Eicosapentaenoic acid influences the pathogenesis of Candida albicans in Caenorhabditis elegans via inhibition of hyphal formation and stimulation of the host immune response.

The intake of omega-3 polyunsaturated fatty acids, including eicosapentaenoic acid (EPA), is associated with health benefits due to its anti-inflammatory properties. This fatty acid also exhibits antifungal properties in vitro. In order to determine if this antifungal property is valid in vivo, we examined how EPA affects Candida albicans pathogenesis in the Caenorhabditis elegans infection model, an alternative to mammalian host models. The nematodes were supplemented with EPA prior to infection, and the influence of EPA on C. elegans lipid metabolism, survival and immune response was studied. In addition, the influence of EPA on hyphal formation in C. albicans was investigated. It was discovered that EPA supplementation changed the lipid composition, but not the unsaturation index of C. elegans by regulating genes involved in fatty acid and eicosanoid production. EPA supplementation also delayed killing of C. elegans by C. albicans due to the inhibition of hyphal formation in vivo, via the action of the eicosanoid metabolite of EPA, 17,18-epoxyeicosatetraenoic acid. Moreover, EPA supplementation also caused differential expression of biofilm-related gene expression in C. albicans and stimulated the immune response of C. elegans. This provides a link between EPA and host susceptibility to microbial infection in this model.

Update on Candida krusei, a potential multidrug-resistant pathogen.

Although Candida albicans remains the main cause of candidiasis, in recent years a significant number of infections has been attributed to non-albicans Candida (NAC) species, including Candida krusei. This epidemiological change can be partly explained by the increased resistance of NAC species to antifungal drugs. C. krusei is a diploid, dimorphic ascomycetous yeast that inhabits the mucosal membrane of healthy individuals. However, this yeast can cause life-threatening infections in immunocompromised patients, with hematologic malignancy patients and those using prolonged azole prophylaxis being at higher risk. Fungal infections are usually treated with five major classes of antifungal agents which include azoles, echinocandins, polyenes, allylamines, and nucleoside analogues. Fluconazole, an azole, is the most commonly used antifungal drug due to its low host toxicity, high water solubility, and high bioavailability. However, C. krusei possesses intrinsic resistance to this drug while also rapidly developing acquired resistance to other antifungal drugs. The mechanisms of antifungal resistance of this yeast involve the alteration and overexpression of drug target, reduction in intracellular drug concentration and development of a bypass pathway. Antifungal resistance menace coupled with the paucity of the antifungal arsenal as well as challenges involved in antifungal drug development, partly due to the eukaryotic nature of both fungi and humans, have left researchers to exploit alternative therapies. Here we briefly review our current knowledge of the biology, pathophysiology and epidemiology of a potential multidrug-resistant fungal pathogen, C. krusei, while also discussing the mechanisms of drug resistance of Candida species and alternative therapeutic approaches.

Synthesis and function of fatty acids and oxylipins, with a focus on Caenorhabditis elegans.

Polyunsaturated fatty acids (PUFAs) exhibit a diverse range of important biological functions in most biological systems. These PUFAs can be oxygenated via enzymatic or free radical-mediated reactions to form bioactive oxygenated lipid mediators termed oxylipins. Eicosanoids are broad class of oxylipins that are transient and locally synthesized signalling molecules, including prostaglandins, leukotrienes, lipoxins and thromboxanes, which mediate various physiological responses, such as inflammation. In addition to arachidonic acid-derived eicosanoids, current developments in lipidomic methodologies have brought attention to vast number of oxylipins produced from other PUFAs, including omega-3. Although, the molecular mechanisms of how PUFAs and oxylipins contribute to majority of the fundamental biological processes are largely unclear, a model organism Caenorhabditis elegans remains a powerful model for exploring lipid metabolism and functions of PUFAs and oxylipins. For instance, the ability of C. elegans to modify fatty acid composition with dietary supplementation and genetic manipulation enables the dissection of the roles of omega-3 and omega-6 PUFAs in many biological processes that include aging, reproduction, and neurobiology. However, much remains to be elucidated concerning the roles of oxylipins, but thus far, C. elegans is well-known for the synthesis of vast set of cytochrome (CYP) eicosanoids. These CYP eicosanoids are extremely susceptible to changes in the relative bioavailability of the different PUFAs, thus providing a better insight into complex mechanisms connecting essential dietary fatty acids to various biological processes. Therefore, this review provides an overview of the synthesis and function of PUFAs and oxylipins in mammals. It also focusses on what is known regarding the production of PUFAs and oxylipins in C. elegans and their functions.

