Genome-wide functional analysis in Candida albicans.

Candida albicans is an important etiological agent of superficial and life-threatening infections in individuals with compromised immune systems. To date, we know of several overlapping genetic networks that govern virulence attributes in this fungal pathogen. Classical use of deletion mutants has led to the discovery of numerous virulence factors over the years, and genome-wide functional analysis has propelled gene discovery at an even faster pace. Indeed, a number of recent studies using large-scale genetic screens followed by genome-wide functional analysis has allowed for the unbiased discovery of many new genes involved in C. albicans biology. Here we share our perspectives on the role of these studies in analyzing fundamental aspects of C. albicans virulence properties.

Pseudomonas aeruginosa produces aspirin insensitive eicosanoids and contributes to the eicosanoid profile of polymicrobial biofilms with Candida albicans.

The interaction of clinically relevant microorganisms is the focus of various studies, e.g. the interaction between the pathogenic yeast, Candida albicans, and the bacterium, Pseudomonas aeruginosa. During infection both release arachidonic acid, which they can transform into eicosanoids. This study evaluated the production of prostaglandin E2, prostaglandin F2α and 15-hydroxyeicosatetraenoic acid by biofilms of P. aeruginosa and C. albicans. The influence of co-incubation, acetylsalicylic acid and nordihydroguaiaretic acid on biofilm formation and eicosanoid production was evaluated. Acetylsalicylic acid decreased colony forming units of P. aeruginosa, but increased metabolic activity and eicosanoid production of the cells. In contrast to prostaglandin E2, prostaglandin F2a production by C. albicans was insensitive to acetylsalicylic acid, indicating that different enzymes are responsible for their production in this yeast. Nordihydroguaiaretic acid inhibited biofilm formation by P. aeruginosa, however co-incubation provided protection against this inhibitor. Production of these eicosanoids could affect pathogen-clearance and infection dynamics and this previously uncharacterized facet of interaction could facilitate novel therapeutic intervention against polymicrobial infection.

Candida albicans and Pseudomonas aeruginosa Interaction, with Focus on the Role of Eicosanoids.

Candida albicans is commonly found in mixed infections with Pseudomonas aeruginosa, especially in the lungs of cystic fibrosis (CF) patients. Both of these opportunistic pathogens are able to form resistant biofilms and frequently infect immunocompromised individuals. The interaction between these two pathogens, which includes physical interaction as well as secreted factors, is mainly antagonistic. In addition, research suggests considerable interaction with their host, especially with immunomodulatory lipid mediators, termed eicosanoids. Candida albicans and Pseudomonas aeruginosa are both able to utilize arachidonic acid (AA), liberated from the host cells during infection, to form eicosanoids. The production of these eicosanoids, such as Prostaglandin E2, by the host and the pathogens may affect the dynamics of polymicrobial infection and the outcome of infections. It is of considerable importance to elucidate the role of host-produced, as well as pathogen-produced eicosanoids in polymicrobial infection. This review will focus on in vitro as well as in vivo interaction between C. albicans and P. aeruginosa, paying special attention to the role of eicosanoids in the cross-talk between host and the pathogens.

Virulence of South African Candida albicans strains isolated from different clinical samples.

Candida albicans is a dimorphic opportunistic pathogenic yeast that is commonly isolated from different anatomical sites and clinical samples. It possesses several virulence factors, including secretion of hydrolytic enzymes, the ability to adhere to abiotic surfaces and cells, and the ability to penetrate tissues. We determined the level of in vitro expression of virulence factors by South African clinical C. albicans strains and the correlation among them. Furthermore, we determined whether there is a correlation between the levels of virulence factors expressed by a strain and the anatomical site from which it was isolated. The overall virulence of strains expressing different levels of these virulence factors in vitro was examined using a chorioallantoic membrane (CAM) chicken embryo model of infection, with variations observed in the production of hydrolytic enzymes. Most strains were able to produce in vitro high levels of protease and phospholipase and medium levels of lipase. Using the quantitative agar invasion assay, most strains were found to be highly invasive. No relationships of virulence factors produced in vitro were observed, except for a weak negative correlation between protease activity and invasiveness, as well as protease activity and cell surface hydrophobicity. There was no indication that the in vitro differences in virulence factors were correlated with virulence in the CAM model. However, we found that the infection model is sensitive enough to distinguish different virulence levels of strains.

Phenothiazine is a potent inhibitor of prostaglandin E2 production by Candida albicans biofilms.

