New structures reveal how phage enzymes attack tuberculosis bacteria

Mycobacteria cause major global illness, from tuberculosis and leprosy to infections by emerging and opportunistic species such as Mycobacterium abscessus. Treatments for these diseases are hard to develop and are increasingly challenged by antimicrobial resistance, creating an urgent need for new kinds of therapies. One promising avenue is the use of enzymes from bacteriophages — viruses that infect bacteria — called endolysins. Mycobacteriophage LysA endolysins are complex, multi-domain peptidoglycan hydrolases with reported antimicrobial relevance and the potential to treat mycobacterial infections. Despite that promise, how these enzymes actually bind to and break down the tough mycobacterial cell wall has been poorly understood. In a study led by corresponding author Inmaculada Pérez‐Dorado, researchers carried out a comprehensive structural and functional analysis of the catalytic parts of two LysA endolysins encoded by bacteriophages D29 and DS6A, which are known to infect pathogenic mycobacteria including Mycobacterium tuberculosis. The team characterized the four catalytic domains present in both endolysins — D29 N4/ D29 GH19 and DS6A GH19/ DS6A Ami2B — testing each domain alone and bound to PG analogues to map their activities.

To dissect how these catalytic domains work, the researchers combined protein engineering, X-ray crystallography, small-angle X-ray scattering, and in silico tools. They engineered and purified the individual hydrolase domains and then solved their three-dimensional arrangements using X-ray crystallography, complemented by small-angle X-ray scattering data that describe the proteins in solution, and computational analyses to interpret interactions with peptidoglycan. The study reports the first experimental structures for mycobacteriophage endolysins, providing direct visual information about how the catalytic domains are organized. By studying complexes with PG analogues, the team revealed key aspects of peptidoglycan binding and hydrolysis by D29 LysA and DS6A LysA lysins. These structures also illuminate features likely shared by other homologous LysAs and their hydrolase domains, offering a clearer picture of the molecular contacts and catalytic elements that allow these enzymes to break down mycobacterial cell walls.

The findings represent a significant step forward in understanding how mycobacterial cell-wall hydrolysis occurs by this important class of endolysins. With experimentally determined structures of catalytic domains in hand, researchers now have concrete templates to guide the design of improved enzymes. This structural information opens the door to exploring therapeutic applications of LysA enzymes as enzybiotics — enzyme-based antimicrobials — aimed at pathogens such as Mycobacterium tuberculosis, Mycobacterium leprae and Mycobacterium abscessus. Importantly, the work promises to enable rational, a la carte design of enzymes with optimized lytic properties against mycobacterial pathogens, moving from basic molecular understanding toward practical strategies for new treatments that could complement or replace existing drugs in the face of rising resistance.