Genomic, Proteomic and Physiological Characterization of a T5-like Bacteriophage for Control of Shiga Toxin-Producing Escherichia coli O157:H7

Abstract

Despite multiple control measures, Escherichia coli O157:H7 (STEC O157:H7) continues to be responsible for many food borne outbreaks in North America and elsewhere. Bacteriophage therapy may prove useful for controlling this pathogen in the host, their environment and food. Bacteriophage vB_EcoS_AKFV33 (AKFV33), a T5-like phage of Siphoviridae lysed common phage types of STEC O157:H7 and not non-O157 E. coli . Moreover, STEC O157:H7 isolated from the same feedlot pen from which the phage was obtained, were highly susceptible to AKFV33. Adsorption rate constant and burst size were estimated to be 9.31×10 −9 ml/min and 350 PFU/infected cell, respectively. The genome of AKVF33 was 108,853 bp (38.95% G+C), containing 160 open reading frames (ORFs), 22 tRNA genes and 32 strong promoters recognized by host RNA polymerase. Of 12 ORFs without homologues to T5-like phages, 7 predicted novel proteins while others exhibited low identity (<60%) to proteins in the National Centre for Biotechnology Information database. AKVF33 also lacked the L-shaped tail fiber protein typical of T5, but was predicted to have tail fibers comprised of 2 novel proteins with low identity (37–41%) to tail fibers of E. coli phage phiEco32 of Podoviridae , a putative side tail fiber protein of a prophage from E. coli IAI39 and a conserved domain protein of E. coli MS196-1. The receptor-binding tail protein ( pb5 ) shared an overall identify of 29–72% to that of other T5-like phages, with no region coding for more than 6 amino acids in common. Proteomic analysis identified 4 structural proteins corresponding to the capsid, major tail, tail fiber and pore-forming tail tip ( pb2 ). The genome of AKFV33 lacked regions coding for known virulence factors, integration-related proteins or antibiotic resistance determinants. Phage AKFV33 is a unique, highly lytic STEC O157:H7-specific T5-like phage that may have considerable potential as a pre- and post-harvest biocontrol agent.

Insights into Bacteriophage T5 Structure from Analysis of Its Morphogenesis Genes and Protein Components

“…We report the first comprehensive description of T5 morphogenesis genes and the topology of their products in viral particles and assigned functions, and we highlight major new structural features of T5, which is the prototype of a large family of lytic siphophages infecting Gram-negative hosts (4)(5)(6)(7). New similarities between T5 and several families of Siphoviridae and Myoviridae are also identified, providing additional examples of the evolutionary connections between related viruses.…”

Salmonella Phages and Prophages: Genomics, Taxonomy, and Applied Aspects

“…Phages in this genus have collinear genomes, as well as high genome conservation (approx. 80 % of nucleotide identity) [ 51 ]. The pre-early region contains genes encoding host enzyme inhibition proteins, the early region contains genes encoding proteins involved in DNA replication, transcription, DNA metabolism, and cell lysis, and the late region contains genes encoding structural proteins (e.g., capsid, tail) [ 49 ].…”

“…Interestingly, T5-like-viruses bind reversible fi rst to the LPS, and then the different members of this genus bind irreversibly to different outer membrane proteins (e.g., FepA, FluA, BtuB) [ 50 -52 ]. The binding to different surface proteins could be related with the host range of these phages; while some of them infect E. coli and Salmonella (SPC35, EPS7), other are specifi c to E. coli O157:H7 (AKFV33) [ 51 ].…”

Identification of the ssDNA-binding protein of bacteriophage T5

“…In addition, the purely lytic life cycle, high burst rate, as well as complete absence of pathogenicity islands, antibiotic resistance genes, and integrases suggest that T5-like phages are promising candidates for the development of medical applications. 26,27 Together, these features may lead to a renewed interest in this classical but still relatively poorly characterized bacteriophage.…”