AUTHOR=Nair Gokul , Jain Vikas 

TITLE=Separation of Mycobacterium smegmatis From a Mixed Culture Using the Cell Wall Binding Domain of D29 Mycobacteriophage Endolysin

JOURNAL=Frontiers in Microbiology

VOLUME=11

YEAR=2020

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2020.01119

DOI=10.3389/fmicb.2020.01119

ISSN=1664-302X

ABSTRACT=<p>Pathological infection caused by <italic>Mycobacterium tuberculosis</italic> is still a major global health concern. Traditional diagnostic methods are time-consuming, less sensitive, and lack high specificity. Due to an increase in the pathogenic graph of mycobacterial infections especially in developing countries, there is an urgent requirement for a rapid, low cost, and highly sensitive diagnostic method. D29 mycobacteriophage, which is capable of infecting and killing <italic>M. tuberculosis</italic>, projects itself as a potential candidate for the development of novel diagnostic methods and phage therapy of mycobacterial infections. In our previous study, we showed that the cell wall binding domain [C-terminal domain (CTD)] located at the C-terminal end of the D29 mycobacteriophage LysA endolysin very selectively binds to the peptidoglycan (PG) of <italic>Mycobacterium smegmatis</italic> and <italic>M. tuberculosis</italic>. Here, by using <italic>M. smegmatis</italic> as model organism and by exploiting the PG binding ability of CTD, we have developed a method to isolate <italic>M. smegmatis</italic> cells from a mixed culture <italic>via</italic> magnetic separation. We show that green fluorescent protein (GFP)-tagged CTD (CTD-GFP) can bind to <italic>M. smegmatis</italic> cells <italic>in vitro</italic> after treatment with non-ionic detergent Triton X-100. Fluorescence-based assays show that CTD-GFP binding to <italic>M. smegmatis</italic> cells is highly specific and stable, and is not disrupted by an excess of either GFP or BSA. We further fused CTD with glutathione-S-transferase (GST) to generate CTD-GST protein and carried out an anti-GST antibody-mediated coating of CTD-GST on Dynabeads. This allowed us to perform successful magnetic separation of <italic>M. smegmatis</italic> from a mixed culture of bacteria having both Gram-negative and Gram-positive bacteria. Furthermore, the separated cells could be confirmed by a simple PCR. Thus our assay allows us to separate and identify <italic>M. smegmatis</italic> from a mixed culture.</p>