bioRxiv2019
Filamentous vibriophages represent a massive repertoire of virulence factors which can be transferred across species boundaries, leading to the emergence of deadly pathogens. All filamentous vibriophages that were characterized until today were isolated from human pathogens. Considering frequent horizontal gene transfer among vibrios, we predict that other environmental isolates, including non-human pathogens also carry filamentous phages, of which some may encode virulence factors. The aim of this study was to characterize the phage repertoire, consisting of prophages and filamentous phages, of a marine pathogen, Vibrio alginolyticus. To do so, we sequenced eight different V. alginolyticus strains, isolated from different pipefish and characterised their phage repertoire using a combination of morphological analyses and comparative genomics. We were able to identify a total of five novel phage regions (three different Caudovirales and two different Inoviridae), whereby only those two loci predicted to correspond to filamentous phages (family Inoviridae) represent actively replicating phages. Unique for this study was that all eight host strains, which were isolated from different eukaryotic hosts have identical bacteriophages, suggesting a clonal expansion of this strain after the phages had been acquired by a common ancestor. We further found that co-occurrence of two different filamentous phages leads to within-host competition resulting in reduced phage replication by one of the two phages. One of the two filamentous phages encoded two virulence genes (Ace and Zot), homologous to those encoded on the V. cholerae phage CTX{Phi}. The coverage of these zot-encoding phages correlated positively with virulence (measured in controlled infection experiments on the eukaryotic host), suggesting that this phages is an important virulence determinant. Impact statementMany bacteria of the genus Vibrio, such as V. cholerae or V. parahaemolyticus impose a strong threat to human health. Often, small viruses, known as filamentous phages encode virulence genes. Upon infecting a bacterial cell, these phages can transform a previously harmless bacterium into a deadly pathogen. While filamentous phages and their virulence factors are well-characterized for human pathogenic vibrios, filamentous phages of marine vibrios, pathogenic for a wide range of marine organisms, are predicted to carry virulence factors, but have so far not been characterized in depth. Using whole genome sequencing and comparative genomics of phages isolated from a marine fish pathogen V. alginolyticus, we show that also environmental strains harbour filamentous phages that carry virulence genes. These phages were most likely acquired from other vibrios by a process known as horizontal gene transfer. We found that these phages are identical across eight different pathogenic V. alginolyticus strains, suggesting that they have been acquired by a common ancestor before a clonal expansion of this ecotype took place. The phages characterized in this study have not been described before and are unique for the Kiel V. alginolyticus ecotype. Data SummaryO_LIThe GenBank accession numbers for all genomic sequence data analysed in the present study can be found in Table S1. C_LIO_LIAll phage regions identified by PHASTER analysis of each chromosome and the respective coverage of active phage loci are listed in Table S2. C_LIO_LIGenBank files were deposited at NCBI for the two actively replicating filamentous phages VALG{Phi}6 (Accession number: MN719123) and VALG{Phi}8 (Accession number: MN690600) C_LIO_LIThe virulence data from the infection experiments have been deposited at PANGAEA: Accession number will be provided upon acceptance of the manuscript. C_LI Data statementAll supporting data have been provided within the article or through supplementary data files. Four supplementary tables and six supplementary figures are available with the online version of this article.