Browsing by Author "Alrafaie, Alhassan"

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    Bacteriophage and Tail associated lysins of enterococci
    (Saudi Digital Library, 2023-10-10) Alrafaie, Alhassan; Stafford, Graham; Shepherd, Joey
    Enterococcal species especially E. faecalis and E. faecium are among the most commonly isolated pathogens that can resist several classes of antibiotics. To mitigate the issue of AMR, bacterial viruses (phages) can be used to infect and kill these pathogens. The first step in this process involves adsorption to specific host receptors followed by genome ejection into the host. To achieve this step, specific phage proteins named Tail-associated lysins (TAL) facilitate phage infection to their hosts via locally degrading bacterial layers. Therefore, we first aimed to bioinformatically investigate the TALs in 506 enterococcal phage and prophage genomes. Prophage genomes were identified using the PHASTER web server. The identification of TALs was carried out using Pfam, NCBI domain database and PHYRE2. Using these tools, we identified various TALS: Endopeptidase, lytic transglycosylase, Pectinesterase, New Lipoprotein C/Protein of 60-kDa (NLPC/P60) and Glycerophosphodiester phosphodiesterase (GPDP). The most common TAL identified in both phage and prophage genomes was endopeptidase followed by lytic transglycosylase. The identified TALs have different targets with endopeptidase and lytic transglycosylase targeting the bacterial peptidoglycan structure, GPDP degrading teichoic acids and pectinesterase possibly attacking enterococcal polysaccharide antigen (EPA). Interestingly, the identified NLPC/P60 proteins were correlated to the phage genomic classifications and host specificity. Phages are found almost everywhere and they have been isolated from several sources such as wastewater. Therefore, we collected and processed wastewater samples in our lab for phage isolation. To assess these samples, TEM was used to visualise various phage morphologies. Using these samples, 8 phages were isolated that E. faecalis and E. faecium strains in enrichment experiments, including clinical strains isolated from patients with diabetic foot ulcers. Following isolation, phage morphology was assessed via TEM with sipho-, myo- and podovirus phages observed. Since phages are very specific in terms of infecting bacteria, we have tested the ability of the isolated phages to infect 36 E. faecalis and E. faecium strains. A genomic analysis was then performed which revealed 5 novel strictly lytic phages. Since phiSHEF13 showed the broadest host range, we aimed to further characterise this phage. An experiment to assess phage host receptors was carried out which revealed that the variable region (epaV) of the E. faecalis strain V583 is essential for phiSHEF13 successful infection. As three different phages were isolated using the E. faecium E1071 strains, killing assays were conducted which revealed quick bacterial inhibition at different MOI. Phage-resistant mutants were also observed and investigated. The last chapter in this thesis is about assessing TALs In vitro by cloning and expressing candidate proteins. First, we selected one example from each identified lysin (a total of five proteins) and the candidate proteins were subcloned into vectors possessing His or GST tags and expressed using BL21(DE3) or C41(DE3). After expression, two TAL proteins namely pectinesterase and NLPC/P60 were successfully expressed and purified. To conclude, the isolation of novel lytic phages would facilitate phage therapy targeting antibiotic-resistant enterococci. Moreover, the identification of various lytic proteins would also provide alternative use of phage to tackle antibiotic-resistant bacteria.