RESUMO
Bacteriophages ΦeiAU and ΦeiDWF are lytic to the catfish pathogen Edwardsiella (Edw.) ictaluri. The Edw. ictaluri host factors that modulate phage-host interactions have not been described previously. This study identified eleven unique Edw. ictaluri host factors essential for phage infection by screening a transposon mutagenized library of two Edw. ictaluri strains for phage-resistant mutants. Two mutants were isolated with independent insertions in the ompLC gene that encodes a putative outer membrane porin. Phage binding and efficiency of plaquing assays with Edw. ictaluri EILO, its ompLC mutant and a complemented mutant demonstrated that OmpLC serves as a receptor for phage ΦeiAU and ΦeiDWF adsorption. Comparison of translated OmpLCs from 15 Edw. ictaluri strains with varying degrees of phage susceptibility revealed that amino acid variations were clustered on the predicted extracellular loop 8 of OmpLC. Deletion of loop 8 of OmpLC completely abolished phage infectivity in Edw. ictaluri. Site-directed mutagenesis and transfer of modified ompLC genes to complement the ompLC mutants demonstrated that changes in ompLC sequences affect the degree of phage susceptibility. Furthermore, Edw. ictaluri strain Alg-08-183 was observed to be resistant to ΦeiAU, but phage progeny could be produced if phage DNA was electroporated into this strain. A host-range mutant of ΦeiAU, ΦeiAU-183, was isolated that was capable of infecting strain Alg-08-183 by using OmpLC as a receptor for adsorption. The results of this study identified Edw. ictaluri host factors required for phage infection and indicated that OmpLC is a principal molecular determinant of phage susceptibility in this pathogen.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Bacteriófagos/fisiologia , Peixes-Gato/microbiologia , Edwardsiella ictaluri/metabolismo , Edwardsiella ictaluri/virologia , Doenças dos Peixes/microbiologia , Especificidade de Hospedeiro , Porinas/metabolismo , Sequência de Aminoácidos , Animais , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Bacteriófagos/genética , Edwardsiella ictaluri/química , Edwardsiella ictaluri/genética , Dados de Sequência Molecular , Porinas/química , Porinas/genética , Alinhamento de SequênciaRESUMO
BACKGROUND: The bacterial pathogen Edwardsiella ictaluri is a primary cause of mortality in channel catfish raised commercially in aquaculture farms. Additional treatment and diagnostic regimes are needed for this enteric pathogen, motivating the discovery and characterization of bacteriophages specific to E. ictaluri. RESULTS: The genomes of three Edwardsiella ictaluri-specific bacteriophages isolated from geographically distant aquaculture ponds, at different times, were sequenced and analyzed. The genomes for phages eiAU, eiDWF, and eiMSLS are 42.80 kbp, 42.12 kbp, and 42.69 kbp, respectively, and are greater than 95% identical to each other at the nucleotide level. Nucleotide differences were mostly observed in non-coding regions and in structural proteins, with significant variability in the sequences of putative tail fiber proteins. The genome organization of these phages exhibit a pattern shared by other Siphoviridae. CONCLUSIONS: These E. ictaluri-specific phage genomes reveal considerable conservation of genomic architecture and sequence identity, even with considerable temporal and spatial divergence in their isolation. Their genomic homogeneity is similarly observed among E. ictaluri bacterial isolates. The genomic analysis of these phages supports the conclusion that these are virulent phages, lacking the capacity for lysogeny or expression of virulence genes. This study contributes to our knowledge of phage genomic diversity and facilitates studies on the diagnostic and therapeutic applications of these phages.
Assuntos
Bacteriófagos/classificação , Bacteriófagos/genética , Biologia Computacional , Edwardsiella ictaluri/virologia , Genoma Viral/genética , Filogenia , Animais , Ictaluridae/microbiologia , Fases de Leitura Aberta , Alinhamento de Sequência , Proteínas Virais/genéticaRESUMO
AIMS: To identify and characterize bacteriophages specific for Edwardsiella ictaluri, the causative agent for enteric septicemia of catfish (ESC). METHODS AND RESULTS: Two bacteriophages were isolated that infect Edw. ictaluri. They both produce clear plaques, have icosahedral heads with a non-rigid tail, and are tentatively classified as Siphoviridae. Phages PhieiDWF and PhieiAU are dsDNA viruses with approximate genome sizes of 40 and 45 kb, respectively. The addition of 500 micromol l(-1) CaCl(2) enhanced phage titres. Both phages have a latent period of 40 min and an estimated burst size of 270. Every Edw. ictaluri strain tested was susceptible to phage infection with variable plaquing efficiencies and with no evidence of lysogeny, with no plaques detected on other bacterial species. CONCLUSIONS: Two unique bacteriophages were isolated that show host-specificity for Edw. ictaluri, have temperature and metal cation-dependent infectivity, and are tentatively placed within the family Siphoviridae. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of bacteriophages specific to Edw. ictaluri, an important fish pathogen affecting farm-raised channel catfish. Initial characterization of these bacteriophages has demonstrated their potential use as biotherapeutic and diagnostic agents associated with ESC.
