Auger-architectomics: introducing a new nanotechnology to infectious disease.

In 2010, we developed a new imaging nanotechnology called Auger-architectomics, to study drug biosensors in nano-detail. We succeeded in applying Auger atom electron physics coupled to scanning electron microscopy (SEM) and Argon-etching to cell structure exploration, thereby exposing a new dimension in structure and element composition architecture. Auger-architectomics was used to expose the fate and effect of drugs on cells. This technology should now be expanded to diseased cells. This paper will outline the development, proof of concept, and application of this imaging nanotechnology. A virtual tour is available at: http://vimeo.com/user6296337 .

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.

Oxidized fatty acids as inter-kingdom signaling molecules.

Oxylipins or oxidized fatty acids are a group of molecules found to play a role in signaling in many different cell types. These fatty acid derivatives have ancient evolutionary origins as signaling molecules and are ideal candidates for inter-kingdom communication. This review discusses examples of the ability of organisms from different kingdoms to "listen" and respond to oxylipin signals during interactions. The interactions that will be looked at are signaling between animals and plants; between animals and fungi; between animals and bacteria and between plants and fungi. This will aid in understanding these interactions, which often have implications in ecology, agriculture as well as human and animal health.

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.

Trichosporon vanderwaltii sp. nov., an asexual basidiomycetous yeast isolated from soil and beetles.

During a survey of unidentified yeast isolates deposited in the UNESCO-MIRCEN Biotechnological Yeast Culture Collection housed at the Department of Microbial, Biochemical and Food Biotechnology of the University of the Free State, one isolate obtained from soil in South Africa showed 100 % identity in D1/D2 rDNA sequence with undescribed basidiomycetous yeasts isolated from the gut of beetles from the United States of America and forest soil from Taiwan in the NCBI sequence database. Phylogenetic analyses using sequences of the D1/D2 rDNA and ITS regions indicated that all these isolates form a well-supported sub-clade that is the sister clade to the Brassicae plus Porosum clades of Trichosporon in the order Trichosporonales. Subsequent phenotypic tests revealed that asexual reproduction by budding is rare but dominated by arthroconidia resulting from segmentation of hyphae and that fusiform giant cells are characterized by budding from a broad base. These findings further suggest that these isolates belong to a single tremellomycetous yeast species for which the name Trichosporon vanderwaltii CBS 12124(T) (=NRRL Y-48732(T), =UOFS Y-1920(T)) is proposed.

Gas bubble formation in the cytoplasm of a fermenting yeast.

Current paradigms assume that gas bubbles cannot be formed within yeasts although these workhorses of the baking and brewing industries vigorously produce and release CO(2) gas. We show that yeasts produce gas bubbles that fill a significant part of the cell. The missing link between intracellular CO(2) production by glycolysis and eventual CO(2) release from cells has therefore been resolved. Yeasts may serve as model to study CO(2) behavior under pressurized conditions that may impact on fermentation biotechnology.

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.

Cryptococcus cyanovorans sp. nov., a basidiomycetous yeast isolated from cyanide-contaminated soil.

Eighteen yeast strains were isolated and identified from cyanide-contaminated soil in South Africa. According to sequence-based analyses using the D1/D2 region of the large ribosomal subunit and ITS region, three of these strains were found to be identical and represent a novel species. Phylogenetic analysis based on the combined dataset of the D1/D2 and ITS regions revealed a grouping with Cryptococcus curvatus, representing a defined clade (Curvatus) in the order Trichosporonales. The three strains were demarcated from Cryptococcus curvatus by standard physiological tests such as assimilation of lactose, xylitol, 5-keto-D-gluconate, succinate and citrate as well as growth on media containing 10 % (w/v) NaCl and 5 % (w/v) glucose. In addition, it was established that these strains could utilize up to 10 mM NaCN as sole carbon source on solid media and as sole nitrogen source in liquid media. On the basis of these findings, it is suggested that the three strains represent a novel species for which the name Cryptococcus cyanovorans sp. nov. is given (type strain CBS 11948(T) = NRRL Y-48730(T)).

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.

The effects of palm oil breakdown products on lipid turnover and morphology of fungi.

The oleaginous fungi Cryptococcus curvatus and Mucor circinelloides were used to determine the effect of palm oil breakdown products, measured as polymerized triglycerides (PTGs), on lipid turnover and on fungal growth and morphology. In M. circinelloides, we found after 7 days of growth, a decrease in biomass and in lipid utilization and accumulation at increased PTG levels, both at low and neutral pH. In C. curvatus, there was also a decrease in lipid utilization and biomass production at increased PTG levels, at both low and neutral pH. However, an increase in oil accumulation was observed at low pH while it remained similar at neutral pH for all PTG levels tested. Hairy and warty protuberances on the cell surface were observed when C. curvatus was grown on oils with 15% and 45% PTGs, respectively. Using nano scanning Auger microscopy, we found no evidence to suggest a difference in elemental composition of the surfaces of the warty protuberances compared with the rest of the cell wall surface. We conclude that the warty protuberances are outgrowths of cell walls and that the changes observed in lipid turnover in both fungi are due to the presence of palm oil breakdown products.

