Characterisation of a novel protein toxin family secreted by the animal and human pathogen Staphylococcus aureus

The Gram-positive bacterium, Staphylococcus aureus, is a mammalian pathogen. It is a major cause of skin and soft tissue infections, and a frequent cause of bovine mastitis, resulting in a significant reduction in milk production. The organism can jump between host species, and at least two multi-drug resistant genetic subtypes that are endemic in people have been traced back to cattle.
In order to cause disease S. aureus must first colonise the host. To do this it must effectively compete with the resident microbiota to establish a niche. The PI's lab has demonstrated that S. aureus uses its Type VII protein secretion system (T7SS) to secrete nuclease and membrane-depolarizing toxins that target other bacteria. S. aureus is protected from the action of its own toxins by the co-production of immunity proteins that neutralize toxic activity. To date, all characterised substrates of the S. aureus T7SS fall into three protein families: the WXG100 proteins, the YeeF domain proteins and the LXG domain proteins. Very recently the PI has identified a fourth family of substrate proteins through proteomic and genomic analysis that have not yet been described in the literature. S. aureus encodes two of these novel substrates, but the protein family is found across many other Gram-positive bacteria. The aim of this project is to characterise these new substrates of the S. aureus T7SS and to define their toxic activities through molecular analysis.

Approaches to be used: To test for toxic activity, both substrates will be produced in E. coli and in S. aureus under a regulatable promoter. Targeting sequences will be added to the toxin domains to direct them to the extracellular side of the membrane (to determine whether their targets reside in this compartment). Immunity proteins are usually encoded adjacently to their toxin partners3. Genes in the immediate neighbourhood of each toxin will be tested to see whether they protect from toxic activity. Direct interaction between toxins and immunity proteins will be detected using bacterial two hybrid and co-immunoprecipitation approaches. Modelling approaches will be used to predict structure/function of toxins, and candidate active site substitutions will be designed and tested in toxicity assays. Toxins and immunity proteins will be overproduced separately and in complex (it may be necessary to use an inactive toxin variant for expression in the absence of an immunity partner). Purified proteins will be used for structural analysis. A range of phenotypic experiments will be undertaken to identify the cellular target each toxin. This will be supported by biochemical experiments to demonstrate biological activities in vitro.
Individual chromosomal deletions of candidate toxins and immunity proteins will be constructed for use in bacterial competition experiments. Interbacterial competition will be assessed using a novel zebra fish embryo colonisation model that has been established in the PI's laboratory. The hindbrain provides a sterile compartment where two bacterial strains can be co-inoculated. The T7SS is highly active under these in vivo conditions and killing of target strains is observed after 9 hours and can be quantitated using bacterial counts. This model will be used to assess the roles of each toxin in the killing of (i) closely related S. aureus strains (ii) more diverse Staphylococci associated with the bovine mammary gland (e.g. S. haemolyticus, S. epidermidis and S. hominis) (iii) other mammary gland-associated species such as Bacteroides and Bifidobacteria.

The work proposed here maps closely to the BBSRC's strategic priority in agriculture and food security. A key aspect of this priority is to support research in areas that have profound implications for food security and food safety such as animal health and welfare.