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  1. 1. Could you begin by introducing the work of the Pathogenic Yeast Research Group? The Pathogenic Yeast Research Group focuses on diseases caused by yeasts, especially those belonging to the Candida and Cryptococcus genera. My focus is the role of bioactive lipids produced by Candida species. I am interested in understanding how fatty acids, including those obtained from the host, are used by these yeasts to produce lipid metabolites (oxylipins) that contribute to their survival in the host, as well as to tissue damage caused by the yeast during infection. What is the immunological role of bioactive lipids in hosts of these diseases? Certain oxylipins can modulate the mammalian immune system and often have several different functions in distinct cell types. One of the best-studied oxylipins produced by both the host and pathogenic yeasts is prostaglandin E2 (PGE2 ), which can shift host immune responses in favour of the pathogen. It can inhibit the type of immune response needed to protect against Candida infection and induce an immune response that may lead to associated chronic or disseminated diseases. It can also stimulate another type of immune response that may cause uncontrolled inflammation and damage to the host. Thus the production of PGE2 during infections can benefit the pathogen. Another important effect caused by PGE2 is tissue eosinophilia leading to tissue damage, which is characteristic of some chronic fungal infections. Interestingly, it is not only the host that is affected: PGE2 has biological activity on yeast by stimulating germ tube formation in Candida species. This change in morphology, from unicellular to filamentous, is considered the start of biofilm formation and is associated with an increased ability to invade tissue. Interestingly, biofilms also produce more PGE2 than unicellular yeast forms. Can you highlight some of the roles that lipids have as ‘inter-kingdom’ signalling molecules in yeast-associated multispecies infections? This is an exciting new research focus for us. It is increasingly being recognised that many infections are not caused by one organism. Interaction between pathogen and host may be influenced by other non-pathogenic organisms. Although it is known that lipid metabolites can act as signals between organisms – even those belonging to different kingdoms – very little work has been done on the bioactive lipids produced by mixed communities. An example of a multispecies interaction is the biofilms consisting of Candida albicans and the Pseudomonas aeruginosa bacterium in the lungs of patients with cystic fibrosis. Are there particularly advanced imaging techniques that you incorporate in your studies? Most studies of different antimicrobial effects start with the production of standardised biofilms, which are notoriously resistant to antifungal treatment. To assess the effectiveness of potential antifungal drugs, biofilms are stained for viability and visualised using a confocal laser scanning microscope. The potential of drugs to decrease biofilm viability or metabolic activity is assessed in an XTT-assay based on the ability of mitochondrial dehydrogenases to convert a substrate to a coloured formazan. The effect of compounds on the ultrastructure – including surface characteristics and organelle structure – is examined by electron microscopy. Our group pioneered the use of nano-scanning Auger microscopy (Nano-SAM) on yeast cells. With Nano-SAM, nanometre-thin slices of a yeast cell enable observation of its 3D structure. The cell elements can also be mapped. Similarly, time-of-flight secondary ion mass spectrometry (TOF-SIMS) is used to map the distribution of certain compounds, including oxylipins, in cells. We also use various assays to measure oxidative stress. Has collaboration with other research groups aided your research? My own speciality is enhanced by that of Dr Olihile Sebolai who focuses on Cryptococcus species and immunology. Dr Chantel Swart is an expert in ultrastructural analyses and was a leading member of the group that pioneered the application of Nano-SAM to yeast research. In this, we also collaborate with our university’s physics department. The fourth member of the group is Professor J Albertyn, who is an expert in yeast molecular biology and yeast signal transduction. To date, has your work drawn any interesting conclusions? An obvious conclusion is that the lipids of pathogenic yeasts are much more than structural compounds or reserve material; like mammalian and plant lipids, they have other functions. It has also become obvious that much of the knowledge regarding metabolic pathways and enzymes responsible for production of mammalian oxylipins does not translate directly to yeasts. With the completion of each project, we end up with more questions – the study of these bioactive compounds is always exciting. Dr Carolina Pohl describes the work of her research team, one of few groups in the world that are exploring the roles of bioactive fatty acids in the development of yeast infections The lipid connection 70 INTERNATIONAL INNOVATION DR CAROLINA POHL
