The Impact of Cholesterol on Staphylococci: Cell Responses and Membrane Dynamics.

The outer surface of human skin has cells that produce a complex mixture of water insoluble lipids including cholesterol. These molecules serve a variety of functions, including producing the water-resistant barrier of skin and contributing to its antimicrobial properties by limiting the types of bacteria which can persist. One of the major groups of bacteria found on skin are members of the genus Staphylococcus, including abundant species such as S. epidermidis and S. hominis. Some species that colonise more intermittently, such as S. aureus are widely known for their association with human diseases, notably MRSA. There are still many unanswered questions regarding the success of staphylococcal colonisation of human skin and our experimental plans are aimed at increasing our understanding of skin survival mechanisms. By examining the response of the staphylococci to components of the lipid matrix, which is proposed to restrict the numbers of colonising bacteria, we have begun to question how staphylococci respond to individual lipids. The response of staphylococci to the lipid cholesterol has received little attention previously and our data leading to this application suggests it has a major effect on Staphylococcus aureus membranes. S. aureus is named after the golden colour of its colonies when grown in the laboratory and we have shown that addition of cholesterol at concentrations found on skin cause the bacterium to stop producing the pigment and become colourless. The pigment has been demonstrated to be important for membrane stability and it protects the cell from oxidising agents.

The described research study will investigate the response of S. aureus to cholesterol to answer key questions: Why does cholesterol reduce the presence of the golden pigment in its membrane? What effect does cholesterol have if it does enter the S. aureus membrane? What are the consequences of not having the pigment and it is replaced by cholesterol? If we add the purified pigment to S. aureus does it have similar effects to adding cholesterol i.e. do they have similar roles? We will examine the consequences of cholesterol challenge on S. aureus membrane composition to examine if it changes. We have identified several gene mutants that have increased survival in the presence of cholesterol and we will investigate why survival is altered to help determine the effect of cholesterol on the cell. We will generate gene expression datasets from three species of staphylococci (S. epidermidis, S. hominis and S. aureus) that are know to differ in their abundance on skin and their production of membrane staphyloxanthin. The datasets that are generated will be compared to analyse cross-species similarities and differences in gene expression to build a picture of their mechanisms and responses to skin lipids.

By identifying differences between staphylococci considered normal skin residents and disease-causing species, this study could generate fundamental data leading to the design of new cosmetics for underarm odour or dandruff, which are important research areas for the project partner Unilever Plc. Similarly the data could lead to target identification for future design of novel therapeutics, including antibiotics.

The genus Staphylococcus comprises species that are able to colonise human skin with varying success and they are found in different sites in varying abundance. The skin lipid matrix provides key properties to the skin such as its permeability barrier due to the presence of a structured combination of fatty acids, ceramide and cholesterol that actually form an array in 1:1:1 proportions. The components of the lipid matrix contribute to antimicrobial defence, yet we have not identified the effects of each component molecule.In this study, we will examine the responses of several species of staphylococci to physiological relevant levels of cholesterol and mimicking those levels found on skin. We have identified that challenge with cholesterol results in decreased transcription of both the crt operon for biosynthesis of the eponymous carotenoid pigment of S. aureus, called staphyloxanthin, and the sigmaB-encoding gene responsible for crt operon transcription. To identify how global transcription and membrane function is affected, we will use RNA-Seq to generate the transcriptional signature of the response to staphyloxanthin and analyse changes to membrane properties resulting from reduced staphyloxanthin, which acts to control fluidity. We will characterise mutants that have lost the ability to use cholesterol for protection from antimicrobial fatty acids that increase fluidity, and test the hypothesis that cholesterol in staphylococcal membranes acts to decrease fluidity in a manner similar to its role in eukaryotes. We will also generate the transcriptional response signatures from S. epidermidis (produces no staphyloxanthin) and S. hominis (produces staphyloxanthin), because these two species are ubiquitous skin commensals in contrast to S. aureus, which resides only transiently on skin. We will also characterise experimentally evolved mutants of these species from culture in growth-limiting levels of cholesterol to identify components interacting with cholesterol.

The research study that is proposed will have academic and commercial impacts via close interactions between Unilever Plc researchers in the UK and University of Liverpool microbiologists. The outcomes of the research will produce large datasets that will help to inform the development of new healthcare and cosmetic products, a core business priority of Unilever. The research outputs and interactions explain their eagerness to collaborate and commit financial support via this Industrial Partnership Award. Therefore, the primary aims of the research have clear potential to generate impacts through a working relationship between the Liverpool study group and scientists working on product design and development. We will monitor how our impact activities progress at six-monthly meetings between Unilever and the Liverpool study group, and measure success by targeting continued relationships via new grants and contracts emanating from our research outcomes.

The planned research study will generate datasets with wide user interest; raw data will be submitted at the earliest opportunity to databases in order to maximise their impact on the research of other scientists. The key results will be published in peer-reviewed journals and presented at scientific conferences that have academic and clinical attendees, providing for wide delivery. An important impact output is to communicate aspects of our scientific study and the wider biology of skin microbiology to the public. This interaction will be achieved by targeting schoolchildren at the Liverpool World Museum via a new annual one-day workshop and continued visits to local schools. We will gather feedback from teachers on how successful our activities are to ensure maximum impact. The University of Liverpool will generate publicity of the work using its established links with media outlets and the use of online videos that allow public feedback; we will present at our annual Institute Research Day attended by local MPs, business leaders and BBC media Salford.

The researcher (PDRA) appointed to the study will be trained in state-of-the-art molecular genetics and bioinformatics to enhance their career and they will actively interact in the laboratories with postgraduate and undergraduate scientists, who will benefit from their training. The ability of the PDRA to interact with Unilever scientists will broaden their own research and skills training, with obvious benefits for career progression.

The research proposal will also have impact on the ability of the BBSRC to meet its own agenda, particularly the strategic theme of 'basic bioscience underpinning health' and the enabling themes of knowledge exchange, innovation and working with business.