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PreprintCited by 4Bacteriophages and microbial interactionsEnzyme Structure and FunctionTuberculosis Research and Epidemiology

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

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Ceballos-Zúñiga, Fernando, Gálvez-Larrosa, Laura, Muñoz, Inés G., Infantes, Lourdes, Pérez-Dorado, Inmaculada·2025·International Journal of Biological Macromolecules
DOI 10.1101/2025.01.22.634383

Preprint — published as 10.1016/j.ijbiomac.2025.148896 · Paper page

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Abstract

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.

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Metadata

Authors
Ceballos-Zúñiga, F., Gálvez-Larrosa, L., Muñoz, I. G., Infantes, L., & Pérez-Dorado, I.
Affiliations
  • Consejo Superior de Investigaciones Científicas (ES)
  • Department of Crystallography and Structural Biology, Institute of Physical Chemistry Blas Cabrera, Spanish Research Council, Serrano 119, 28006 Madrid, Spain
  • Spanish National Cancer Research Centre (ES)
ORCID
Venue
International Journal of Biological Macromolecules
Publisher
openRxiv
Published
2025-01-24
Volume
334
Issue
Pt 1
Pages
148896-148896
ISSN
0141-8130 (ISSN-L)
DOI
10.1101/2025.01.22.634383
OpenAlex
W4416009356
Cited by
4
References
1
Citations by year
2026: 4
FWCI
2.13
Type
journal article
Language
en
Open access
Licence
Locations
  • International Journal of Biological Macromolecules (publishedVersion, cc-by-nc)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • DIGITAL.CSIC (Spanish National Research Council (CSIC)) (acceptedVersion, other-oa)
Funders
  • Diamond Light Source
  • Comunidad de Madrid (2019-T1/BMD-14774)
  • European Commission
  • European Synchrotron Radiation Facility
  • Consejo Superior de Investigaciones Científicas (202380E208, MMT24-IQF-01, 2024ICT153)
Topics
  • Life Sciences › Immunology and Microbiology › Virology › Bacteriophages and microbial interactions
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Structural Biology › Enzyme Structure and Function
  • Health Sciences › Medicine › Infectious Diseases › Tuberculosis Research and Epidemiology
Keywords
peptidoglycan hydrolase, catalytic domain, substrate binding site, peptidoglycan recognition, conformational change, phage therapy, tuberculosis, antimicrobial resistance, mycobacteria
MeSH terms
Cell Wall, Hydrolysis, Models, Molecular, Mycobacteriophages, Mycobacterium, Endopeptidases, Peptidoglycan, Viral Proteins, Crystallography, X-Ray, Catalytic Domain

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CoChat. (2026). Citation check: Dissecting the molecular basis underlying mycobacterial cell-wall hydrolysis by the catalytic domains of D29 LysA and DS6A LysA phage endolysins. https://app.cochat.ai/papers/10.1101/2025.01.22.634383
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Ceballos-Zúñiga, F., Gálvez-Larrosa, L., Muñoz, I. G., Infantes, L., & Pérez-Dorado, I. (2025). Dissecting the molecular basis underlying mycobacterial cell-wall hydrolysis by the catalytic domains of D29 LysA and DS6A LysA phage endolysins. International Journal of Biological Macromolecules. https://doi.org/10.1101/2025.01.22.634383