extrapulmonary TB, and in human immunodeficiency virus-TB coinfection. Results: Analysis of immune response to these purified antigens by indirect and sandwich enzyme-linked immunosorbent assay (ELISA) using sensitive penicillinase enzyme-immuno assay, showed ES-31 antigen as having good diagnostic potential in pulmonary TB and in certain groups of extrapulmonary TB, in particular tuberculous lymphadenopathy, tuberculous meningitis, whereas ES-41 was found to be more seroreactive in abdominal and bone and joint TB. ES-43 antigen was primarily recognized by serum antibodies in relapse cases, while ES-6 was useful in contacts. Antigen assay was found to be more sensitive than antibody-based assay for detecting TB with human immunodeficiency virus coinfection. Immunomonitoring for the presence of antigens in TB patients under antitubercular treatment showed that ES-31 antigen assay was useful in determining the effectiveness of therapy and the patient's compliance. User-friendly peroxidase ELISA has been standardized for the detection of circulating mycobacterial ES-31 serine protease (free antigen and immune-complexed antigen) with 70–75% sensitivity and 90% specificity and with a limit of detection of antigen at 1 ng/2 μL (0.5 μg/mL serum). In-house developed SEVA TB ELISA assay using a cocktail of antigens (ES-31+EST-6) and a cocktail of specific antibodies is being routinely done for screening of patients suspected of TB attending Kasturba Hospital—a tertiary health care center. The characterization of ES-31 antigen protein showed that ES-31 is a 31-kDa protein antigen with zinc containing serine protease as well as lipase activities and shown to be a chymotrypsin-like protein which has the catalytic triad responsible for both the activities. Addition of serine protease inhibitors: (1) pefabloc; (2) 3,4 dichloroisocoumarin; (3) phenyl methyl sulfonyl fluoride (53–76%); and metallo-protease inhibitors: (1) EDTA; (2) 1,10 phenanthroline (46–61%), lipase inhibitor, orlistat (61%), or anti-ES-31 serine protease antibody (89%) inhibited the Mycobacterium tuberculosis (MTB) H37Ra growth in axenic culture which is further confirmed by a decreased amount of ES-31 protein secreted in the culture filtrate. The importance of excretory secretory ES-31 protein for the survival of MTB H37Ra and H37Rv bacilli has been shown by 77% and 78% growth inhibition in macrophage culture by protease inhibitor pefabloc and was further confirmed by the enhancement of growth of TB bacilli in the presence of ES-31. Conclusion: Inhibition of ES-31 leads to the growth inhibition of MTB bacilli, suggesting that ES-31 is important for entry and multiplication of bacilli and an important drug target for exploring new drugs for TB based on protease and lipase activities of ES-31 protein.

Keywords

• Drug target
• ES-31
• Excretory secretory protein antigens
• Lipase
• Serine protease