Eukaryotic opportunists dominate the deep-subsurface biosphere in South Africa.

Following the discovery of the first Eukarya in the deep subsurface, intense interest has developed to understand the diversity of eukaryotes living in these extreme environments. We identified that Platyhelminthes, Rotifera, Annelida and Arthropoda are thriving at 1.4 km depths in palaeometeoric fissure water up to 12,300 yr old in South African mines. Protozoa and Fungi have also been identified; however, they are present in low numbers. Characterization of the different species reveals that many are opportunistic organisms with an origin due to recharge from surface waters rather than soil leaching. This is the first known study to demonstrate the in situ distribution of biofilms on fissure rock faces using video documentation. Calculations suggest that food, not dissolved oxygen is the limiting factor for eukaryal population growth. The discovery of a group of Eukarya underground has important implications for the search for life on other planets in our solar system.

Candida albicans mutant construction and characterization of selected virulence determinants.

Candida albicans is a diploid, polymorphic yeast, associated with humans, where it mostly causes no harm. However, under certain conditions it can cause infections ranging from superficial to life threatening. This ability to become pathogenic is often linked to the immune status of the host as well as the expression of certain virulence factors by the yeast. Due to the importance of C. albicans as a pathogen, determination of the molecular mechanisms that allow this yeast to cause disease is important. These studies rely on the ability of researchers to create deletion mutants of specific genes in order to study their function. This article provides a critical review of the important techniques used to create deletion mutants in C. albicans and highlights how these deletion mutants can be used to determine the role of genes in the expression of virulence factors in vitro.

Haemophilus paragallinarum haemagglutinin: role in adhesion, serotyping and pathogenicity.

It is suggested that Haemophilus paragallinarum requires at least three haemagglutinins for adhesion during infection. This paper reports the partial purification and characterization of the HA-L haemagglutinin from H. paragallinarum strain 46-C3, a heat sensitive, trypsin sensitive haemagglutinin that has been shown to be the serovar specific haemagglutinin in this organism. Using the pl and molecular mass obtained, it was shown that this protein shares similarities with other types of adhesins found in Gram-negative bacteria. The haemagglutination assay conditions were optimized at pH 7.5 at 37 degrees C. It was also shown that activity is enhanced by the addition of Ca2+ and Mn2+ ions.

Cycloheximide resistance in the Lipomycetaceae revisited.

Cycloheximide (CYH) is a heterocyclic, glutarimide antibiotic that is a potent inhibitor of protein biosynthesis in most eukaryotes. This study demonstrated that yeasts from all species of the Lipomycetaceae, with the exception of Dipodascopsis spp., can grow in the presence of up to 5 g.L(-1) CYH -- a concentration that is five times higher than the accepted "highest" concentration of 1 g.L(-1) used in physiological tests for yeast identification. Lipomycetaceous yeasts are known to utilize heterocyclic nitrogen-containing compounds such as thymine as sole nitrogen source. CYH contains a glutarimide ring, which is chemically similar to thymine. We investigated the possibility that CYH resistance in the Lipomycetaceae might be due to an ability to degrade CYH and use it as the sole nitrogen source. However, we were unable to demonstrate significant growth on CYH as sole nitrogen source. When thymine was used as positive control, we could demonstrate its utilization as sole nitrogen source.

Genetic diversity of the Rep gene of beak and feather disease virus in South Africa.

A study on the genetic variation of Beak and feather disease virus (BFDV) isolates in South Africa was performed by amplifying and sequencing a region within the ORF1 of the genome. Six different BFDV isolates were found in 15 psittacine species from 6 regions within South Africa, representing three unique isolates and three isolates that clustered into a budgerigar lineage (BG) previously described.

Beak and feather disease virus haemagglutinating activity using erythrocytes from African Grey parrots and Brown-headed parrots.

