Phage infection of bacterial biofilm

Descripción del proyecto

Uso de un enemigo natural de las bacterias para eliminar biopelículas resistentes aantibióticos

«Staphylococcus aureus» es una bacteria común que mucha gente tiene, sin saberlo, en la piel y las membranas mucosas. Por lo general, no provoca infecciones en la piel sana, pero las infecciones invasivas y enfermedades relacionadas, específicamente en entornos sanitarios, pueden ser graves e incluso mortales. Cuando se produce la infección, las bacterias producen una biopelícula que requiere un tratamiento con dosis elevadas de antibióticos a los cuales el patógeno se ha vuelto resistente en buena medida. El tratamiento con bacteriófagos, utilizando virus bacterianos para tratar las infecciones bacterianas, es una opción prometedora, pero requiere un conocimiento más profundo de los mecanismos subyacentes. El proyecto BioPhage, financiado con fondos europeos, analizará la propagación de los bacteriófagos en una biopelícula, la inmunidad de rebaño frente a la infección con bacteriófagos y la replicación de los bacteriófagos en las células en condiciones biológica y clínicamente relevantes.

Objetivo

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 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 the effects of phage infection on a biofilm are not sufficiently characterized. We propose to study the dynamics of the propagation of Herelleviridae phage phi812 in a 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 the 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 the fixation of biofilm segments for subsequent correlative imaging by serial block-face scanning electron microscopy to identify the 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 the 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. This proposal’s biological significance lies in its focus on the as-yet uncharacterized interactions of phages and bacteria under biologically and clinically relevant conditions. Our analyses of phage spread in a biofilm, herd immunity against phage infection, and phage replication in cells may identify approaches for making phage therapy more effective.