Opportunistic Yeast Candida Albicans Research Articles

Candida albicans β-1,2-mannosyltransferase Bmt3 prompts the elongation of the cell-wall phosphopeptidomannan.

β-1,2-Linked mannosides are expressed on numerous cell-wall glycoconjugates of the opportunistic pathogen yeast Candida albicans. Several studies evidenced their implication in the host-pathogen interaction and virulence mechanisms. In the present study, we characterized the in vitro activity of CaBmt3, a β-1,2-mannosyltransferase involved in the elongation of β-1,2-oligomannosides oligomers onto the cell-wall polymannosylated N-glycans. A recombinant soluble enzyme Bmt3p was produced in Pichia pastoris and its enzyme activity was investigated using natural and synthetic oligomannosides as potential acceptor substrates. Bmt3p was shown to exhibit an exquisite enzymatic specificity by adding a single terminal β-mannosyl residue to α-1,2-linked oligomannosides capped by a Manβ1-2Man motif. Furthermore, we demonstrated that the previously identified CaBmt1 and CaBmt3 efficiently act together to generate Manβ1-2Manβ1-2[Manα1-2]n sequence from α-1,2-linked oligomannosides onto exogenous and endogenous substrates.

Candida albicans Flu1-Mediated Efflux of Salivary Histatin 5 Reduces Its Cytosolic Concentration and Fungicidal Activity

Histatin 5 (Hst 5) is a salivary human antimicrobial peptide that is toxic to the opportunistic yeast Candida albicans. Fungicidal activity of Hst 5 requires intracellular translocation and accumulation to a threshold concentration for it to disrupt cellular processes. Previously, we observed that total cytosolic levels of Hst 5 were gradually reduced from intact cells, suggesting that C. albicans possesses a transport mechanism for efflux of Hst 5. Since we identified C. albicans polyamine transporters responsible for Hst 5 uptake, we hypothesized that one or more polyamine efflux transporters may be involved in the efflux of Hst 5. C. albicans FLU1 and TPO2 were found to be the closest homologs of Saccharomyces cerevisiae TPO1, which encodes a major spermidine efflux transporter, indicating that the products of these two genes may be involved in efflux of Hst 5. We found that flu1Δ/Δ cells, but not tpo2Δ/Δ cells, had significant reductions in their rates of Hst 5 efflux and had significantly higher cytoplasmic Hst 5 and Hst 5 susceptibilities than did the wild type. We also found that flu1Δ/Δ cells had reduced biofilm formation compared to wild-type cells in the presence of Hst 5. Transcriptional levels of FLU1 were not altered over the course of treatment with Hst 5; therefore, Hst 5 is not likely to induce FLU1 gene overexpression as a potential mechanism of resistance. Thus, Flu1, but not Tpo2, mediates efflux of Hst 5 and is responsible for reduction of its toxicity in C. albicans.

High resolution antifungal HPLC profiling for the early identification of compounds in plants from French Polynesia

In order to rapidly localize antifungal compounds in crude plant extracts, micro-dilution assays with the opportunistic yeast Candida albicans were used in combination with semi preparative HPLC micro-fractionation [1]. Using this approach, HPLC antifungal profiles of selected active crude methanolic extracts of plants from French Polynesia were efficiently recorded.

How Do Microbial Pathogens Make CENs?

In a variety of pathogens, drug resistance and aneuploidy areintimately associated. Anueploidy is believed to alter the dosage ofcertain genes that can impart drug resistance. Generation of a newchromosome by duplication of chromosome segments followed bytelomere addition in a pathogenic yeast Candida glabrata [1] andisochromosome formation by breakage of chromosome 5 at thecentromere followed by joining two identical arms of chromosome5 in an opportunistic yeast Candida albicans [2] have been shown tooccur frequently in drug-resistant isolates. Fluconazole-resistantstrains of another pathogenic fungus, Cryptococcus neoformans, havebeen shown to be disomic for certain chromosomes [3].Experimental evidence suggests that acquisition of chromosomesby the plant pathogenic fungus Fusarium can convert a non-pathogenic strain to a pathogenic one [4]. Anueploidy has beenshown to be the cause of drug resistance in the protozoan parasiteLeishmania as well [5].Improper chromosome segregation is one route to aneuploidy.The centromere–kinetochore complex facilitates interaction be-tween a chromosome and the spindle microtubules to ensure equalsegregation of chromosomes from a mother to daughter cells. Inaddition to serving as sites of protein assembly to formkinetochores, centromeres also hold two sister chromatids togetheruntil the onset of anaphase by balancing opposing forces acting onthem—a pole-ward (outward) force generated by spindle micro-tubules depolymerizing toward opposite poles, and a cohesive(inward) force between two sister chromatids. Paradoxically, inspite of performing the conserved function of chromosomesegregation, centromere (CEN) DNA sequence and organizationof CEN DNA elements vary widely among eukaryotes. In contrast,several kinetochore proteins are evolutionarily conserved, al-though sometimes this conservation is restricted to a group oforganisms. For example, the Dam1 complex, an outer kinetochoreprotein complex, is fungus specific and is essential for viability in C.albicans [6,7]. Thus, this complex may be a suitable target fordevelopment of anti-fungal drugs. However, a CEN-specifichistone H3 variant of the CENP-A/Cse4 family is found to beuniversally associated with the formation of specialized and uniquechromatin at all functional CENs despite seemingly diverse CENDNA sequences.

