Mechanisms of interbacterial competition during Staphylococcus aureus colonisation

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. Palmer and Horsburgh have complementary research programmes studying the role of small molecules (Horsburgh) and secreted anti-bacterial toxins (Palmer) on interbacterial competition. The aim of this project is to characterise protein toxins secreted by the Type VII secretion system (T7SS) of S. aureus, to decipher their toxic activities and how they interplay with small secreted antimicrobials to kill rival bacteria.

Our bioinformatics and proteomic analysis has identified four novel secreted substrates of the T7SS. Initially substrates will be produced in E. coli and in S. aureus under a regulatable promoter to score for toxic activity. These assays will also be used to identify neutralising anti-toxins (usually encoded in the immediate genomic vicinity of the cognate toxin). A range of phenotypic experiments will be undertaken to identify the cellular targets of up to two toxins. This will be supported by biochemical experiments to demonstrate biological activities in vitro.
Individual chromosomal deletions of candidate toxins and antitoxins will be constructed for use in bacterial competition experiments. These will be combined with deletions of xx antimicrobial gene clusters identified by the Horsburgh group. Interbacterial competition will be assessed using a novel zebra fish embryo colonisation model. 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 relative roles of toxins and antibacterials 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. It is recognised that endemic diseases including zoonoses remain key challenges, and that they also contribute to the global burden of antimicrobial resistance. Staphylococcus aureus is a major cause of mastitis in UK dairy herds. Bovine mastitis results in reduced level of milk production, and costs the UK dairy industry around £170 M per year. Its control and treatment in the UK relies on the use of around 12 million doses of therapeutic and prophylactic antibiotic annually. This project will examine mechanisms used by S. aureus to kill bacterial competitors, an essential process to allow the organism to colonise its host. Characterisation of a secreted proteins involved in colonisation and persistence of S. aureus has the potential to pave the way for the development of this proteins as a novel vaccine candidates, or for use in diagnostics.