Periodic Reporting for period 1 - BioPhage (Phage infection of bacterial biofilm)
Reporting period: 2023-01-01 to 2025-06-30
Antibiotic-resistant Staphylococcus aureus (S. aureus) infections pose a major health challenge, with biofilm-associated infections being complicated to treat. Biofilms protect bacterial cells from antibiotics and immune responses. Traditional antibiotic treatments are often ineffective, necessitating the search for new therapeutic strategies. One alternative is phage therapy, but its clinical application remains limited due to an incomplete understanding of how phages interact with biofilms and replicate in bacterial cells. Project BioPhage aims to fill this knowledge gap by visualizing the infection dynamics of Herelleviridae phage phi812 in S. aureus biofilms and determining the molecular mechanisms of phage replication in bacterial cells. Using a unique combination of light-sheet fluorescence microscopy (LSFM), correlative light-electron microscopy, and cryo-electron tomography (cryo-ET), we will: (i) Characterize phage infection dynamics in biofilms, identifying how biofilm structure, extracellular matrix, and metabolically dormant cells contribute to phage resistance and herd immunity. (ii) Determine molecular mechanisms of phage replication in individual bacterial cells, revealing key steps in phage genome ejection, assembly, and maturation.
This research will advance our fundamental understanding of phage-biofilm interactions and may provide insights for improving phage therapy. By identifying biofilm vulnerabilities and mechanisms that limit phage efficacy, our findings could inform strategies to enhance phage penetration and replication, increasing the therapeutic potential of phage-based treatments against antibiotic-resistant infections. Ultimately, this work contributes to the broader goal of developing novel, effective alternatives to antibiotics, addressing a critical public health need.
This research will advance our fundamental understanding of phage-biofilm interactions and may provide insights for improving phage therapy. By identifying biofilm vulnerabilities and mechanisms that limit phage efficacy, our findings could inform strategies to enhance phage penetration and replication, increasing the therapeutic potential of phage-based treatments against antibiotic-resistant infections. Ultimately, this work contributes to the broader goal of developing novel, effective alternatives to antibiotics, addressing a critical public health need.
The project focused on investigating the infection dynamics of Herelleviridae phage phi812 in Staphylococcus aureus biofilms and the molecular mechanisms of phage replication in bacterial cells. A combination of live-cell imaging, correlative electron microscopy, and in situ structural studies was used to address knowledge gaps in phage-biofilm interactions. A microfluidic system integrated with light-sheet fluorescence microscopy (LSFM) was developed to enable multi-day observation of biofilms. This approach allowed for tracking the progression of biofilm development. To study the replication cycle of phi812 within S. aureus cells, focused ion beam milling (FIBM) and cryo-electron tomography (cryo-ET) were used. This high-resolution structural approach enabled the visualization of key phage infection intermediates, including phage genome ejection, capsid assembly, DNA packaging, and particle formation. The study may provide insights into the role of bacterial membranes and scaffolding proteins in assembling phage heads and tails, contributing to a deeper understanding of the molecular mechanisms governing phage replication. These findings contribute to a broader effort to develop alternative strategies for combating antibiotic-resistant bacterial infections.
The project’s results have implications for fundamental microbiology research and the development of phage therapy against antibiotic-resistant Staphylococcus aureus infections. Structural characterization of phage phi812 provided insights into genome retention, ejection, and assembly, clarifying key molecular mechanisms governing phage replication. These findings enhance our understanding of phage-bacteria interactions and may inform strategies to improve the therapeutic use of phages. For further uptake, additional research is needed to optimize genetically modified phages to infect biofilms. Testing phi812’s efficacy in eradicating biofilms within infection models will be crucial for preclinical development. Regulatory approval and commercialization require standardization of phage production, intellectual property considerations, and collaboration with industry partners.