Amit Rahi1, Sumit Kumar Matta2, Alisha Dhiman3, Jaishree Garhyan4, Monisha Gopalani5, Subhash Chandra6, Rakesh Bhatnagar7. 1. Laboratory of Molecular Biology and Genetic Engineering, School of Biotechnology, Jawaharlal Nehru University, New Delhi, India. Electronic address: amitrahi84@gmail.com. 2. Laboratory of Molecular Biology and Genetic Engineering, School of Biotechnology, Jawaharlal Nehru University, New Delhi, India. Electronic address: sumitmatta@yahoo.co.in. 3. Laboratory of Molecular Biology and Genetic Engineering, School of Biotechnology, Jawaharlal Nehru University, New Delhi, India. Electronic address: alisha.dhiman1986@gmail.com. 4. Laboratory of Molecular Biology and Genetic Engineering, School of Biotechnology, Jawaharlal Nehru University, New Delhi, India. Electronic address: jgarhyan@gmail.com. 5. Laboratory of Molecular Biology and Genetic Engineering, School of Biotechnology, Jawaharlal Nehru University, New Delhi, India. Electronic address: monishasbt@gmail.com. 6. Laboratory of Molecular Biology and Genetic Engineering, School of Biotechnology, Jawaharlal Nehru University, New Delhi, India. Electronic address: scjnu@yahoo.co.in. 7. Laboratory of Molecular Biology and Genetic Engineering, School of Biotechnology, Jawaharlal Nehru University, New Delhi, India. Electronic address: rakeshbhatnagar@jnu.ac.in.
Abstract
BACKGROUND: Enolase, a glycolytic enzyme, has long been studied as an anchorless protein present on the surface of many pathogenic bacteria that aids in tissue remodeling and invasion by binding to host plasminogen. METHODS: Anti-Mtb enolase antibodies in human sera were detected using ELISA. Immunoelectron microscopy, immunofluorescence microscopy and flow cytometry were used to show surface localization of Mtb enolase. SPR was used to determine the affinity of enolase-plasminogen interaction. Plasmin formation upon plasminogen binding to enolase and Mtb surface was measured by ELISA. Mice challenge and histopathological studies were undertaken to determine the protective efficacy of enolase immunization. RESULTS: Enolase of Mtb is present on its surface and binds human plasminogen with high affinity. There was an average of 2-fold increase in antibody mediated recognition of Mtb enolase in human sera from TB patients with an active disease over control individuals. Substitution of C-terminal lysine to alanine in rEno decreased its binding affinity with human plasminogen by >2-folds. Enolase bound plasminogen showed urokinase mediated conversion into plasmin. Binding of plasminogen to the surface of Mtb and its conversion into fibrinolytic plasmin was significantly reduced in the presence of anti-rEno antibodies. Immunization with rEno also led to a significant decrease in lung CFU counts of mice upon infection with Mtb H37Rv. CONCLUSIONS: Mtb enolase is a surface exposed plasminogen binding protein which upon immunization confers significant protection against Mtb challenge. GENERAL SIGNIFICANCE: Plasminogen binding has been recognized for Mtb, however, proteins involved have not been characterized. We show here that Mtb enolase is a moonlighting plasminogen binding protein. Copyright Â
BACKGROUND: Enolase, a glycolytic enzyme, has long been studied as an anchorless protein present on the surface of many pathogenic bacteria that aids in tissue remodeling and invasion by binding to host plasminogen. METHODS: Anti-Mtb enolase antibodies in human sera were detected using ELISA. Immunoelectron microscopy, immunofluorescence microscopy and flow cytometry were used to show surface localization of Mtb enolase. SPR was used to determine the affinity of enolase-plasminogen interaction. Plasmin formation upon plasminogen binding to enolase and Mtb surface was measured by ELISA. Mice challenge and histopathological studies were undertaken to determine the protective efficacy of enolase immunization. RESULTS: Enolase of Mtb is present on its surface and binds human plasminogen with high affinity. There was an average of 2-fold increase in antibody mediated recognition of Mtb enolase in human sera from TBpatients with an active disease over control individuals. Substitution of C-terminal lysine to alanine in rEno decreased its binding affinity with human plasminogen by >2-folds. Enolase bound plasminogen showed urokinase mediated conversion into plasmin. Binding of plasminogen to the surface of Mtb and its conversion into fibrinolytic plasmin was significantly reduced in the presence of anti-rEno antibodies. Immunization with rEno also led to a significant decrease in lung CFU counts of mice upon infection with MtbH37Rv. CONCLUSIONS:Mtb enolase is a surface exposed plasminogen binding protein which upon immunization confers significant protection against Mtb challenge. GENERAL SIGNIFICANCE: Plasminogen binding has been recognized for Mtb, however, proteins involved have not been characterized. We show here that Mtb enolase is a moonlighting plasminogen binding protein. Copyright Â
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