Psittacine beak and feather disease (PBFD) is a common viral disease of wild and captive psittacine birds characterized by symmetric feather loss and beak deformities. The causative agent, beak and feather disease virus (BFDV), is a small, circular single-stranded DNA virus that belongs to the genus Circovirus. BFDV can be detected by PCR or the use of haemagglutination (HA) and haemagglutination inhibition (HI) assays that detect antigen and antibodies respectively. Erythrocytes from a limited number of psittacine species of Australian origin can be used in these tests. In South Africa, the high cost of these birds makes them difficult to obtain for experimental purposes. Investigation into the use of erythrocytes from African Grey parrots and Brown-headed parrots yielded positive results showing the haemagglutinating activity of their erythrocytes with purified BFDV obtained from confirmed clinical cases of the disease. The HA activity was further confirmed by the demonstration of HI using BFDV antiserum from three different African Grey parrots previously exposed to the virus and not showing clinical signs of the disease.

The testing and modification of a commercially available transport medium for the transportation of pure cultures of Haemophilus paragallinarum for serotyping.

Haemophilus paragallinarum, the causative agent of infectious coryza in poultry, is an extremely fastidious organism requiring specific growth conditions for isolation. For complete control of the disease in regions where more that one of the serovars of the different serogroups occurs, it is essential that the bacterium causing the problem be isolated and serotyped. This work describes the modification and testing of transport media, which will ensure the survival of the causative agents in suspected infectious coryza cases for transport to a laboratory where the bacterium can be isolated and serotyped. The various transport media used are based on commercially available Amies Transport Medium supplemented with the different supplements used for the growth of H. paragallinarum. It was established that the bacterium remains viable for up to 18 days in Amies Transport Medium containing all the supplements when stored at 4 degrees C or 37 degrees C. At room temperature or 25 degrees C, there was no difference in the survival of H. paragallinarum in commercial Amies Transport Medium (without charcoal) and Amies Transport Medium with supplements.

Genetic organisation of the capsule transport gene region from Haemophilus paragallinarum.

The region involved in export of the capsule polysaccharides to the cell surface of Haemophilus paragallinarum was cloned and the genetic organisation determined. Degenerate primers designed from sequence alignment of the capsule transport genes of Haemophilus influenzae, Pasteurella multocida and Actinobacillus pleuropneumoniae were used to amplify a 2.6 kb fragment containing a segment of the H. paragallinarum capsule transport gene locus. This fragment was used as a digoxigenin labelled probe to isolate the complete H. paragallinarum capsule transport gene locus from genomic DNA. The sequence of the cloned DNA was determined and analysis revealed the presence of four genes, each showing high homology with known capsule transport genes. The four genes were designated hctA, B, C and D (for H. paragallinarum capsule transport genes) and the predicted products of these genes likely encode an ATP-dependent export system responsible for transport of the capsule polysaccharides to the cell surface, possibly a member of a super family designated ABC (ATP-binding cassette) transporters.

Psittacine beak and feather disease virus in budgerigars and ring-neck parakeets in South Africa.

Psittacine beak and feather disease (PBFD) is a common disease of the psittacine species and is caused by the psittacine beak and feather disease virus (PBFDV). In this study the occurrence of the disease in ring-neck parakeets and budgerigars in South Africa suffering from feathering problems, using polymerase chain reaction as a diagnostic test was investigated. The genetic variation between viral isolates was also studied. Results indicate that PBFDV can be attributed to being the cause of feathering problems in some of the ring-neck parakeets and budgerigars in South Africa. Genetic variation of isolates occurs between species and individuals. A cheap and easy to use method of blood sample collection on filter paper for diagnostic purposes was also evaluated. It proved to be less stressful to the birds and did not inhibit further processes.

Cluster headache and the sympathetic nerve.

