Bacteriophage control of host cell DNA transactions by small ORF proteins
Lead Research Organisation:
University of Bristol
Department Name: Biochemistry
Abstract
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.
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.
Technical Summary
This research will improve our understanding of how bacteriophage control bacterial DNA transactions through the evolution of small open reading frame (smORF) proteins which bind to and inhibit specific targets in the host cell. This field of research has wide ranging implications for structural biology, synthetic biology, biotechnology and medicine, but is most immediately relevant to the identification and validation of new targets for anti-bacterial drugs.
Specifically, this proposal is focussed on the E. coli RecBCD complex and its role as a bacterial immunity hub. RecBCD is best-characterised as a DNA repair enzyme, but its ability to discriminate between host and foreign DNA also enables it to degrade phage DNA sequences (a form of innate immunity) and catalogue them within CRISPR libraries to help prevent further infections (a form of acquired immunity). Naturally, phage have evolved to overcome the immunity functions of RecBCD by producing small anti-RecBCD proteins of which we know of three distinctive classes; Gam, gp5.9 and Abc2. Remarkably, bacterial cells have subsequently responded to the challenge of anti-RecBCD proteins by sensing RecBCD inhibition as a proxy for infection, and then using this as a cue for programmed cell suicide (a mechanism described as abortive infection).
Our proposal addresses three hypotheses, each of which is related to the mechanism of anti-RecBCD proteins and the strategies bacteria have developed to counteract them. It builds upon, (1) our recently-published structures of anti-RecBCD proteins bound to their target, (2) our unpublished and unprecedented discovery of a mechanism for control of chromosome segregation by a phage-encoded smORF protein and (3) seminal studies on bacterial retrons which have finally unveiled their function in anti-phage defence approximately 40 years after their discovery.
Specifically, this proposal is focussed on the E. coli RecBCD complex and its role as a bacterial immunity hub. RecBCD is best-characterised as a DNA repair enzyme, but its ability to discriminate between host and foreign DNA also enables it to degrade phage DNA sequences (a form of innate immunity) and catalogue them within CRISPR libraries to help prevent further infections (a form of acquired immunity). Naturally, phage have evolved to overcome the immunity functions of RecBCD by producing small anti-RecBCD proteins of which we know of three distinctive classes; Gam, gp5.9 and Abc2. Remarkably, bacterial cells have subsequently responded to the challenge of anti-RecBCD proteins by sensing RecBCD inhibition as a proxy for infection, and then using this as a cue for programmed cell suicide (a mechanism described as abortive infection).
Our proposal addresses three hypotheses, each of which is related to the mechanism of anti-RecBCD proteins and the strategies bacteria have developed to counteract them. It builds upon, (1) our recently-published structures of anti-RecBCD proteins bound to their target, (2) our unpublished and unprecedented discovery of a mechanism for control of chromosome segregation by a phage-encoded smORF protein and (3) seminal studies on bacterial retrons which have finally unveiled their function in anti-phage defence approximately 40 years after their discovery.
Organisations
People |
ORCID iD |
| Mark Dillingham (Principal Investigator) |