(Co-)Evolution between Vibrio spp. and temperate Phages alter Phage Host Range as well as Bacteria Virulence

Field: Immunology and Microbiology (Life Sciences)

The antagonistic coevolution between hosts and parasite can create strong selective forces that may be responsible for an increase in parasite virulence and host immune defense. While such antagonistic interactions between two species are well understood, the interaction between more than two players is very complex and largely unexplored. A model system to go beyond typical two-species interactions is given by studying a tripartite species interaction consisting of temperate bacteriophages (viruses that infect bacteria), bacteria and final animal hosts. First an evolution experiment was conducted. Thereby one Vibrio strain was propagated with 3 generalist and 3 specialist phages and by means of 2 different batch cultures. In one culture (further described as evolution treatment) only the phages are able to evolve and in the second culture (further described as coevolution treatment) both players were allowed to evolve. After 24 h the first samples containing bacteria and phage isolates were collected and this sampling procedure was subsequently repeated every 48 h. This evolution experiment lasts 13 days (312 h) and results in 7 transfers. The evolved and (co-) evolved phages and the co-evolved bacteria were subsequently used for further experiments to determine correlated fitness responses during phage-bacteria coevolution. Phage fitness was estimated as infectivity against 75 different Vibrio isolates. Bacterial correlated responses were estimated by means of controlled infection experiments on the final animal host, the broad-nosed pipefish Syngnathus typhle and assessed by measuring pipefish mortality, total amount of colony forming units (CFU) and gene expression of selected pipefish immune genes. Furthermore the bacterial growth rate was measured, to determine whether it is restricted as a cost of resistance against phages. In addition, to determine the underlying selective force of phage-bacteria (co-)evolution, phage infectivity as well as bacteria resistance was tested against past, contemporary and future bacteria (phages, respectively) by using a time-shift-assay. Phage host-range increases rapidly during the 24 hours of (co-)evolution. This increase includes infectivity to more bacteria from the same Vibrio clade and also to former resistant bacteria from another clade. Furthermore I observed a time-lagged antagonistic correspondence between both players, with increasing phage infectivity and bacteria resistance over evolutionary time. This indicates that this coevolution is driven by directional selection, where recurrent selective sweeps lead to the accumulation of beneficial mutations. Controlled infection experiments with evolved Vibrio/phage combinations on pipefish revealed that Vibrio virulence has changed during antagonistic coevolution between Vibrio and phages. Virulence, estimated as gene expression of selected immune genes and pipefish mortality rate first decreased during (co-)evolution followed by an increase towards the end of the experiment. Furthermore CFU and bacterial growth decrease with increasing time of (co-)evolution. These results indicate that gaining resistance against phages includes a loss of virulence as well as decreasing growth rate. Those results emphasize the great complexity between more than two players and call for an integrative view that goes beyond classical two-way systems to be able understand the evolution of host-parasite interactions.