Candida albicans is an important opportunistic yeast pathogen of humans and has the ability to form drug-resistant biofilms, with increased expression of multidrug ATP-binding cassette (ABC) transporters. These biofilms are also capable of secreting immune-modulating prostaglandin E2 (PGE2 ) from host-derived arachidonic acid (AA). Phenothiazine, an aromatic amine, and its derivatives display broad activity as inhibitors and antioxidants. These compounds have fungistatic and fungicidal activity against planktonic C. albicans and can inhibit ABC transporters of C. albicans. This study investigated the effect of phenothiazine on biofilm formation, ABC transporters and PGE2 production by C. albicans. This was carried out by growing C. albicans biofilms in the presence of AA and phenothiazine and measuring the biomass as well as reduction of 2,3-bis(2-methoxy-4-nitro-5-sulphophenyl)-5[(phenylamino) carbonyl]-2H tetrazolium hydroxide. The effect on ABC transporters was determined by rhodamine 6G efflux, and the concentration of PGE2 was determined by a monoclonal PGE2 enzyme-linked immunosorbent assay and LC/MS/MS. Our results indicate that phenothiazine can cause a reduction in both the metabolic activity and biomass of C. albicans biofilms, without affecting biofilm morphology or ABC transporters. However, it is a potent inhibitor of PGE2 production by C. albicans biofilms.

Arachidonic acid metabolites in pathogenic yeasts.

Although most of what is known about the biology and function of arachidonic acid metabolites comes from the study of mammalian biology, these compounds can also be produced by lower eukaryotes, including yeasts and other fungi. It is also in this group of organisms that the least is known about the metabolic pathways leading to the production of these compounds as well as the functions of these compounds in the biology of fungi and yeasts. This review will deal with the discovery of oxylipins from polyunsaturated fatty acids, and more specifically the arachidonic acid derived eicosanoids, such as 3-hydroxy eicosatetraenoic acid, prostaglandin F2α and prostaglandin E2, in yeasts starting in the early 1990s. This review will also focus on what is known about the metabolic pathways and/or proteins involved in the production of these compounds in pathogenic yeasts. The possible roles of these compounds in the biology, including the pathology, of these organisms will be discussed.

Polyunsaturated fatty acids cause apoptosis in C. albicans and C. dubliniensis biofilms.

BACKGROUND: Polyunsaturated fatty acids (PUFAs) have antifungal properties, but the mode by which they induce their action is not always clear. The aim of the study was to investigate apoptosis as a mode of action of antifungal PUFAs (stearidonic acid, eicosapentaenoic acid and docosapentaenoic acid) which are inhibitory towards biofilm formation of C. albicans and C. dubliniensis. METHODS: Candida biofilms were grown in the absence or presence of 1mM PUFAs (linoleic acid, stearidonic acid, eicosapentaenoic acid, docosapentaenoic acid) for 48h at 37°C. The effect of these PUFAs on the membrane fatty acid profile and unsaturation index, oxidative stress, mitochondrial transmembrane potential and apoptosis was evaluated. RESULTS: When biofilms of C. albicans and C. dubliniensis were exposed to certain PUFAs there was an increase in unsaturation index of the cellular membranes and accumulation of intracellular reactive oxygen species (ROS). This resulted in apoptosis, evidenced by reduced mitochondrial membrane potential and nuclear condensation and fragmentation. The most effective PUFA was stearidonic acid. CONCLUSIONS: The resultant cell death of both C. albicans and C. dubliniensis is due to apoptosis. GENERAL SIGNIFICANCE: Due to the increase in drug resistance, alternative antifungal drugs are needed. A group of natural antifungal compounds is PUFAs. However, understanding their mechanisms of action is important for further use and development of these compounds as antifungal drugs. This paper provides insight into a possible mode of action of antifungal PUFAs.

Sciadonic acid modulates prostaglandin E2 production by epithelial cells during infection with C. albicans and C. dubliniensis.

Candida albicans is an important opportunistic pathogen in humans. During infection, arachidonic acid (ω6) is released from host phospholipids, leading to the production of host and yeast derived prostaglandin E(2) (PGE(2)). This stimulates yeast hyphal formation, is immunomodulatory and causes cell damage during infection. Although supplementation of mammalian cells with ω3 fatty acids has received attention due to their immunomodulatory and anti-inflammatory activities, increased production of ω3 fatty acid metabolites could lower the host's ability to combat infections. Since mammalian cells cannot produce PGE(2) from sciadonic acid (SA), a non-methylene interrupted ω6 fatty acid (NMIFA), supplementation of cells with SA may decrease the production of PGE(2) without increasing levels of ω3 fatty acid metabolites. Our study evaluated PGE(2) production by SA supplemented epithelial cells in response to Candida albicans and C. dubliniensis. We show that PGE(2) production during infection can be modulated by incorporation of SA into host lipids and that this does not influence the levels of ω3 fatty acids in the epithelial cells.

