Molecular dissection of phage endolysin: an interdomain interaction confers host specificity in Lysin A of Mycobacterium phage D29.

Mycobacterium tuberculosis has always been recognized as one of the most successful pathogens. Bacteriophages that attack and kill mycobacteria offer an alternate mechanism for the curtailment of this bacterium. Upon infection, mycobacteriophages produce lysins that catalyze cell wall peptidoglycan hydrolysis and mycolic acid layer breakdown of the host resulting in bacterial cell rupture and virus release. The ability to lyse bacterial cells make lysins extremely significant. We report here a detailed molecular dissection of the function and regulation of mycobacteriophage D29 Lysin A. Several truncated versions of Lysin A were constructed, and their activities were analyzed by zymography and by expressing them in both Escherichia coli and Mycobacterium smegmatis. Our experiments establish that Lysin A harbors two catalytically active domains, both of which show E. coli cell lysis upon their expression exclusively in the periplasmic space. However, the expression of only one of these domains and the full-length Lysin A caused M. smegmatis cell lysis. Interestingly, full-length protein remained inactive in E. coli periplasm. Our data suggest that the inactivity is ensued by a C-terminal domain that interacts with the N-terminal domain. This interaction was affirmed by surface plasmon resonance. Our experiments also demonstrate that the C-terminal domain of Lysin A selectively binds to M. tuberculosis and M. smegmatis peptidoglycans. Our methodology of studying E. coli cell lysis by Lysin A and its truncations after expressing these proteins in the bacterial periplasm with the help of signal peptide paves the way for a large scale identification and analysis of such proteins obtained from other bacteriophages.