Inhibition of Mycobacterium tuberculosis secretory serine protease blocks bacterial multiplication both in axenic culture and in human macrophages

To study the possible importance of mycobacterial ES-31 serine protease for bacterial cell growth, the effect of serine and metalloprotease inhibitors, anti-tubercular drugs such as isoniazid and anti-ES-31 antibody, was evaluated on mycobacterial ES-31 serine protease in vitro and on bacilli in axenic and macrophage cultures. Serine protease inhibitors such as pefabloc, 3,4 dichloroisocoumarin, phenyl methyl sulfonyl fluoride (PMSF) and metalloprotease inhibitors such as ethylene diamine tetracetic acid (EDTA) and 1,10 phenanthroline inhibited 65–92% serine protease activity in vitro. Isoniazid showed 95% inhibition on mycobacterial ES-31 serine protease. These inhibitors also showed decreased bacterial growth in axenic culture and inhibition was further confirmed by a decreased amount of ES-31 serine protease in culture filtrate. In human macrophage culture, highly inhibitory pefabloc, 1,10 phenanthroline and isoniazid inhibited infectivity of virulent as well as avirulent M. tuberculosis bacilli to macrophages. It was observed that addition of mycobacterial ES-31 serine protease to macrophage culture enhanced the entry of bacilli and their multiplication in human macrophages. However, the addition of anti-ES-31 serine protease antibody strongly inhibited the mycobacterial growth as observed by decreased CFU count, showing the importance of mycobacterial ES-31 serine protease for entry of bacilli and their multiplication.

[1]  J. Keane,et al.  Bystander Macrophage Apoptosis after Mycobacterium tuberculosis H37Ra Infection , 2007, Infection and Immunity.

[2]  Satish Kumar,et al.  A cocktail of affinity-purified antibodies reactive with diagnostically useful mycobacterial antigens ES-31, ES-43, and EST-6 for detecting the presence of Mycobacterium tuberculosis. , 2006, Diagnostic microbiology and infectious disease.

[3]  S. Gupta,et al.  Detection of free and immune-complexed serine protease and its antibody in TB patients with and without HIV co-infection. , 2005, The international journal of tuberculosis and lung disease : the official journal of the International Union against Tuberculosis and Lung Disease.

[4]  R. North,et al.  Immunity to tuberculosis. , 2003, Annual review of immunology.

[5]  N. C. Gey van Pittius,et al.  Mycosin-1, a subtilisin-like serine protease of Mycobacterium tuberculosis, is cell wall-associated and expressed during infection of macrophages , 2002, BMC Microbiology.

[6]  N. Mendelson,et al.  The mechanisms responsible for 2-dimensional pattern formation in bacterial macrofiber populations grown on solid surfaces: fiber joining and the creation of exclusion zones , 2002, BMC Microbiology.

[7]  S. Banerjee,et al.  Purification and characterization of a 31 kDa mycobacterial excretory-secretory antigenic protein with a diagnostic potential in pulmonary tuberculosis. , 2001, The Indian journal of chest diseases & allied sciences.

[8]  N. C. Gey van Pittius,et al.  The mycosins of Mycobacterium tuberculosis H37Rv: a family of subtilisin-like serine proteases. , 2000, Gene.

[9]  H. Marrakchi,et al.  InhA, a target of the antituberculous drug isoniazid, is involved in a mycobacterial fatty acid elongation system, FAS-II. , 2000, Microbiology.

[10]  M. Horwitz,et al.  An Inhibitor of Exported Mycobacterium tuberculosis Glutamine Synthetase Selectively Blocks the Growth of Pathogenic Mycobacteria in Axenic Culture and in Human Monocytes: Extracellular Proteins as Potential Novel Drug Targets , 1999, The Journal of experimental medicine.

[11]  P. Barnes,et al.  Growth of Virulent and AvirulentMycobacterium tuberculosis Strains in Human Macrophages , 1998, Infection and Immunity.

[12]  G. Besra,et al.  Role of the major antigen of Mycobacterium tuberculosis in cell wall biogenesis. , 1997, Science.

[13]  S. Zolla-Pazner,et al.  Human humoral responses to antigens of Mycobacterium tuberculosis: immunodominance of high-molecular-mass antigens , 1997, Clinical and diagnostic laboratory immunology.

[14]  H. Maeda.  Role of Microbial Proteases in Pathogenesis , 1996, Microbiology and immunology.

[15]  J. Potempa,et al.  Are bacterial proteinases pathogenic factors? , 1995, Trends in microbiology.

[16]  H. Maeda,et al.  Effects of protease inhibitors on growth of Serratia marcescens and Pseudomonas aeruginosa. , 1991, Microbial pathogenesis.

[17]  J. Bennedsen,et al.  Proteins released from Mycobacterium tuberculosis during growth , 1991, Infection and immunity.

[18]  J. Jenne,et al.  Growth Inhibition of Mycobacterium tuberculosis After Single-Pulsed Exposures to Streptomycin, Ethambutol, and Rifampin , 1970, Infection and immunity.

[19]  A. Lind,et al.  Studies on the precipitinogenic pattern of mycobacteria cultivated at various temperatures , 1970, Pneumonologie.

[20]  A. Gomashe,et al.  Isolation, characterisation and kinetic studies on SEVA TB ES-31 antigen, a metallo-serine protease of interest in serodiagnosis. , 2009, The Indian journal of tuberculosis.

[21]  B. C. Harinath,et al.  Peroxidase enzyme immunoassay for circulating SEVA TB ES-31Antigen in pulmonary tuberculosis sera , 2008 .

[22]  Swati Banerjee,et al.  Isolation ofMycobacterium Tuberculosis 31 kDa antigen protein of diagnostic interest from culture filtrate using anti-ES-31 antibody by affinity chromatography , 2008, Indian Journal of Clinical Biochemistry.

[23]  J. Palomino,et al.  Tuberculosis 2007; from basic science to patient care , 2007 .

[24]  F. Portaels,et al.  Drug resistance and drug resistance detection , 2007 .

[25]  S. Banerjee,et al.  Isolation and characterization of a 31 kDa mycobacterial antigen from tuberculous sera and its identification with in vitro released culture filtrate antigen of mtb H37Ra bacilli. , 2000, Scandinavian journal of infectious diseases.

[26]  P. Narang,et al.  INCREASED YIELD OF EXCRETORY-SECRETORY ANTIGEN WITH THYROXEVE SUPPLEMENT IN IN VITRO CULTURE OF TUBERCLE BACILLI* , 1997 .

[27]  Chaparas Sd.  Immunity in tuberculosis. , 1982, Bulletin of the World Health Organization.