Novel anti-bacterial toxins: mode of action and delivery between cells

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Many bacterial pathogens use the Type VI secretion system (T6SS) nanomachine to fire toxic 'effector' proteins directly into target cells. It is becoming increasingly apparent that the T6SS plays a key role in the virulence and competitiveness of diverse Gram-negative bacteria, including important human pathogens. Pathogens can use T6SSs to directly target eukaryotic organisms, as classical virulence factors. Alternatively, many pathogens can use T6SSs to target other bacterial cells, killing or inhibiting rivals. 'Anti-bacterial' T6SSs thus provide a competitive mechanism to allow pathogens to proliferate in polymicrobial infection sites or environmental reservoirs and ultimately cause disease. Anti-bacterial T6SSs inject toxic proteins into target bacterial cells, efficiently killing them or inhibiting their growth. Recent work in the field, including from our lab, suggests that different T6SSs secrete multiple, diverse and novel anti-bacterial toxins. Whilst several broad classes have been functionally characterised to date, particularly enzymic toxins attacking the bacterial cell wall, nucleic acid or cell membrane, many other toxins appear unrelated to known proteins. It is important to discover the vulnerable cellular targets of such toxins since they may reveal new targets for antibiotic or other drug design; the toxins may also represent valuable research tools to understand basic bacterial physiology.In the SJC lab, we have identified new T6SS-secreted anti-bacterial toxins which do not fall into the previously described classes. Therefore their mode of action and cellular target in susceptible cells is unknown. The focus of this PhD project will be to study one (or more) of these new toxins in order to determine: (1) its mode of toxicity and cellular target in susceptible cells; (2) its pathway of secretion and any cellular functions of the target cell which are required in order for the toxin to exert its effect; (3) how self-resistance or 'immunity' to the toxin in the secreting cell is achieved; and (4) distribution of related toxins in different organisms. In order to tackle part (1) and inform on the remaining parts, the molecular bacteriology expertise of the SJC lab will be complemented by the target discovery expertise of the SW lab, who utilise state-of-the-art chemical biology and other approaches to determine the mode of action of new anti-parasite and anti-microbial compounds.This project will provide research training in a broad-spectrum of molecular microbiological techniques, including: molecular biology and genetic analyses; protein purification and biochemical assays; microscopy and other cell biological approaches. The student will further utilise state-of-the-art chemical biology strategies, including thermal shift proteomics - an unbiased, label-free method to search for the targets of drugs or in this instance T6SS-secreted anti-bacterial toxins. Advanced transcriptomics and genomics approaches are also available in the host or collaborating labs, as required. The student will have many opportunities to present their work at internal and external meetings and to interact with other research groups.