Effect of inhibitors of arachidonic acid metabolism on prostaglandin E2 production by Candida albicans and Candida dubliniensis biofilms.

Arachidonic acid (AA) is released from infected host cells during Candida albicans infection and may serve as carbon source for yeast growth and as precursor for the production of biologically active eicosanoids, such as prostaglandin E2 (PGE2) by C. albicans. However, the mechanism involved in this production is still unclear. Therefore, it was of interest to investigate the effect of different arachidonic acid metabolism inhibitors on PGE2 production by biofilms of C. albicans and the closely related C. dubliniensis. This was done by growing Candida biofilms in the presence of AA as well as cytochrome P450 (CYP), multicopper oxidase, cyclooxygenase or lipoxygenase inhibitors. The concentration of PGE2 was determined by a monoclonal PGE2 enzyme-linked immunosorbent assay and verified with LCMS/MS. The results obtained indicate the ability of C. albicans and C. dubliniensis biofilms to produce PGE2 from exogenous AA. The use of different inhibitors suggested that CYPs and multicopper oxidases are involved in PGE2 production by these Candida biofilms.

Candida albicans or Candida dubliniensis?

Candida albicans is increasing as an opportunistic pathogen causing candidemia and candidiasis worldwide. In addition, other non-albicans Candida species are now also associated with pertinent infections. These include the closely related C. dubliniensis, which shares many phenotypic similarities with C. albicans. These similarities pose problems in the identification of isolates and have previously led to misidentification of these species. As a result, several identification techniques based on phenotypic and genotypic characteristics have been developed to differentiate between these Candida species. This review will focus on the similarities and differences between these two Candida species highlighting different identification methods and their advantages and disadvantages.

Effect of marine polyunsaturated fatty acids on biofilm formation of Candida albicans and Candida dubliniensis.

The effect of marine polyunsaturated fatty acids on biofilm formation by the human pathogens Candida albicans and Candida dubliniensis was investigated. It was found that stearidonic acid (18:4 n-3), eicosapentaenoic acid (20:5 n-3), docosapentaenoic acid (22:5 n-3) and docosahexaenoic acid (22:6 n-3) have an inhibitory effect on mitochondrial metabolism of both C. albicans and C. dubliniensis and that the production of biofilm biomass by C. dubliniensis was more susceptible to these fatty acids than C. albicans. Ultrastructural differences, which may be due to increased oxidative stress, were observed between treated and untreated cells of C. albicans and C. dubliniensis with formation of rough cell walls by both species and fibrillar structures in C. dubliniensis. These results indicate that marine polyunsaturated fatty acids may be useful in the treatment and/or prevention of biofilms formed by these pathogenic yeasts.

Arachidonic acid increases antifungal susceptibility of Candida albicans and Candida dubliniensis.

OBJECTIVES: During Candida albicans infection, arachidonic acid (AA) is released from phospholipids of infected host cell membranes and used by C. albicans as the sole carbon source and for production of eicosanoids. AA can be incorporated into the phospholipids of yeasts, influencing the saturation level and fluidity of yeast cell membranes. It is suggested that the effectiveness of polyene (e.g. amphotericin B) and imidazole (e.g. clotrimazole) antifungals may depend upon the level of unsaturation and ergosterol in the membrane. Therefore, the aim of this study was to evaluate the effect of AA on the cell membrane and susceptibility of C. albicans and Candida dubliniensis biofilms towards amphotericin B and clotrimazole. METHODS: Both yeasts were grown in the presence and absence of AA and the effect of amphotericin B and clotrimazole was examined by confocal laser scanning microscopy, determination of mitochondrial metabolism, unsaturation index of the phospholipid fractions and ergosterol content of the membranes. RESULTS: AA had no effect on the viability of the cells in the biofilm; however, there was an increase in ergosterol levels as well as antifungal susceptibility of biofilms grown in the presence of AA. CONCLUSIONS: AA influences phospholipid unsaturation and ergosterol content of both yeasts C. albicans and C. dublininensis, increasing susceptibility towards the antifungals. Pretreatment of biofilms with polyunsaturated fatty acids may result in the reduction in antifungal dose needed to inhibit biofilms.