Dissecting the molecular basis underlying mycobacterial cell-wall hydrolysis by the catalytic domains of D29 LysA and DS6A LysA phage endolysins

Mycobacteria encompass a broad range of microorganisms that cause infections with a significant impact on human health, resulting in millions of deaths each year. From tuberculosis and leprosy, caused by Mycobacterium tuberculosis and Mycobacterium leprae , respectively, to infections caused by emerging/opportunistic pathogens such as Mycobacterium abscessus . The battle to combat this health burden is further challenged by limitations in the treatments currently available and the rise of antimicrobial resistance. This underscores the need for new therapeutic strategies to combat these infections. Mycobacteriophage LysA endolysins are complex, multi-domain peptidoglycan hydrolases with reported antimicrobial relevance and the potential to treat mycobacterial infections. However, despite the therapeutic prospects of LysAs, our understanding of their mechanism of action remains limited. This study provides a comprehensive structural-functional analysis of the catalytic domains of two LysA endolysins encoded by the bacteriophages D29 and DS6A , which are known to infect pathogenic mycobacteria, including M. tuberculosis . As part of this work, we have characterized the four catalytic domains present in both endolysins ( D29 N4/ D29 GH19 and DS6A GH19/ DS6A Ami2B) both alone and in complex with PG analogues. To achieve this, we combined protein engineering, X-ray crystallography, small-angle X-ray scattering, and in silico tools. To our knowledge, this has led to the first experimental structures reported for mycobacteriophage endolysins, which reveals key aspects of peptidoglycan binding and hydrolysis by D29 LysA and DS6A LysA lysins, as well as other homologous LysAs, including the hydrolase domains similar to those examined here. Altogether, this represents a significant step forward in understanding how mycobacterial cell-wall hydrolysis occurs by this important class of endolysins and opens the door to their future use in therapeutic applications as enzybiotics. Information that will allow the rational design of a la carte enzymes with optimized lytic properties against mycobacterial pathogens.