Transposon Insertion Sequencing Elucidates Novel Gene Involvement in Susceptibility and Resistance to Phages T4 and T7 in Escherichia coli O157

Abstract

Experiments using bacteriophage (phage) to infect bacterial strains have helped define some basic genetic concepts in microbiology, but our understanding of the complexity of bacterium-phage interactions is still limited. As the global threat of antibiotic resistance continues to increase, phage therapy has reemerged as an attractive alternative or supplement to treating antibiotic-resistant bacterial infections. Further, the long-used method of phage typing to classify bacterial strains is being replaced by molecular genetic techniques. Thus, there is a growing need for a complete understanding of the precise molecular mechanisms underpinning phage-bacterium interactions to optimize phage therapy for the clinic as well as for retrospectively interpreting phage typing data on the molecular level. In this study, a genomics-based fitness assay (TraDIS) was used to identify all host genes involved in phage susceptibility and resistance for a T4 phage infecting Shiga-toxigenic Escherichia coli O157. The TraDIS results identified both established and previously unidentified genes involved in phage infection, and a subset were confirmed by site-directed mutagenesis and phenotypic testing of 14 T4 and 2 T7 phages. For the first time, the entire sap operon was implicated in phage susceptibility and, conversely, the stringent starvation protein A gene (sspA) was shown to provide phage resistance. Identifying genes involved in phage infection and replication should facilitate the selection of bespoke phage combinations to target specific bacterial pathogens.

High-throughput mapping of the phage resistance landscape inE. coli

“…For example, we used two laboratory E. coli strains that have rough LPS architecture where core oligosaccharide is the terminal part of the LPS. However, it is known that the genetic and structural diversity of LPS and the repeat structure O-polysaccharide attached to LPS (to form smooth LPS) is very large in pathogenic and environmental isolates of Enterobacteriaceae, and may impact phage infectivity [47,57,[199][200][201][202]. The genetic screens presented in this work may aid in filling the knowledge gap on phage interaction with different O-antigens and its impact on phage infectivity and resistance.…”

“…Any changes in levels or structure of these receptors can compromise efficient phage infection, thereby leading to an improvement in host fitness in the presence of phages. Consequently, receptor mutants are the most commonly found candidates that display phage resistance [48,49,52,57,87,89]. To confirm the validity of our approach, we looked for receptors recognized by many of the canonical phages used in this study for which there is published data available [49,50,[90][91][92][93][94][95][96].…”

“…There have been few attempts to use genetic approaches for studying genome-wide host factors essential in phage infection. These loss-of-function (LOF) genetic screens broadly use bacterial saturation mutagenesis [48,53,[57][58][59][60] or an arrayed library of single-gene deletion strains for studying phage-host interactions [49,50,52,61,62]. Consequently, these studies have generally involved laborious experiments on relatively few phages and their hosts, and scaling the approach to characterize hundreds of phages is challenging.…”

Discovering the Molecular Determinants ofPhaeobacter inhibenssusceptibility toPhaeobacterphage MD18

“…We sought to characterize the relationship between Phaeobacter phage MD18 and P. inhibens by identifying host gene products that confer susceptibility to infection. Recent work has used transposon insertion sequencing to rapidly perform reverse-genetic screens in hosts and identify genes that contribute to phage susceptibility (25,26). We selected for P. inhibens transposon insertion mutants with decreased susceptibility to phage by exposing a previously constructed, barcoded transposon mutant library (27) to phage MD18 ( Figure 3A).…”

The Genetic Basis of phage susceptibility, cross-resistance and host-range in Salmonella

“…Metagenomics and isolation studies from these complex samples have identified numerous Salmonella phages, but they often lack substantial characterization 13,[20][21][22] . Metabolic, stress-response, and regulatory hostfactor requirements beyond receptors are known for individual phages [23][24][25][26] , but have not yet entered the conversation for therapeutic design.…”