The genotypic and phenotypic impacts of Shiga toxin encoding bacteriophage interactions with their host cells: consequences for food borne zoonoses

In 1982, the first recorded outbreak of a novel, but deadly, E. coli serogroup, O157:H7 was recorded in North America, and such episodes were subsequently repeated across the world. Though foodborne outbreaks of this pathogen are limited in number, the severity of the disease is high and results in death or life-long debilitation in a significant proportion of infected individuals, particularly in young children. The major effector for the severity of disease is Shiga toxin. To date, more than 500 serogroup variants of E. coli have been reported to produce Shiga toxin as well as a few other related, and even unrelated, bacterial species, demonstrating the ongoing emergence of Shigatoxigenic potential across a variety of bacterial species. The genes enabling Shiga toxin production are carried by viruses known as bacteriophages, more specifically Stx phages. These viruses are directly responsible for the spread of shigatoxigenic potential, and though mush is known about shigatoxigenic E. coli (STEC), we know little about the Stx phages, which act as transmissible 'survival capsules' for Shiga toxin genes. We have recently demonstrated that Stx phages can actually infect a single bacterial cell multiple times. This increases the number of Shiga toxin genes within a bacterial cell, driving increased expression of Shiga toxin, enabling a multiply infected host to potentially cause more severe disease. The proposed project addresses several aspects of Stx phage biology that have been uncovered by our group. In identifying how Stx phages can multiply infect a single host cell, two miss-annotated genes of unknown function were identified. Preliminary mutant analyses demonstrate that these genes are essential to efficient virus production, and more importantly to Shiga toxin production. We intend to further understand how these genes ae controlled and function as they are crucial to virulence of these foodborne pathogens. Secondly, we have identified that a novel enzyme, which drives the ability of our model Stx phage to multiply infect its host cell, is a very promiscuous integrase. It has multiple recognitions sites in the E. coli chromosome, drives a unidirectional recombination reaction and has no close characterised relatives. Because of the these properties it has the potential to be a very valuable molecular tool for both standard laboratory techniques but also for use in gene therapy and other second generation molecular medicine techniques. The last two objectives focus on understanding how the bacterial virus and its host cell interact. To these ends, using tools we have obtained in previous work, we intend to examine how the bacterial cell responds to the virus it carries and how the viruses manipulates its host cell by examining all gene expression in response to single and double virus carriage with and without viral induction. We can now easily do this through second generation sequencing technologies (SOLiD), which will provide identification of all transcripts and their relative densities. This information will be directly informative, but it will also inform subsequent analyses that we have begun to examine the function of viral genes expressed by the host cell. Using qPCR we have been able to identify viral gene expression that is linked to viral replication and to the stable infection state where the viral genome is supposedly carried silently. This silent state is associated with increased resistance to a variety of environmental perturbations. We have begun to address the role these expressed genes play in the lifestyle of the bacterial host cell. These data will allow us to gain some understanding of the benefits of viral carriage and identify factors that provie a selective advantage to the host cell, driving the further spread and emergence of Shigatoxigenic potential. This will underpin our progress towards strategies to limit the future expansion and spread of these Shiga toxin producing zoonotic pathogens.

Shiga toxin-encoding bacteriophages (Stx phages) drove the emergence of Shigatoxigenic bacterial pathogens in 1982, of which the E. coli O157:H7 is the most notorious. The adsorption target recognized by most Stx phages is a highly conserved and essential surface protein. High levels of recombination, promoted by the unusual ability of some Stx phages to multiply infect a host, has driven the dissemination of shigatoxigenic potential. Multiple infection events also enable higher production levels of Stx, enabling more severe disease. Here, we focus on trying to fully understand those phage genes that impact the biology of the bacterial lysogen. We will examine how Ant and Roi-like proteins promote full expression of Stx and production of new phage particles using mutant analyses, DNA binding and protein interaction assays. Using our tri-partite in situ integration assay and qPCR we will determine the minimum requirements for integration driven by the Phi24B integrase, which itself is likely to become a useful molecular reagent. We will use the SOLiD platform to undertake transcriptomic analyses of the impact of single and double prophage carriage on the host cell, both during lysogen growth and induction. Through a combination of experimental molecular genetic experiments we will test hypotheses based on sophisticated bioinformatic predictions of genes identified by former expression and current transcriptomic analyses, which conventional genome annotation most often fails to identify, in order to attempt to establish their roles in the biology of the host cell, providing insights into their evolutionary significance in the emergence and success of zoonotic shigatoxigenic pathogens. We have already successfully done this for a few phage-encoded proteins. These data will yield insights into the rapid evolution and dissemination of these zoonotic pathogens that emerged <30 years ago, providing information relevant to the development of control/intervention strategies.

This project is a basic science project, and as such may not immediately result in translational output, however, understanding how Stx phages affect their bacterial host is essential if we are to both predict and model the spread and emergence of STEC and STEC-like pathogens. This information will have direct relevance to DEFRA, the HPA and possibly the Environment Agency, and may become more important in a changing global environment. We have already demonstrated that we can associate and assign biological activity to previously hypothetical proteins of unknown function and this project will provide further function analyses of hypothetical proteins of bacteriophage and bacterial origin. As we already know that many of the genes encoding these hypothetical proteins are disseminated across many different phages, this project will impact heavily on our ability to annotate genomic databases and better assign protein function to sequences based upon novel domain identifications. These data will of course be of major international significance as currently ~90% of bacteriophage genes are hypothetical, and indeed a significant number of bacterial genes are also hypothetical. It is expected that the integrase characterised during this project will be very useful for the development of general molecular tools, based upon the level of activity it possesses and its ability to target a variety of sequences. This group already has a track record in making presentations not only to academic audiences but also to business and policy makers. This will continue, and in addition data from our work will inform presentations made within our local communities at school level and will inform work with Honours students, linking these future graduates with the teaching profession. Better understanding of the biology of STEC and related organisms is urgently required (i.e. the Griffin report following the Godstone petting farm outbreak 2009-2010). The data generated by the experiments proposed here will provide more information on the spread and emergence of shigatoxigenic pathogens and will have an indirect effect on agricultural practices and the safe handling of livestock (farming, pet ownership and public access to zoos and farms).