Co-infecting phages impede each other's entry into the cell

Bacteriophage lambda tunes its propensity to lysogenize based on the number of viral genome copies inside the infected cell. Viral self-counting is believed to serve as a way of inferring the abundance of available hosts in the environment. This interpretation is premised on an accurate mapping between the extracellular phage-to-bacteria ratio and the intracellular multiplicity of infection (MOI). However, here we show this premise to be untrue. By simultaneously labeling phage capsids and genomes, we find that, while the number of phages landing on each cell reliably samples the population ratio, the number of phages entering the cell does not. Single-cell infections, followed in a microfluidic device and interpreted using a stochastic model, reveal that the probability and rate of individual phage entries decrease with MOI. This decrease reflects an MOI-dependent perturbation to host physiology caused by phage landing, evidenced by compromised membrane integrity and loss of membrane potential. The dependence of phage entry dynamics on the surrounding medium is found to result in a strong impact of environmental conditions on the infection outcome, while the protracted entry of co-infecting phages increases the cell-to-cell variability in infection outcome at a given MOI. Our findings demonstrate the previously unappreciated role played by entry dynamics in determining the outcome of bacteriophage infection.