Study On The Genome Of Yersinia Pestis Phage Yep-phi And Identification Of Its Receptors

Yersinia pestis, the causative agent of plague, belongs to the familyEnterobacteriaceae genus Yersinia. Yersinia pestis has killed hundreds of millions ofpeople in the three major historical plague pandemics. The World HealthOrganization (WHO) has classified plague as a re-emerging infectious disease in2000.Y. pestis has gained much attention as a possible biowarfare agent that could bemisused by bioterrorists. Plague still poses a significant threat to human health. Thesepticaemic and pneumonic forms are always lethal if untreated in time. The use ofantibiotics such as streptomycin and tetracycline for treating plague has beenembraced by the World Health Organization Expert Committee on Plague as the ’goldstandard’ treatment scheme. However, concerns regarding the development ofantibiotic-resistant Y. pestis strains have led to the exploration of alternatives toantibiotics. In1995, multiple drug-resistant strains of Y. pestis have been isolatedfrom patients with bubonic plague. One of them carried genes of high-level resistanceto12antibiotics, including virtually all of the drugs commonly used for plagueprophylaxis and treatment. Y. pestis phages play an important role in clinicaldiagnosis and epidemiological investigation due to their high specificity andsensitivity to its host. With the emergence of multiple drug-resistant Yersinia pestis,phages could be used as alternatives to antibiotics for the prevention and treatment ofplague.Yep-phi, a lytic phage of Y. pestis and a member of the T7family, was isolatedin China and is routinely used as a diagnostic phage for the identification of theplague pathogen. Yep-phi infects Y. pestis grown at both20°C and37°C, and isinactive in other Yersinia species irrespective of the growth temperature. Yep-phi hasan isometric hexagonal head containing dsDNA and a short non-contractile conicaltail. In this study, we sequenced the Yep-phi genome (GenBank accession no.HQ333270) and performed proteomics analysis. The genome consists of38,611bp ofDNA, including direct terminal repeats of217bp, and is predicted to contain45ORFs.The G+C content of the Yep-phi genome is47.12%, similar to that (47.6%) of itshost Y. pestis. Compared with the three available genome sequences of lytic phagesfor Y. pestis, the phages could be divided into two subgroups. Yep-phi displaysmarked homology to the bacteriophages Berlin (GenBank accession no. AM183667)and Yepe2(GenBank accession no. EU734170), and these comprise one subgroup.The other subgroup is represented by bacteriophage PhiA1122(GenBank accession no. AY247822). Homologous recombination is a powerful source of geneticvariability and a potent evolutionary mechanism for closely related bacteriophages.Recombination analysis detected four potential recombination events in the Yep-phisubgroup.Phage’s host specificity depends largely on the recognition and adsorption of thephage to its host surface receptors. Therefore, it is critical to understand the process ofphage lysis by exploring the mechanism of phage-receptor interactions. The receptorsof eight Y. pestis phages have been identified (L-413C, P2vir1, PhiJA1, PhiA1122,Pokrovskaya, T7Yp, Y and PST). All these phages employ different components oflipopolysaccharide (LPS) as a specific receptor. As far as we know, all closely relatedT7-like phages (T7, T3, PhiA1122and PhiYeO3-12) utilize the LPS core as theirreceptors.In this study, the phage receptor-binding protein and its receptors were identifiedfor Y. pestis diagnostic phage Yep-phi. Based on phage adsorption, phage plaqueformation, affinity chromatography and Western Blotting assays, the outer membraneproteins of Y. pestis, namely, Ail and OmpF, were identified to act as the receptors ofYep-phi in addition to the rough lipopolysaccharide. The plaguing efficiencies ofphage were evaluated on Yersinia pestis mutants of deleting different potentialreceptor genes singly or in different combinations and their trans-complementedstrains; and the necessary domains responsible for the interactions were identified andvalidated for phage tail fiber protein. The510-529residues of the distal portion of thephage tail fiber protein were sufficient in allowing binding to the OMP receptors invitro. Residues518N,519N, and523S of this protein are essential for the interactionwith OmpF, whereas residues518N,519N,522C, and523S are essential for theinteraction with Ail. This study is the first to demonstrate membrane-bound proteinsto act as receptors for a T7-related bacteriophage. Yep-phi has a unique hostspecificity that can be attributed to its tail fiber gp17protein. The observationshighlight the importance of the tail fiber protein in the evolution and function ofvarious complex phage systems. These findings are critical in developing phage-basedtherapeutic strategy to treat Y. pestis infection and diagnostic agent for identifying Y.pestis, and in providing insights into phage-bacterium interaction.