Separation of <i>Mycobacterium smegmatis</i> From a Mixed Culture Using the Cell Wall Binding Domain of D29 Mycobacteriophage Endolysin

Pathological infection caused by Mycobacterium tuberculosis 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 M. tuberculosis , 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 Mycobacterium smegmatis and M. tuberculosis . Here, by using M. smegmatis as model organism and by exploiting the PG binding ability of CTD, we have developed a method to isolate M. smegmatis cells from a mixed culture via magnetic separation. We show that green fluorescent protein (GFP)-tagged CTD (CTD-GFP) can bind to M. smegmatis cells in vitro after treatment with non-ionic detergent Triton X-100. Fluorescence-based assays show that CTD-GFP binding to M. smegmatis 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 M. smegmatis 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 M. smegmatis from a mixed culture.