In medical settings, biofilms cause devastating damage during chronic and acute infections; indeed, bacteria are often viewed as agents of human disease. However, bacteria themselves suffer from diseases, most notably in the form of viral pathogens termed bacteriophages, which are the most abundant replicating entities on Earth. Phage-biofilm encounters are undoubtedly common in the environment, but the mechanisms that determine the outcome of these encounters are unknown. Although there is a rich literature on the co-evolution of bacteria and their viruses, little is known about the interactions between biofilms and phages. While a number of studies have determined the outcome of biofilms exposed to phages, this work has not yet provided an understanding of the mechanisms that govern phage-biofilm interactions. To address this open question, we developed a method for visualizing infection and spread of lytic T7 phages in living Escherichia coli biofilms at single-cell resolution.
Figure: Bacteriophages, viral predators of bacteria, cannot enter bacterial biofilms if these biofilms contain curli amyloid fibers as an extracellular matrix component. Phages are shown in cyan, bacterial cells in yellow, and curli amyloid fibers are labeled in pink.
Biofilm size, matrix composition, internal architecture, and cellular physiology can vary dramatically during biofilm growth, so we hypothesized that phage susceptibility may vary as a function of biofilm developmental stage. To test this possibility, biofilms of varying ages – grown in microfluidic flow chambers – were exposed to a continuous influx of phages and imaged by confocal microscopy. We discovered that biofilms that had grown for 48 hours or less were rapidly eradicated as a result of phage exposure. By contrast, biofilms that had grown for 60 hours or more were collectively protected from phage-mediated killing. Phage resistance therefore depends on the developmental state of these bacterial biofilm communities.
To understand the mechanism by which this biofilm-specific phage resistance occurs, we investigated biofilms grown from a range of different matrix mutants and found that only mutants that lacked curli amyloid fibers were susceptible to phages, while other matrix mutations did not have an effect on phage protection. For resolving the detailed mechanisms by which curli fibers protect biofilms from phages, we constructed minimal synthetic biofilms composed of purified curli fibers and fluorescent microbeads with a size similar to bacterial cells. Remarkably, when beads were incubated with in vitro polymerized curli fibers, they spontaneously formed clusters embedded within the curli mesh, and each curli-embedded bead cluster – like a wild type biofilm – prevented the diffusion of phages into its inner volume. Electron microscopy revealed that curli fibers localize in the pore-space between beads and can directly capture phage virions, implying that even a sparse distribution of curli fibers in the pores is sufficient to prevent phage diffusion through biofilm pores, which is a collective cell level phage protection mechanism.
Separately, some of the deletion mutants of the individual biofilm matrix components permitted phage mobility into the biofilm, but did not allow for phage infection. We therefore hypothesized that curli fibers might additionally protect cells individually. Consistent with this idea, we found that cells that are completely covered by curli in shaking liquid cultures were protected from phage infection, while neighboring daughter cells that were not producing curli became infected and lysed.
Curli fibers therefore mediate protection of E. coli biofilms form phage predation via two separate mechanisms: the curli fibers fill the pores between cells and bind phages, which is a collective cell level protection mechanism. But curli fibers can also wrap around cells and provide a protection at the individual cell level. Investigating the general relationship between biofilm matrix production and biofilm-phage interactions, as well as modifying the diffusivity and infectivity of phages in biofilms, are therefore important directions for developing the next generation of therapeutic phages, for editing microbiomes, and for a fundamental understanding of phage-bacteria co-evolution in nature.
HFSP funding through a Career Development Award was important for establishing an image analysis software platform in Knut Drescher’s lab that is now used for analyzing all 3D biofilm microscopy data, including the data for this project.
Reference
Dynamics biofilm architecture confers individual and collective mechanisms of viral protection. Vidakovic L, Singh PK, Hartmann R, Nadell CD, Drescher K. Nature Microbiology 3, 26-31 (2018). doi:10.1038/s41564-017-0050-1.