Understanding and exploiting phage-host interactions for enhanced phage therapy
Hannah X Zhu 1,2, Shahla A Kangachar 3, Bradley W Wright 1,2, Dominic Y Logel 1,2, Ellina Trofimova 1,2, Karen D Weynberg 3, Mark P Molloy 4, Paul R Jaschke 1,2
- School of Natural Sciences, Macquarie University, Sydney, NSW, Australia
- ARC Centre of Excellence in Synthetic Biology, Macquarie University, Sydney, NSW, Australia
- Australian Centre for Ecogenomics, School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, QLD, Australia
- Kolling Institute, Northern Clinical School, The University of Sydney, Sydney, NSW, Australia
Antimicrobial resistance (AMR) presents a significant global health, economic, and social burden. With traditional antibiotics losing effectiveness against drug-resistant bacterial pathogens, phage therapy, which uses bacteriophages (phages) to treat bacterial infection, offers a promising alternative. The success of phage therapy relies on productive interactions between phages and their hosts. Thus, understanding these complex interactions remains an important goal, as it will enable us to select natural phages more efficiently for a given task, and engineer phages with enhanced capabilities. We present progress towards this goal through the investigation of three different model phage-host systems (Microviridae, Autographiviridae, and Mesyanzhinovviridae) and two diverse classes of genes (small heat shock proteins and anti-phage defence systems and their effect on phage susceptibility.
First, we determined the role of two small heat shock proteins (sHsps) – IbpA and IbpB – during Microviridae φX174 replication. sHsps are a family of molecular chaperones produced by bacterial cells under stress, which prevent the irreversible aggregation of proteins. IbpA and IbpB, a class of holding modulators or "holdases", bind partially folded proteins and await ATP-driven folding chaperones for refolding. These two proteins have recently been shown to be highly upregulated during φX174 infection of Escherichia coli C. Here, using a hybrid approach of CRISPR interference (CRISPRi) and genomic knockouts to disrupt the ibpA/B genes, we show that surprisingly, IbpA and IbpB appear nonessential for φX174 infection.
Next, we focused on uropathogenic E. coli (UPEC) as a model system to holistically investigate the role of host factors on phage susceptibility. We assembled a set of 35 multidrug-resistant (MDR) clinical UPEC strains from 15 sequence types and a panel of eight recently isolated UPEC phages. We experimentally determined the host-range of the phages and assessed their attachment efficiency. We found that most of the non-infection events could be attributed to failed adsorption, with the action of host defence systems likely accounting for the remaining cases. Next, we built a bioinformatics pipeline integrating PADLOC-DB, DefenseFinder, and CRISPRDetect to determine anti-phage defence systems involved in phage susceptibility. We identified 41 putative anti-phage defence systems in our UPEC collection. We found that one strain contained as many as 12 defence systems and was resistant to seven UPEC phages. To extend this work further and determine its generalisability across the UPEC strains, we identified 74 anti-phage defence systems in the genomes of 409 UPEC isolates from the Refseq database. We also compared the prevalence of defence systems in UPEC to Non-UPEC, and we discovered significant differences in several systems, such as CRISPR/Cas.
In parallel, we are further developing methods to clone phage into yeast to enable their manipulation and augmentation with heterologous genetic functions to expand their host-range. Examples of modified phages using yeast recombineering and OrthoRep in vivo continuous evolution system will be presented.
In summary, by leveraging the knowledge of phage-host interactions and applying genetic engineering approaches, we will optimise the therapeutic potential of phages, improving the overall efficacy of phage therapy.