Candida albicans virulence and drug-resistance requires the O-acyltransferase Gup1p

BackgroundGUP1 gene was primarily identified in Saccharomyces cerevisiae being connected with glycerol uptake defects in association with osmotic stress response. Soon after, Gup1p was implicated in a complex and extensive series of phenotypes involving major cellular processes. These include membrane and wall maintenance, lipid composition, bud-site selection, cytoskeleton orientation, vacuole morphology, secretory/endocytic pathway, GPI anchors remodelling, and lipid-ordered domains assembly, which is compatible with their inclusion in the Membrane Bound O-acyl transferases (MBOAT) family. In mammals, it has been described as a negative regulator of the Sonic hedgehog pathway involved in morphogenesis, differentiation, proliferation, among other processes.ResultsWe show that Candida albicans Gup1p strongly interferes with the capacity of cells to develop hyphae, to adhere, to invade, and to form a biofilm, all of which are significant virulence factors. Furthermore, the mutant colonies exhibited an aberrant morphology/differentiation pattern. Identically to S. cerevisiae, Cagup1Δ null mutant was more resistant to antifungals like fluconazole, ketoconazole, and clotrimazole, and displayed an abnormal even sterol distribution at the plasma membrane.ConclusionsThis work is the first study in the opportunistic yeast Candida albicans, showing a role for the GUP1 gene in virulence as well as in the mechanisms underlying antifungal resistance. Moreover, its impact is even more significant since these results, taken together with all the knowledge about GUP1 gene (from S. cerevisiae and mammals) give consistence to the possibility that Gup1p may be part of a yeast morphogenic pathway parallel to the mammalian Hedgehog.

Factor H and Factor H-Related Protein 1 Bind to Human Neutrophils via Complement Receptor 3, Mediate Attachment to Candida albicans, and Enhance Neutrophil Antimicrobial Activity

The host complement system plays an important role in protection against infections. Several human-pathogenic microbes were shown to acquire host complement regulators, such as factor H (CFH), that downregulate complement activation at the microbial surface and protect the pathogens from the opsonic and lytic effects of complement. Because CFH can also bind to host cells, we addressed the role of CFH and CFH-related proteins as adhesion ligands in host-pathogen interactions. We show that the CFH family proteins CFH, CFH-like protein 1 (CFHL1), CFH-related protein (CFHR) 1, and CFHR4 long isoform bind to human neutrophil granulocytes and to the opportunistic human-pathogenic yeast Candida albicans. Two major binding sites, one within the N-terminus and one in the C-terminus of CFH, were found to mediate binding to neutrophils. Complement receptor 3 (CD11b/CD18; alpha(M)beta2 integrin) was identified as the major cellular receptor on neutrophils for CFH, CFHL1, and CFHR1, but not for CFHR4 long isoform. CFH and CFHR1 supported cell migration. Furthermore, CFH, CFHL1, and CFHR1 increased attachment of neutrophils to C. albicans. Adhesion of neutrophils to plasma-opsonized yeasts was reduced when CFH binding was inhibited by specific Abs or when using CFH-depleted plasma. Yeast-bound CFH and CFHR1 enhanced the generation of reactive oxygen species and the release of the antimicrobial protein lactoferrin by human neutrophils, and resulted in a more efficient killing of the pathogen. Thus, CFH and CFHR1, when bound on the surface of C. albicans, enhance antimicrobial activity of human neutrophils.

Genotoxicity of Tryptophol in a Battery of Short‐Term Assays on Human White Blood Cells in vitro