  2. 2. Oxylipins are central elements in adaptation and survival; in some instances they aid organisms to defend themselves, propagate or prompt symbiotic relationships, whereas in other situations they can induce pathogenesis A RICH AND diverse population of bacteria and fungi live in a mutualistic fashion within the human body. Unfortunately, when their normal balance is disturbed, their populations can respond with rapid growth, giving rise to infection. Because such disturbances typically follow an event of ill health, the impact of such infections may have severe consequences, especially for already immune-compromised individuals. For example, fungal infections caused by the Candida yeast are common among individuals who are HIV-positive and can inhibit their nutrition. Candida species infections can also exacerbate infection by the tuberculosis bacterium or lead to deterioration in patients with cystic fibrosis or head and neck cancers. Similarly, infection by pathogenic Cryptococcus causes serious fungal infections that can result in pneumonia- like symptoms, infertility, central nervous system damage, encephalitis or death. In the Republic of South Africa – where the incidence of tuberculosis is high, low birth weight and infant mortality are prevalent, sexually-transmitted infections are widespread and HIV infection is at epidemic proportions – pathogenic yeasts lie at the intersection of a number of priority health issues: women and children’s health, and infectious and noncommunicable diseases. ADDRESSING THE PROBLEM The Pathogenic Yeast Research Group at the University of the Free State in South Africa is focused on the role of bioactive lipids, the fatty molecules fundamental to cellular growth in both hosts and pathogenic yeasts, with the aim of furthering development of new drugs and treatments to counteract yeast infections. The Group is particularly interested in the role of these lipids in multispecies infections, as found in cystic fibrosis, peritonitis and tuberculosis, and in cancer-related and female reproductive tract infections. In addition, they aim to discover more about the vulnerability of yeasts to antimicrobial compounds, in order to counteract the growing problem of multidrug-resistant yeast strains. This Group was the first to discover yeast oxylipins, or oxidised fatty acids, including prostaglandins. One member, Dr Carolina Pohl, is an expert in lipid metabolism in yeasts and has been involved in the discovery of the antifungal effect of acetylsalicylic acid – an oxylipin that is a mitochondrial inhibitor and anti-inflammatory compound. Since making these findings, her work has mainly concentrated on biofilm formation and subsequent infection by the Candida species C. albicans and C. dubliniensis – highly related strains that behave in similar ways, which makes them ideal as model systems of pathogenic yeasts. “These two species both have the ability to produce the lipid signalling molecule prostaglandin E2 (PGE2 ), and react in the same way to this chemical by producing germ tubes,” Pohl explains. “With both, the formation of biofilms, and subsequent increase in ability to form PGE2 , is an important disease-causing mechanism through the stimulation of inflammation.” THE ROLE OF OXYLIPINS Oxylipins have ancient origins as signalling molecules, elements of which are conserved across different kingdoms of life. Pohl recently undertook a review of oxylipin mediation of diverse inter-organism communication that leads to beneficial or deleterious interaction: between plants, animals, fungi and bacteria. Although the mechanism by which yeasts ‘sense’ the presence of bioactive lipids in their environment or host remains unclear, the review has led Pohl to conclude that oxylipins Research into the molecular mechanisms of common yeasts and bacteria at the University of the Free State in South Africa seeks to exploit the roles of lipid molecules as instigators of disease symptoms towards development of new therapeutics and antifungal agents Pathways of infection Ultrastructure Antifungal drugs Molecular biology Immunology Bioactive lipids 71
