Ceballos-Zuniga, F. | bioRxiv Science

bioRxiv2025

Dissecting the molecular basis underlying mycobacterial cell-wall hydrolysis by the catalytic domains of D29LysA and DS6ALysA 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 (D29N4/D29GH19 and DS6AGH19/DS6AAmi2B) 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 D29LysA and DS6ALysA 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.

biochemistry↗

bioRxiv2024

New insights into the domain of unknown function DUF of EccC5, the pivotal ATPase providing the secretion driving force to the ESX5 secretion system

Type VII secretion (T7S) systems, also referred to as ESAT6 secretion (ESX) systems, are molecular machines that have gained great attention due to their implication in cell homeostasis and host pathogen interactions in mycobacteria. The latter include important human pathogens such as Mycobacterium tuberculosis (Mtb), the etiological cause of human tuberculosis and a pandemic accounting for more than 1 million deaths every year. The ESX5 system is exclusively found in slow-growing pathogenic mycobacteria, where it mediates the secretion of a large family of virulence factors, the PE and PPE proteins. The secretion driving force is provided by EccC5, a multidomain ATPase operating through four globular cytosolic domains, an N-terminal domain of unknown function (EccCDUF) and three FtsK/SpoIIIE ATPase domains. Recent structural and functional studies of ESX3 and ESX5 systems have revealed EccCDUF as an ATPase-like fold domain with potential ATPase activity, and whose functionality is essential for secretion. Here we report the crystal structure of MtbEccC5DUF domain at 2.05 [A] resolution, which unveils a nucleotide-free structure with degenerated cis-acting and trans-acting elements involved in ATP-binding and hydrolysis. Our crystallographic study, together with a biophysical assessment of MtbEccC5DUF interaction with ATP/Mg2+, supports the absence of ATPase activity proposed for this domain. We show that this degeneration is also present in DUF domains of other ESX and ESX-like systems, which are likely to exhibit poor or null ATPase activity. Moreover, and based on an in-silico model of MtbEccC5 N-terminal region, we propose that MtbEccC5DUF is a degenerated ATPase domain that may have retained the ability to hexamerise. Observations that call the attention on DUF domains as structural elements with potential implications in the opening and closure of the membrane pore during the secretion process.

microbiology↗