Therapeutic and diagnostic potential of endolysins targeting gram-positive pathogens
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Date
2024
Authors
Murray, Ellen
Journal Title
Journal ISSN
Volume Title
Publisher
University College Cork
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Abstract
Infection as a result of antibiotic resistant microbes (AMR) is a growing health concern, creating a need for the development of novel antimicrobial and diagnostic strategies. Bacteriophage-derived lytic enzymes, endolysins, could offer a promising additional weapon in the battle to overcome AMR. This thesis investigates the therapeutic and diagnostic potential of endolysins targeting gram-positive pathogens Cutibacterium acnes, Ruminococcus gnavus, Enterococcus faecium, and Clostridioides difficile. The research conducted focuses on several key objectives: the isolation of novel endolysins, the identification of their catalytic and cell wall binding domains, the characterisation of lytic activity, the assessment of resistance mechanisms and their potential for diagnostics.
Chapter 1 describes the advantages and challenges of using endolysins in a clinical setting. This review explores the potential of bacteriophage-derived endolysinsas novel antimicrobials. The advantages discussed here include specificity, synergistic effects with other antimicrobials and the ability to engineer endolysins for a specific gain. Endolysins have shown efficacy against several topical pathogens, including Methicillin-resistant Staphylococcus aureus (MRSA), Listeria monocytogenes and Pseudomonas aeruginosa. Challenges facing the future of endolysin therapy include regulatory hurdles, production and stability, and delivery methods.
C. acnes is a commensal skin bacterium that is associated with the skin condition acne vulgaris. Acne is estimated to affect 9.4% of the world’s population, making it the eighth most prevalent disease worldwide. Current treatment plans are inefficient and include the use of antibiotics and harsh topical treatments that are paired with undesirable side effects. In this thesis, six novel phages infecting C. acnes were isolated, and their genomes were sequenced. The endolysin genes of these phages were selected for their potential biotechnological applications. The six endolysin genes were determined to be identical. The endolysin LysCa5A2, derived from phage 005A2, was expressed. This study revealed that LysCa5A2 is a highly insoluble protein, and its expression in E. coli BL21(DE3) led to the formation of inclusion bodies. In Chapter 2, we describe the largely unsuccessful attempts made to recover a native bioactive protein.The human microbiome plays an important role in human health and disease. While assessing the potential of a new therapeutic where selectivity is often postulated as an important attribute, it is vital to ensure that the candidate drug is specifically targeting the microbe of choice.
In Chapter 3, we describe the isolation of LysH1 – a novel endolysin derived from an E. faecium phage. Structural analysis of this endolysin revealed a two-domain structure composed of a catalytic domain and a previously uncharacterised domain. Binding assays with a green fluorescent protein (GFP) fusion protein allowed us to functionally characterise this unknown domain as the cell wall binding domain of LysH1. While initially investigated for its potential to manage vancomycin-resistant enterococci (VRE), host range analysis showed that LysH1 also has cross-species activity against R. gnavus. To assess the therapeutic potential of LysH1, a simplified human intestinal microbiota (SIHUMI) was established. Here, we show that LysH1 can selectively target R. gnavus without impacting the other members of the consortium. Endolysin therapy is commended for many reasons, but one of particular note is the reported lack of resistance mechanisms.
In Chapter 4, we isolated endolysins that could target Ruminococcus gnavus, a commensal gut bacterium associated with inflammatory bowel disease (IBD). Five novel endolysins were successfully cloned and recombinantly expressed, and one was selected for further testing (LysIBDN1). LysIBDN1 initially demonstrated effective lytic activity against R. gnavus, but further analysis revealed the emergence of endolysin resistant (ER) mutants of R. gnavus. Genomic analysis revealed a series of SNPs, deletions, and insertions in the genomes of mutant strains, largely associated with the stringent stress response. Phenotypic analysis demonstrated significant fitness costs of resistance. This study provides
insights into the mechanisms of endolysin resistance.
The functional characterisation of a novel receptor binding domain from a Clostridiodes difficile bacteriophage tail-associated lysin is described in Chapter 5. C. difficile infection (CDI) is a significant nosocomial infection. To reduce the transmission of CDI in healthcare settings, it is important to be able to rapidly monitor C. difficile levels to inform treatment options and to control infection. In this study, we assess the potential of this receptor binding protein (CdRBP) as a means of identifying C. difficile. Functional analysis with a GFP-fusion protein revealed that the domain can effectively bind C. difficile strains. However, analysis with more complex samples revealed that CdRBP displays off-target binding.
This thesis contributes to the growing body of knowledge of endolysin therapy. Critical analysis of the endolysins described in this thesis indicates that, while some candidates show promise as therapeutics, we must be aware of challenges, including the emergence of resistance and off-target effects. Here, we offer insights into the specificity of binding domains, emphasise the need for microbiome-driven studies, and discuss potential resistance mechanisms and their consequences.
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Keywords
Endolysins , Bacteriophage , Antimicrobial resistance , Biotechnology , Microbiome
Citation
Murray, E. 2024. Therapeutic and diagnostic potential of endolysins targeting gram-positive pathogens. PhD Thesis, University College Cork.