OBJECTIVE: To determine the effect of a sympathetic block at C7 on cluster headache. BACKGROUND: Eleven patients presenting to a pain control unit with cluster headache were included in the study after giving informed consent. METHODS: In all patients, a mixture of 5 mL of 0.5% bupivacaine hydrochloride and 1 cc of methylprednisolone acetate was injected onto the base of the C7 transverse process. RESULTS: The injection was applied during the acute phase of headache in 6 patients and all experienced immediate and complete relief. The other 5 patients received the injection between attacks. Of the 11 patients treated, 8 went into remission by aborting the cluster. In some patients, repeated injections were given before the cluster was aborted. Three patients did not respond to treatment. One patient with chronic paroxysmal hemicrania experienced pain relief of the acute attack after treatment, but the procedure did not abort the subsequent attacks. A surgical sympathectomy removing the stellate ganglion rendered him pain-free for 15 months after which he was lost to follow-up. CONCLUSION: Blocking the sympathetic nerve aborts an acute attack of cluster headache and may play a major role in aborting the cluster. Although only one patient with chronic paroxysmal hemicrania responded to surgical sympathectomy, this procedure may be considered as an alternative if there is poor response to oral medication or a sympathetic block.

Virulence of South African isolates of Haemophilus paragallinarum. Part 3: experimentally produced NAD-independent isolate.

A NAD-dependent isolate 46 (C-3) of Haemophilus paragallinarum, which was previously demonstrated to be of high virulence, was transformed to NAD independence using a plasmid isolated from a naturally occurring NAD-independent isolate of H. paragallinarum. The transformation was performed by two different methods and the identity of all of the isolates, before and after transformation was confirmed using a H. paragallinarum-specific PCR test. The transformed NAD-independent serovar C-3 isolate and the wild-type serovar C-3 isolate were used to experimentally infect vaccinated layer chickens. It was shown that the transformation to NAD independence significantly altered the virulence of the serovar C-3 isolate that was used in the transformation experiment. The mechanisms responsible for a decrease in virulence are not clear, but may be related to the pathology of the transformed isolate in the sinus of the chickens.

Transcriptional repression of ADH2-regulated beta-xylanase production by ethanol in recombinant strains of Saccharomyces cerevisiae.

The regulation of endo-beta-(1,4)-xylanase production by two different strains of Saccharomyces cerevisiae, each transformed with the XYN2 gene from Trichoderma reesei under control of the promoter of the alcohol dehydrogenase II (ADH2) gene of S. cerevisiae, was investigated. In batch culture, the rate of xylanase production was severely reduced by the pulse addition of 390 mmol ethanol l(-1). Pulses of 190-630 mmol ethanol l(-1) into aerobic glucose-limited steady-state continuous cultures reduced the xylanase activity about five-fold and showed that ethanol repressed the ADH2 promoter, as was evident from Northern blot analyses. Derepression of the ADH2-regulated xylanase gene occurred at ethanol concentrations below approximately 50 mmol l(-1).

Fps1p controls the accumulation and release of the compatible solute glycerol in yeast osmoregulation.

The accumulation of compatible solutes, such as glycerol, in the yeast Saccharomyces cerevisiae, is a ubiquitous mechanism in cellular osmoregulation. Here, we demonstrate that yeast cells control glycerol accumulation in part via a regulated, Fps1p-mediated export of glycerol. Fps1p is a member of the MIP family of channel proteins most closely related to the bacterial glycerol facilitators. The protein is localized in the plasma membrane. The physiological role of Fps1p appears to be glycerol export rather than uptake. Fps1 delta mutants are sensitive to hypo-osmotic shock, demonstrating that osmolyte export is required for recovery from a sudden drop in external osmolarity. In wild-type cells, the glycerol transport rate is decreased by hyperosmotic shock and increased by hypo-osmotic shock on a subminute time scale. This regulation seems to be independent of the known yeast osmosensing HOG and PKC signalling pathways. Mutants lacking the unique hydrophilic N-terminal domain of Fps1p, or certain parts thereof, fail to reduce the glycerol transport rate after a hyperosmotic shock. Yeast cells carrying these constructs constitutively release glycerol and show a dominant hyperosmosensitivity, but compensate for glycerol loss after prolonged incubation by glycerol overproduction. Fps1p may be an example of a more widespread class of regulators of osmoadaptation, which control the cellular content and release of compatible solutes.

MAP kinase pathways in the yeast Saccharomyces cerevisiae.

