Bacteriophage control of host cell DNA transactions by small ORF proteins

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This research proposal is designed to help us understand how bacterial cells defend themselves from invading viruses, called bacteriophage, and how bacteriophage have evolved to overcome these "immunity systems". Bacteriophage are the most abundant biological entity on the planet. Their survival depends on an ability to invade and manipulate the bacterial host in order to steal the chemical resources and machinery they need to build and release more bacteriophage particles. Upon invading the bacterial cell, bacteriophage deploy a range of small proteins (smORFs) which bind to targets in the host cell in order to take control of their resources and evade their defence systems. Recent research has uncovered a huge diversity of these immune systems which either prevent phage infection or trigger cell suicide in the infected cells as a means to preserve the uninfected population.One such bacterial defence system is RecBCD, a large protein complex which acts as an immunity hub. It functions both to recognise and degrade bacteriophage DNA molecules and to help catalogue the DNA sequences of the invading DNA to form a memory of viral sequences that is used to counteract future infection. In order to evade RecBCD, bacteriophage make smORF proteins which bind to and inhibit RecBCD: the so-called anti-RecBCD proteins. Remarkably, in response to this challenge, bacterial cells have subsequently developed retrons; genes that produce molecules called msDNAs which are capable of sensing anti-RecBCD proteins as a indicator of viral infection and triggering suicide.This project will elucidate the mechanisms of anti-RecBCD proteins and the msDNAs which can sense them. It is driven by the broader concept that the study of smORF proteins has value beyond a purely academic interest in the inner workings of bacterial viruses. By understanding how bacteriophage manipulate and kill bacteria, as well as how bacteria defend themselves from such attack, we can better develop strategies to overcome pathogenic bacteria which cause human disease. For example, we have shown that anti-RecBCD proteins potentiate the effect of fluoroquinolones (an antibiotic) and can even restore the sensitivity of clinically-resistant strains of pathogenic bacteria. Consequently, scientists are now developing drugs that target RecBCD and related complexes.It is well-established that studying the molecular machinery which orchestrates the fight between bacteriophage and bacteria has uncovered a treasure-trove of useful molecules. These include proteins and enzymes representing some of the most important and useful tools available to molecular biologists. For example, all modern gene-editing methods are based on technologies that were discovered through the study of bacterial immunity. This precedent, and the fact that vast numbers of smORF proteins and their cellular targets remain overlooked and uncharacterised, suggests that many more valuable tools await discovery.