Bacterial toxin-antitoxin system functionality and bacteriophage abortive infection: structure function and biology

Bacteria are attacked and killed by specific viruses called bacteriophages (phages). Phages are the most abundant biological entities on Earth and they outnumber their bacterial hosts by 10-1. Despite this superabundance of phages, the bacteria are still ubiquitous and this is because they have evolved remarkable systems to prevent the potentially lethal effects of viral infection. Some of these protective anti-viral systems are activated by the virus after infection in a process called abortive infection (Abi). There are over 20 known Abi systems which are thought to act at different stages in the viral infection and replication process. We discovered a new Abi system encoded by a plasmid in the bacterial plant pathogen, Erwinia - the causative agent of commercially significant potato soft rot disease. This new Abi system works via two components; one a protein (ToxN) that is toxic to the bacterial cell, and the other an RNA molecule (ToxI) that suppresses the toxin under uninfected conditions, but, when inactivated, leads to activation of the ToxN toxin, leading eventually to programmed death of the bacterial host cell. In this way, infected bacterial cells commit 'suicide' and an important net effect is that the virus is trapped and cannot be released to infect the other sibling members of the bacterial population. The group thus survives viral infection of some individual cells in a behavior akin to bacterial altruism. We have shown that this novel Abi system operates in different bacteria and in response to many different phages. We think that this Toxin-Antitoxin (TA) based system is likely to be important in controlling viral propagation in the environment, and for both bacterial and phage evolution. In this project we are trying to dissect the mechanism of action of the TA system. We will determine the structure of the toxin (ToxN) that can kill the bacteria and try to determine how the ToxI (RNA) molecule can suppress the function of the toxin. We will also study the relationship between the structure of the toxin and its biological function (in aborting phage infection and killing bacterial cells). We will investigate the nature of the cellular target(s) of the toxin. We will study mutant viruses that are able to get around the lethal effects of the ToxIN system and, by studying these mutants, we hope to be able to work out what components of the phage act to activate the ToxIN system. We will then try expressing the specific viral components artificially to see if we can switch on the antiviral system or altruistic suicide, to kill bacteria. Developing a deeper structural and mechanistic understanding of the interactions between phages, bacteria and the antiviral defence systems is important in our appreciation of evolution and adaptation of bacteria (particularly to viral attack). However, in addition, this research could lead eventually to the development of novel chemicals that have uses in antibacterial chemotherapy - new antibiotics.

This project will investigate a new class of Toxin-Antitoxin (TA) system from the potato blackleg pathogen, Erwinia, that operates as a phage abortive infection (Abi) system. TA systems drive the host cell into prokaryotic apoptosis - in this case, post-viral infection - thereby limiting phage spread in bacterial populations. Although discovered in Erwinia, this new system (ToxIN) is functional in E. coli and other bacteria and against a wide spectrum of phages. The ToxIN module is comprised of a two gene operon: toxI, toxN with toxI encoding a regulatory RNA and toxN encoding the protein toxin. The toxicity of the ToxN protein to bacteria is suppressed by interaction with ToxI RNA and ToxN may has nuclease activity, although the precise cellular targets are not known. Neither do we know how infecting phages activate the system but we think it possible that phage products act directly (or indirectly) to allow the ToxN protein to be activated e.g. by displacing the ToxI RNA or by alternative routes. Predicted homologues of this new TA system from Erwinia have been identified in diverse bacterial genera. The main themes of this proposal are to solve the structure of the ToxN toxin of Erwinia and some of the homologues from other bacteria possessing similar systems. We intend to analyse how the function of the ToxN protein is affected by ToxI in a mutagenesis programme on both the the ToxN protein and cognate regulatory ToxI RNAs. We will also investigate the physiological effects on the host of artificial and 'natural' (phage-induced) ToxN activation, and identify cellular targets of ToxN activity. We will determine the molecular bases of viral escape from the ToxIN system and we will examine the cellular physiology impacts of ToxIN when specific phage products are artificially expressed.