A cascade of three protein kinases known as a mitogen-activated protein kinase (MAPK) cascade is commonly found as part of the signaling pathways in eukaryotic cells. Almost two decades of genetic and biochemical experimentation plus the recently completed DNA sequence of the Saccharomyces cerevisiae genome have revealed just five functionally distinct MAPK cascades in this yeast. Sexual conjugation, cell growth, and adaptation to stress, for example, all require MAPK-mediated cellular responses. A primary function of these cascades appears to be the regulation of gene expression in response to extracellular signals or as part of specific developmental processes. In addition, the MAPK cascades often appear to regulate the cell cycle and vice versa. Despite the success of the gene hunter era in revealing these pathways, there are still many significant gaps in our knowledge of the molecular mechanisms for activation of these cascades and how the cascades regulate cell function. For example, comparison of different yeast signaling pathways reveals a surprising variety of different types of upstream signaling proteins that function to activate a MAPK cascade, yet how the upstream proteins actually activate the cascade remains unclear. We also know that the yeast MAPK pathways regulate each other and interact with other signaling pathways to produce a coordinated pattern of gene expression, but the molecular mechanisms of this cross talk are poorly understood. This review is therefore an attempt to present the current knowledge of MAPK pathways in yeast and some directions for future research in this area.

Characteristics of Fps1-dependent and -independent glycerol transport in Saccharomyces cerevisiae.

Eadie-Hofstee plots of glycerol uptake in wild-type Saccharomyces cerevisiae W303-1A grown on glucose showed the presence of both saturable transport and simple diffusion, whereas an fps1delta mutant displayed only simple diffusion. Transformation of the fps1delta mutant with the glpF gene, which encodes glycerol transport in Escherichia coli, restored biphasic transport kinetics. Yeast extract-peptone-dextrose-grown wild-type cells had a higher passive diffusion constant than the fps1delta mutant, and ethanol enhanced the rate of proton diffusion to a greater extent in the wild type than in the fps1delta mutant. In addition, the lipid fraction of the fps1delta mutant contained a lower percentage of phospholipids and a higher percentage of glycolipids than that of the wild type. Fps1p, therefore, may be involved in the regulation of lipid metabolism in S. cerevisiae, affecting membrane permeability in addition to fulfilling its specific role in glycerol transport. Simultaneous uptake of glycerol and protons occurred in both glycerol- and ethanol-grown wild-type and fps1delta cells and resulted in the accumulation of glycerol at an inside-to-outside ratio of 12:1 to 15:1. Carbonyl cyanide m-chlorophenylhydrazone prevented glycerol accumulation in both strains and abolished transport in the fps1delta mutant grown on ethanol. Likewise, 2,4-dinitrophenol inhibited transport in glycerol-grown wild-type cells. These results indicate the presence of an Fps1p-dependent facilitated diffusion system in glucose-grown cells and an Fps1p-independent proton symport system in derepressed cells.

Fps1, a yeast member of the MIP family of channel proteins, is a facilitator for glycerol uptake and efflux and is inactive under osmotic stress.

The Saccharomyces cerevisiae FPS1 gene, which encodes a channel protein belonging to the MIP family, has been isolated previously as a multicopy suppressor of the growth defect of the fdp1 mutant (allelic to GGS1/TPS1) on fermentable sugars. Here we show that overexpression of FPS1 enhances glycerol production. Enhanced glycerol production caused by overexpression of GPD1 encoding glycerol-3-phosphate dehydrogenase also suppressed the growth defect of ggs1/tps1 delta mutants, suggesting a novel role for glycerol production in the control of glycolysis. The suppression of ggs1/tps1 delta mutants by GPD1 depends on the presence of Fps1. Mutants lacking Fps1 accumulate a greater part of the glycerol intracellularly, indicating that Fps1 is involved in glycerol efflux. Glycerol-uptake experiments showed that the permeability of the yeast plasma membrane for glycerol consists of an Fps1-independent component probably due to simple diffusion and of an Fps1-dependent component representing facilitated diffusion. The Escherichia coli glycerol facilitator expressed in a yeast fps1 delta mutant can restore the characteristics of glycerol uptake, production and distribution fully, but restores only partially growth of a ggs1/tps1 delta fps1 delta double mutant on glucose. Fps1 appears to be closed under hyperosmotic stress when survival depends on intracellular accumulation of glycerol and apparently opens rapidly when osmostress is lifted. The osmostress-induced High Osmolarity Glycerol (HOG) response pathway is not required for inactivation of Fps1. We conclude that Fps1 is a regulated yeast glycerol facilitator controlling glycerol production and cytosolic concentration, and might have additional functions.