Periodic Reporting for period 1 - PHAGE (Structure and dynamics of PHage Attachment and Genome Ejection (PHAGE))
Reporting period: 2023-08-01 to 2025-07-31
Antimicrobial resistance (AMR) is a growing public health crisis, with nearly 1 million deaths in 2019 and projections of 10 million annually by 2050. Two major AMR pathogens, Staphylococcus aureus and Pseudomonas aeruginosa, form persistent infections often associated with biofilms and medical devices. Phage therapy—using viruses that infect and kill bacteria—offers a promising alternative to antibiotics. However, its clinical use remains limited due to gaps in our understanding of how phages infect bacterial cells.
The PHAGE project aims to elucidate the mechanisms by which two phages, phi812 (S. aureus) and phiKZ (P. aeruginosa), bind to bacterial surfaces and eject their genomes. These phages have long, contractile tails, which undergo major structural changes during infection. The project combines structural biology with advanced imaging to study these processes at high resolution and in real time.
First, I will use cryo-electron microscopy to determine the structure of the phiKZ tail in its native and contracted states. This will reveal how receptor binding triggers tail contraction and genome delivery. Second, I will visualise and quantify genome ejection in vivo using cryo-STEM and holotomography. These techniques will allow, for the first time, the direct observation of single-phage infection events on bacterial cells.
PHAGE will advance fundamental knowledge of phage biology and provide insights essential for the rational design of phage therapies. The findings will support the EU’s One Health Action Plan against AMR and contribute to developing novel treatments for drug-resistant infections.
The PHAGE project aims to elucidate the mechanisms by which two phages, phi812 (S. aureus) and phiKZ (P. aeruginosa), bind to bacterial surfaces and eject their genomes. These phages have long, contractile tails, which undergo major structural changes during infection. The project combines structural biology with advanced imaging to study these processes at high resolution and in real time.
First, I will use cryo-electron microscopy to determine the structure of the phiKZ tail in its native and contracted states. This will reveal how receptor binding triggers tail contraction and genome delivery. Second, I will visualise and quantify genome ejection in vivo using cryo-STEM and holotomography. These techniques will allow, for the first time, the direct observation of single-phage infection events on bacterial cells.
PHAGE will advance fundamental knowledge of phage biology and provide insights essential for the rational design of phage therapies. The findings will support the EU’s One Health Action Plan against AMR and contribute to developing novel treatments for drug-resistant infections.
Phages phi812 and phiKZ were successfully produced and purified in sufficient quantity and quality for structural and imaging studies. Phi812 infection of S. aureus was monitored under the interferometric scattering (iSCAT) microscope. These preliminary observations revealed that tail contraction and genome ejection occur in under one second, suggesting a highly efficient infection mechanism.
For phiKZ, cryo-EM datasets were processed to reconstruct the tail and baseplate structure. The reconstruction reached sufficient resolution to identify and model seventeen different proteins with up to 36 copies per phage. The reconstruction reveals inter-subunit contacts critical for tail architecture and contraction. There are still part of the baseplate reconstruction maps that are not well resolved to model more proteins.
Initial attempts to induce phiKZ tail contraction included treatments with varying urea concentrations, temperatures, pH values, and incubation with purified LPS. While contraction occasionally occurred, it was often accompanied by baseplate detachment. Only incubation with outer membrane vesicles resulted in contracted tails with intact baseplates, making this the most promising approach for capturing the post-infection structural state.
For phiKZ, cryo-EM datasets were processed to reconstruct the tail and baseplate structure. The reconstruction reached sufficient resolution to identify and model seventeen different proteins with up to 36 copies per phage. The reconstruction reveals inter-subunit contacts critical for tail architecture and contraction. There are still part of the baseplate reconstruction maps that are not well resolved to model more proteins.
Initial attempts to induce phiKZ tail contraction included treatments with varying urea concentrations, temperatures, pH values, and incubation with purified LPS. While contraction occasionally occurred, it was often accompanied by baseplate detachment. Only incubation with outer membrane vesicles resulted in contracted tails with intact baseplates, making this the most promising approach for capturing the post-infection structural state.
The PHAGE project has produced the first reconstructions of the tail and baseplate of the jumbophage phiKZ. These structures provide critical insight into the infection machinery of these giant phages, which differ substantially from classical tailed phages in size, and replication strategy. By identifying 17 components of the baseplate-tail complex, this work lays the foundation for understanding how large, contractile tails operate in jumbophages and sets the stage for comparative studies across diverse phage taxa.
Preliminary experiments aimed at visualising genome ejection under near-native conditions, using a single-particle method developed in a collaborating lab, yielded initial observations of sub-second contraction events. Although the method could not be pursued further during this reporting period, these results underscore the need for dynamic approaches in studying phage infection processes at high temporal resolution.
Finally, the project reinforces the importance of basic science in enabling applied outcomes. Phage therapy cannot be scaled safely and effectively without a detailed molecular understanding of how phages recognise, penetrate, and hijack their hosts. These findings contribute essential knowledge that supports the rational selection, engineering, and regulation of therapeutic phages within the One Health framework.
Preliminary experiments aimed at visualising genome ejection under near-native conditions, using a single-particle method developed in a collaborating lab, yielded initial observations of sub-second contraction events. Although the method could not be pursued further during this reporting period, these results underscore the need for dynamic approaches in studying phage infection processes at high temporal resolution.
Finally, the project reinforces the importance of basic science in enabling applied outcomes. Phage therapy cannot be scaled safely and effectively without a detailed molecular understanding of how phages recognise, penetrate, and hijack their hosts. These findings contribute essential knowledge that supports the rational selection, engineering, and regulation of therapeutic phages within the One Health framework.