  3. 3. EFFECT OF POLYUNSATURATED FATTY ACIDS ON PATHOGENICITY OF CANDIDA ALBICANS AND CANDIDA DUBLINIENSIS OBJECTIVES • To understand the metabolism of arachidonic acid to proinflammatory oxylipins by Candida species • To understand the signalling pathways of oxylipins leading to morphological changes in Candida species • To investigate the lipid metabolisms of mixed species biofilms and the influence of the produced oxylipins on the host KEY COLLABORATORS Professor J Albertyn; Dr O Sebolai; Dr C Swart, University of the Free State, South Africa FUNDING South African National Research Foundation CONTACT Dr Carolina Pohl Associate Professor University of the Free State PO Box 339 Bloemfontein 9300 Republic of South Africa T +27 824 540 072 E DR CAROLINA POHL is an associate professor in the Department of Microbial, Biochemical and Food Biotechnology at the University of the Free State, South Africa. Her research focus is Bioactive Lipids in pathogenic Candida species. She had been part of a research group studying the distribution and functions of these compounds in environmental yeasts since completing her PhD in 1999. She started her own research group in 2006, focusing on yeasts that are important human pathogens. INTELLIGENCE are central elements in adaptation and survival; in some instances they aid organisms to defend themselves, propagate or prompt symbiotic relationships, whereas in other situations they can induce pathogenesis. Pohl’s research has shown that some yeast oxylipins work by mimicking host signalling molecules to induce an immune response. They can also bring about structural changes in the yeast that then assist its colonisation activities or help it to evade host defences. Additonally, lipids are involved in cross-talk between different cellular compartments, such as cell walls and mitochondria, which can aid the acquisition of multidrug resistance, but also underlies their potential as antifungal agents. Hence Pohl recently conducted an assessment of the effects of an omega-3 fatty acid on C. albicans and C. dubliniensis growth. In this study, the long-chain stearidonic fatty acid stopped biofilm formation by increasing reactive oxygen species production and the rate of apoptosis. The study also suggested that stearidonic acid can act in synergy with some antifungal compounds. This was the first finding that it is not just medium-chain fatty acids that can stop the growth of pathogenic yeasts. OXYLIPINS IN MULTISPECIES INFECTION Pohl’s investigation into the effects of supplementing levels of an omega-6 fatty acid, sciadonic acid, in epithelial cells on infection with C. albicans and C. dubliniensis, showed changes in PGE2 concentrations, but no reduction in those of benign anti-inflammatory omega-3 fatty acids. Furthermore a trial of the effects of aromatic amine phenothiazine – a constituent of several types of drugs in medical and veterinary use – on C. albicans biofilms, demonstrated significant reductions in biofilm metabolic activity and biomass and large reductions in PGE2 production. Given their abilities to mediate communication between species, bioactive lipids in single- species yeast or host contexts are not, however, Pohl’s only line of research. Multispecies infections, particularly concerning the production of oxylipins and subsequent reactions of the host and pathogens in mixed populations of yeasts and bacteria, are a current key area of study: “Finding out more about the types and levels of oxylipins produced by different organisms when growing together may tell us more about the influence of interaction on each microbe, and the potential effects on the host,” she elaborates. Such information would be particularly relevant for understanding the oxylipin role in generating the symptoms of mixed pathogen diseases, such as cystic fibrosis. TRANSLATING IN VITRO RESULTS In a recent project, Pohl explored the principle of inhibiting the release of arachidonic acid – a necessary component of the PGE2 production pathway. This was achieved by substituting sciadonic acid for arachidonic acid in C. albicans and C. dubliniensis biofilms grown in vitro. Her team found that the levels of PGE2 were indeed reduced and now aim to further validate this result in experiments using in vivo infection models. Potential approaches to mitigating yeast infections currently being tested by Pohl’s team include preventing the production of PGE2 in yeast biofilms, so reducing their proinflammatory effects: “An ideal drug target may be a metabolic pathway that is unique to yeasts and not found in the host,” Pohl muses. She is also working to identify the pathway that allows Candida species to react to the presence of oxylipins produced by either the host or other yeast cells. Pohl is confident that greater knowledge of the role of lipids and the mechanisms of lipid metabolism of pathogenic yeasts will lead to significant advances in the near future. With a focus on their structures and their ability to metabolise fatty acids, she now plans to follow up several candidate enzymes that her team have already identified as being implicated in prostaglandin production and virulence in Candida. With the support of the rest of the Pathogenic Yeast Research Group, Pohl also intends to further explore the influence of arachidonic acid on gene expression of pathogenic yeasts, using genomic and proteomic analysis techniques. She intends for this to deliver as yet unanticipated antifungal drug targets and lead to new applications in diverse fields, from medicine to agriculture. The Pathogenic Yeast Research Group 72 INTERNATIONAL INNOVATION