Browsing by Author "Webber, Paige"

Antibiotic-resistant bacteria present a severe threat to global health. The future treatment of common bacterial infections relies on the identification of novel antibiotics and targets in the present. One area of antimicrobial research is the study of bacteriophage (Petrovic Fabijan et al.) mechanisms and the identification of phage-derived antimicrobials. Sequenced phage genomes are largely (>70%) annotated as “hypothetical proteins of unknown function” (HPUFs) and investigation into HPUFs with a toxic effect on host bacteria (toxHPUFs) aims to reveal new antibacterial targets and antimicrobials. Next-generation sequencing and plating-based toxicity screening of Staphylococcus phage Stab21 HPUFs identified nine HPUFs that incurred toxicity to Escherichia coli. In this study, the tightly controlled tetracycline-inducible plasmid pRAB11N was used as a shuttle vector and verified the toxicity of five out of nine HPUFs to E. coli and revealed that no HPUFs caused toxicity to the Stab21 natural target and clinically relevant Staphylococcus aureus. These results suggest that screening for toxHPUFs should be carried out in closely related bacterial species or the phages’ natural host. The five toxHPUFs of E. coli were further characterised by protein function and structural predictions. Only one toxHPUF, g024, returned a reliable model with homology to Bacillus phage SPO1 homing endonuclease I-HmuI, yet the role of this DNase in bacterial host toxicity is still unknown. To determine the bacterial targets of the toxHPUFs, spontaneous toxin-insensitive mutants of the five toxHPUFs were investigated. For three toxHPUFs, the toxin insensitivity was ascribed to the elimination of the toxin-encoding gene. However, toxin-insensitive g172 and g187 sequences revealed mutations in the tetR gene of pRAB11N that led to the inability of tetracycline binding and thus no induction of gene expression and did not aid in identifying the bacterial targets of these toxHPUFs. This study highlights the experimental complexities of phage-derived antimicrobial research. It also maintains the value of this research strategy, with the verification of HPUFs with a toxic effect on E. coli and accompanied future studies of bacterial target determination having the potential to uncover novel antimicrobial mechanisms that can be exploited for therapeutic application.