Viral jumping of genus and species barriers: engineering phage host range promiscuity for diverse bacteria

Summary (up to 4000 characters)

All living organisms can be infected by viruses, including plants, animals and humans. It has been known for just over a century that bacteria are also susceptible to attack by viruses (called bacteriophages or phages). Phages tend to be very host-specific because they only infect their bacterial hosts. Phages are thought to be the most abundant biological entities on Earth; there are 10 times more bacterial viruses than bacteria on the planet. However, these viruses are obligate intracellular parasites (being utterly dependent on susceptible bacterial hosts for their propagation). Bacteria and their viral parasites have existed for millions of years and their relationship is a perpetual "arms race" - the bacteria evolve strategies to become phage-resistant but the phages can also evolve by mutation to get around the defences of the bacterial cells. The bacteria then evolve to resist the evolved phages, and so on - in perpetuity. This endless biological process is called co-evolution.

Phages have to adsorb to their bacterial hosts before they can infect. Adsorption depends on two things: 1) the bacteria expose a specific cell surface structure that can be "recognised" by the phage, and 2) the viruses have tail structures that allow them to "lock on" to the bacterial surface receptors in a specific "lock and key" mechanism. Only then, the virus can inject its DNA into the bacterial prey. On injection, the viral DNA re-programmes the bacteria, forcing them to make many new virus particles inside the bacterial cells, which burst to release new viruses that then infect more bacteria.

The specificity of the interaction between the virus tail components and the bacterial surface receptor is the first key requirement in the phage-host relationship and that will be exploited in this project. There are some interesting biological similarities between the phage-bacterium interaction and the situation operating between the human coronavirus and the surface receptor of human cells. In the coronavirus case, viral "spike" proteins bind to surface receptor components of human cells - and that interaction is essential for viral adsorption, penetration and eventual replication in human cells.

In this study we will exploit a phage called a "viunalikevirus". The viunalikeviruses are killers of the bacteria that they infect, but we have shown that they also have the capacity to transfer genes between bacteria ("horizontal gene transfer") in a process called generalised transduction. The viunalikeviruses are very specific for their own particular bacterial host species. However, we believe that these particular viruses have the genetic capacity to replicate in a wide range of bacteria but are prevented from doing so simply because of the tight specificity of the virus tail-bacterial host receptor interaction. One aim of this project is to test that hypothesis robustly. We will transfer genes coding for the surface receptor of a viunalikevirus to a spectrum of bacterial hosts in this synthetic biology project. The ability of the virus to infect genetically engineered bacteria will be confirmed and then these engineered bacteria will be tested as donors and recipients for genetic transfer capacity driven by the phage. The organisms to be investigated in this study will include non-pathogenic bacteria related to the original viunalikevirus host but will also include other bacteria that can infect plants, animals and insects. Furthermore, we will expand our approach into testing of taxonomically unrelated bacteria, including bacteria of medical, agricultural, environmental and biotechnological significance. Our aim is to exploit this strategy to provide a facile, innovative generic route to virus-mediated manipulation of diverse bacteria - thereby providing exceptional general utility for exploitation in bacterial genetics and engineering.

Phage infection of a cognate bacterial host begins with viral adsorption (via a receptor binding protein; RBP) to the surface receptor on the bacterium. RBPs are usually phage tail fibre proteins or tail spike proteins. Surface receptors of Gram-negative bacteria include pili, flagella, outer membrane proteins (OMPs), and surface components such as LPS or capsular polysaccharides (CPS). Viunalikeviruses have tail spike proteins as their RBPs and the corresponding bacterial surface receptors are polysaccharides - CPS being an example.

Viunalikeviruses have been isolated on several enterobacterial genera and on Sinorhizobium and Acinetobacter but, despite exceptions, they are specific for their respective host species. We showed that viunalikeviruses are generalised transducers with utility for genetic engineering of their cognate hosts, but the restricted host range of most viunalikeviruses limits their exploitation potential. However, our hypothesis is that the viunalikeviruses will be capable of productive lytic cycle replication in many Gram-negative bacteria but are prevented from doing so because constraints of the viral RBP-host receptor interaction are a demanding barrier to adsorption. That notion will be tested in this project.

We will move bacterial genes coding for a viunalikevirus surface receptor into diverse bacterial hosts. The recombinant bacteria will be tested for CPS production and inheritance of viral sensitivity. The engineered bacteria will be assessed for genetic transfer capacity by phage-mediated transduction. A wide selection of heterologous bacterial hosts will be tested, including a range of enterobacteria that can make antibiotics and infect plants, animals and insects. Taxonomically distant bacteria of medical, agricultural, environmental or biotechnological relevance will also be manipulated as viunalikevirus-permissive. This innovation will open up opportunities for the genetic analysis and engineering of diverse bacteria.