Enzymatic and immunological comparison of Mycobacterium tuberculosis and a clinical isolate of streptomyces.

Abstract

Tuberculosis is a bacterial infection that affects one-third of the global population. The pathogen responsible for the vast majority of these cases is Mycobacterium tuberculosis and the current vaccines are insufficiently effective. The current vaccine against Tuberculosis is the live bacille Calmette-Guérin (BCG) vaccine with efficacy varying between 0% and 60% depending on the population demographics. DNA, cellular fractions of the pathogen, and subunit vaccines failed to provide protection beyond what the BCG vaccine can provide. Streptomyces, phylogenetic relatives of the mycobacteria, have been suggested as heterologous systems to formulate new vaccines against Tuberculosis. The main research objective of this study is to establish a functional relationship between M. tuberculosis and a clinical isolate of streptomyces using enzymatic and immunological profiling. This clinical isolate was confirmed to be related to Streptomyces albus. Enzymatic profiling of the culture filtrate showed that out of a total of 19 enzyme activities investigated, eight were common between S. albus and M. tuberculosis. These were: alkaline phosphatase, esterase lipases (C8), lipase (C14), leucine arylamidase, valine arylamidase, acid phosphatase, naphthol-ASBI- phosphohydrolase, and B-glucosidase. Highest levels of acid phosphatase activity was found in the culture filtrate protein (CFP) fraction of S. albus cultured in media containing glycine as a nitrogen source but was highest in the cytoplasmic fractions of cells grown with nitrate as a nitrogen source. The opposite was true for alkaline phosphatase where the highest activity was detected in the media with asparagine as a nitrogen source. Alanine dehydrogenase, alcohol dehydrogenase, and catalase/peroxidase showed highest levels in the CFP fraction of the media supplemented with nitrate as the nitrogen source whereas it was highest in the cytoplasmic fraction of cells harvested from media with glycine as the nitrogen source. Gelatinase zymography showed that the cytoplasmic fraction of cells grown in Sauton’s media with ammonium chloride and nitrate as nitrogen sources contained the highest activities. The zymograms showed two distinct bands corresponding to approximately 120 kDa and 70 kDa and two minor bands at 48 kDa and 20 kDa. In the CFP fraction, one minor band was visible only in the medium with nitrate as a nitrogen source, corresponding to approximately 50 kDa in size. Additionally, seven monoclonal antibodies specific for seven distinct antigens of M. tuberculosis were used to screen for cross reactivity with the secretory fractions of S. albus. Of the seven antibodies, only one (F181-ID3-2) gave a positive reaction. This is a monoclonal antibody directed at a specific internal amino acid sequence in the secreted acid phosphatase of mycobacteria (SapM). This protein has a size of about 28kDa and is implicated in the pathogenesis of M. tuberculosis.