Vibrio cholerae is a major human pathogen and the causative agent of the life-threatening diarrheal disease, Cholera. V. cholerae"s lifestyle is tightly linked to bacteriophages (or short phages), which are viruses that infect bacteria. Unlike lysogenic phages, such as phage CTXφ, which has a major impact in the development of toxigenic V. cholerae strains, lytic phages control cholera outbreaks by diminishing V. cholerae populations in the environment. One of these lytic phages, the vibriophage N4 (Podoviridae family), is able to infect and lyse various V. cholerae variants, however, the underlying molecular mechanisms are currently unknown.

In this project, we identified and characterized host factors required for N4 infection of V. cholerae, as well as defense mechanisms that inhibited this process. To this end, we employed a recently developed library of synthetic sRNA variants that we harnessed to screen for phage-resistance. Indeed, using high-throughput sequencing, we discovered and validated several sRNAs providing phage-resistance. Follow up experiments identified resistance mechanisms that either inhibited phage adsorption or interfered with different functions of the phage infection process.

Detailed characterization of the interactions of a highly abundant sRNA variant with its respective target transcripts revealed a role of the V. cholerae"s O-antigen lipopolysaccharide (LPS O-antigen) in the N4 phage adsorption process. Additionally, the resistance provided from other sRNA variants indicate their involvement in downstream regulatory processes occurring after phage DNA entry in the cell. Our data demonstrates that synthetic sRNAs are a powerful tool to characterize phage-host interactions and the mechanisms underlying phage resistance.