Anti-inflammatory drugs selectively target sporangium development in Mucor.

It is known that acetylsalicylic acid, an anti-inflammatory and anti-mitochondrial drug, targets structure development and functions of yeasts depending on elevated levels of mitochondrial activity. Using antibody probes, we previously reported that sporangia of Mucor circinelloides also contain increased mitochondrial activity, yielding high levels of 3-hydroxyoxylipins. This was, however, not found in Mortierella alpina (subgenus Mortierella). In this study we report that acetylsalicylic acid (aspirin) also targets sporangium development of Mucor circinelloides selectively, while hyphae with lower levels of mitochondrial activity are more resistant. Similar results were obtained when the anti-inflammatory compounds benzoic acid, ibuprofen, indomethacin, and salicylic acid were tested. The anti-inflammatory drugs exerted similar effects on this dimorphic fungus as found under oxygen-limited conditions. Interestingly, sporangium development of Mortierella alpina was found not to be selectively targeted by these drugs. Mortierella alpina, which could not exhibit dimorphic growth under oxygen-limited conditions, was also more sensitive to the anti-inflammatory drugs when compared with Mucor circinelloides. These results prompt further research to assess the applicability of these antimitochondrial antifungals to protect plants and animals against Mucor infections.

Development of a yeast bio-assay to screen anti-mitochondrial drugs.

Previous studies show that acetylsalicylic acid (aspirin) at low concentrations affects yeast sexual structure development in a similar fashion than oxygen depletion. This is ascribed to its anti-mitochondrial action. In this study, we report the same for other anti-inflammatory (i.e. ibuprofen, indomethacin, salicylic acid, benzoic acid) as well as anticancer (Lonidamine) drugs, also known for inhibiting mitochondrial activity in mammalian cells. This is shown by a unique yeast bio-assay, with the mitochondrion-dependent sexual structure, riboflavin production, and hyphal morphology of the yeast Eremothecium ashbyi serving as indicators. These drugs affect this yeast in a similar way as found under oxygen limitation conditions by inhibiting sexual structure development (most sensitive), riboflavin production, and yielding characteristically wrinkled and granular hyphae, presenting a unique "anoxic" morphological pattern for this yeast. Only drugs associated with anti-mitochondrial activity presented such a pattern. This bio-assay may find application in the screening for novel drugs from various sources with anti-mitochondrial actions.

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.

The yeast Saccharomyces cerevisiae- the main character in beer brewing.

Historically, mankind and yeast developed a relationship that led to the discovery of fermented beverages. Numerous inventions have led to improved technologies and capabilities to optimize fermentation technology on an industrial scale. The role of brewing yeast in the beer-making process is reviewed and its importance as the main character is highlighted. On considering the various outcomes of functions in a brewery, it has been found that these functions are focused on supporting the supply of yeast requirements for fermentation and ultimately to maintain the integrity of the product. The functions/processes include: nutrient supply to the yeast (raw material supply for brewhouse wort production); utilities (supply of water, heat and cooling); quality assurance practices (hygiene practices, microbiological integrity measures and other specifications); plant automation (vessels, pipes, pumps, valves, sensors, stirrers and centrifuges); filtration and packaging (product preservation until consumption); distribution (consumer supply); and marketing (consumer awareness). Considering this value chain of beer production and the 'bottle neck' during production, the spotlight falls on fermentation, the age-old process where yeast transforms wort into beer.

Variation in yeast mitochondrial activity associated with asci.

An increase in mitochondrial membrane potential (DeltaPsim) and mitochondrially produced 3-hydroxy (3-OH) oxylipins was experienced in asci of the nonfermentative yeasts Galactomyces reessii and Lipomyces starkeyi and the fermentative yeasts Pichia farinosa and Schizosaccharomyces octosporus. Strikingly, asci of Zygosaccharomyces bailii showed no increase in mitochondrial activity (DeltaPsim and oxylipin production). As expected, oxygen deprivation only inhibited ascus formation in those yeasts with increased ascus mitochondrial activity. We conclude that ascus formation in yeasts is not always dependent on mitochondrial activity. In this case, fermentation may provide enough energy for ascus formation in Z. bailii.

Oxylipin and mitochondrion probes to track yeast sexual cells.

When oxylipin and mitochondrion probes, i.e., fluorescing antibodies specific for 3-hydroxy fatty acids (3-OH oxylipins) and rhodamine 123 (Rh123), were added to yeast cells, these probes accumulated mainly in the sexual cells (i.e., both associated with ascospores) and not in the vegetative cells. This suggests increased mitochondrial activity in asci, since 3-OH oxylipins are mitochondrially produced and it is known that Rh123 accumulates selectively in functional mitochondria that maintain a high transmembrane potential (Delta Psi m). This increased activity may be necessary for the production and effective release of the many spores found in single-celled asci. These results may be useful in the rapid identification of asci and in yeast sexual spore mechanics, which may find application in yeast systematics as well as hydro-, aero-, and nano-technologies.