In 2017, the World Health Organization declared Staphylococcus aureus to be an antibiotic-resistant pathogen for which new therapeutics are urgently needed. Upon infection, S. aureus forms biofilms that can only be treated by the long-term application of a combination of several antibiotics in high doses or the surgical removal of the infected tissues. An alternative approach, phage therapy, has not been approved for clinical use because phage infection is not sufficiently characterized.
We propose to study the dynamics of the propagation of Myoviridae phage phi812 in S. aureus biofilm and molecular details of phi812 replication in a cell. We integrated a microfluidic system into a light-sheet microscope to enable continuous multi-day observation of phage infection of a biofilm. We will determine how sub-populations of metabolically dormant or phage resistant cells in a biofilm provide herd immunity against phi812 infection. Our system enables fixation of segments of biofilm for subsequent correlative imaging by serial block-face scanning electron microscopy to identify interactions of phages with bacterial cells. We will use focused ion beam milling together with cryo-electron microscopy and tomography to determine high-resolution structures of previously uncharacterized phi812 replication and assembly intermediates in S. aureus cells. We will study the function of bacterial membranes and macromolecular complexes in initiation and completion of phage genome delivery, the assembly of phage portal complexes and heads, and the mechanisms of genome packaging and head-tail attachment.
The biological significance of this proposal lies in its focus on the as-of-yet uncharacterized interactions of phages and bacteria in biologically and clinically relevant conditions. Our analyses of phage spread in a biofilm, the herd immunity against phage infection, and phage replication in cells may identify approaches for making phage therapy more effective.

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