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Identification of Staphylococcus bacteriophage Stab21 toxic gene products using Escherichia coli as a host.

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Title: Identification of Staphylococcus bacteriophage Stab21 toxic gene products using Escherichia coli as a host.
Author(s): Nyhamar, Ellisiv
Contributor: University of Helsinki, Faculty of Agriculture and Forestry
Degree program: Master 's Programme in Microbiology and Microbial Biotechnology
Specialisation: no specialization
Language: English
Acceptance year: 2022
S. aureus infections are prominent worldwide, and with the rapid increase in antimicrobial resistant variants such as methicillin-resistant MRSA, the need for new treatment alternatives is imminent (Monaco et al., 2017). Lytic bacteriophages are continually evolving new methods for the destruction of bacterial cells while avoiding their defence mechanisms. Screening hypothetical proteins of unknown function (HPUFs) from bacteriophages for toxic activity against bacteria may provide new and potentially life-saving approaches to combat bacterial infections (Liu et al., 2004, Singh et al., 2019). The Stab21 phage of Staphylococcus is a recently described lytic phage with over 85 % of its open reading frames annotated as HPUFs (Oduor et al., 2019). The successful identification of potentially toxic gene products could facilitate the discovery of novel bacterial targets for the development of new antimicrobials. It could also provide treatment options to multi-drug resistant S. aureus caused infections where no effective drugs are currently available. To reduce unnecessary screening of phage particle associated yet poorly annotated proteins, total proteins of phage particle were previously identified by LC-MS. Similar studies have previously been performed with Yersinia phage fR1-RT and Klebsiella phage fHe-Kpn01, where a handful of toxic proteins were discovered (Mohanraj et al., 2019, Spruit et al., 2020). To accelerate the screening process, a next-generation sequencing (NGS) high-throughput screening method was further developed by Kasurinen et al. (2021). In this study, 96 true HPUFs were selected and screened for their bactericidal activity in E. coli using the NGS-based approach. Fourteen potentially bacteriotoxic Stab-21 gene products were identified through toxicity screening in E. coli. Of these, three had a particularly low ratio of isolated plasmid after transformation while having a significant number of reads over each joint sequence, indicating their potentially high toxicity. The three most promising candidates were the gene products of g008, g081c and g175 of the Stab21 bacteriophage.
Keyword(s): Bacteriophage antimicrobial resistance virology bacteriology toxicity screening

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