The genotoxic effects of tryptophol (indole-3-ethanol), an aromatic alcohol and known secondary metabolite of the opportunistic yeast Candida albicans and other Candida spp., were studied using a battery of short-term assays on human white blood cells in vitro. The concentration range of tryptophol tested was 0.25 mM to 2.00 mM. Lymphocyte viability and induction of apoptosis/necrosis were studied by simultaneous use of a fluorescent assay with ethidium bromide and acridine orange. Levels of primary DNA damage and dynamics of DNA repair were evaluated using the alkaline comet assay while the levels and nature of residual DNA damage were assessed by the analysis of structural chromosome aberrations, the sister chromatid exchange test and the cytokinesis-block micronucleus assay. The results obtained suggest cytotoxic, cytostatic and genotoxic effects of the tryptophol treatment in vitro that were mainly dose-dependent. The type and the extent of DNA lesions detected in tryptophol-treated samples indicate the possibility that observed damage is mediated by highly reactive aldehyde metabolite and/or free radicals produced by treatment. The results show that mortality of lymphocytes in tryptophol-treated samples was primarily caused by apoptosis. The generation of additional DNA strand breaks and cytogenetic consequences (chromosome aberrations, sister chromatid exchanges and micronuclei), as observed in this study, sustain the possibility that tryptophol toxicity is mediated by the formation of DNA cross-links and aldehyde-protein adducts. In conclusion, this preliminary study elucidates only a part of tryptophol toxicity to human cells. Because current evidence is not sufficient to obtain information relevant for human risk assessment, further in vitro and in vivo studies are essential in order to clarify remaining issues, especially to elucidate the exact mechanisms and nature of the damage produced following treatment as well to estimate possible interindividual variability in genotoxic responses to the chemical.

Chemical Composition and Antimicrobial Activity of Essential Oil of Wild-Growing Salvia officinalis L. from Montenegro

The essential oil composition of wild-growing Salvia officinalis L. (Lamiaceae) from Montenegro was analysed by GC-FID and GC-MS and its antimicrobial activity tested at different oil concentrations. Twenty-five compounds were identified in the essential oil, with major components: α-thujone (29.50%), camphor (22.52%) and 1,8-cineole (12.19%). The amount of several minor compounds in the essential oil was also significant: camphene (5.35%), borneol (4.43%) and limonene (4.21%). Sixteen different microorganisms were used in this study as test organisms, including four reference ATCC strains and twelve clinically isolated strains. These included human pathogens, food poisoning and spoilage bacteria and the blastomycete opportunistic yeast Candida albicans. Results showed that the sage essential oil exhibited a significant antibacterial and antifungal activity. The most sensitive strain was Staphyloccocus aureus, and of Gram-negative species even at the lowest concentration of oil, Providencia stuartii was the most sensitive. The origin of the bacterial strain and the fact of being Gram-positive or Gram-negative appeared to have little influence on the sensitivity of the oil; in general all the bacteria, except Pseudomonas aeruginosa and Salmonella enteridis, were susceptible in various degrees to the different concentration of sage essential oil. Noteworthy is significant activity of sage essential oil against clinically isolated bacterial strains and yeast C. albicans.

Energy Depletion Protects Candida albicans against Antimicrobial Peptides by Rigidifying Its Cell Membrane

Inhibitors of the energy metabolism, such as sodium azide and valinomycin, render yeast cells completely resistant against the killing action of a number of cationic antimicrobial peptides, including the salivary antimicrobial peptide Histatin 5. In this study the Histatin 5-mediated killing of the opportunistic yeast Candida albicans was used as a model system to comprehensively investigate the molecular basis underlying this phenomenon. Using confocal and electron microscopy it was demonstrated that the energy poison azide reversibly blocked the entry of Histatin 5 at the level of the yeast cell wall. Azide treatment hardly induced depolarization of the yeast cell membrane potential, excluding it as a cause of the lowered sensitivity. In contrast, the diminished sensitivity to Histatin 5 of energy-depleted C. albicans was restored by increasing the fluidity of the membrane using the membrane fluidizer benzyl alcohol. Furthermore, rigidification of the membrane by incubation at low temperature or in the presence of the membrane rigidifier Me(2)SO increased the resistance against Histatin 5, while not affecting the energy charge of the cell. In line, azide induced alterations in the physical state of the interior of the lipid bilayer. These data demonstrate that changes in the physical state of the membrane underlie the increased resistance to antimicrobial peptides.

Histatin 5 and derivatives: Their localization and effects on the ultra-structural level

Histatins, a family of cationic peptides present in saliva, are active against the opportunistic yeast Candida albicans. The mechanism of action is still unclear. Histatin 5 and more potent synthetic variants, dhvar4 and dhvar5, were used to study localization and effects on morphology on the ultra-structural level. Although all peptides induced leakage, no association with the plasma membrane, indicative for permanent pores, was observed with immuno-gold-labeling. Freeze-fracturing showed severe changes of the plasma membrane. Together with, for the dhvars, the loss of intracellular integrity, this suggests that leakage may be a secondary effect rather than an effect of formation of permanent pores.

Adhesive and mammalian transglutaminase substrate properties of Candida albicans Hwp1.

The pathogenesis of candidiasis involves invasion of host tissues by filamentous forms of the opportunistic yeast Candida albicans. Morphology-specific gene products may confer proinvasive properties. A hypha-specific surface protein, Hwp1, with similarities to mammalian small proline-rich proteins was shown to serve as a substrate for mammalian transglutaminases. Candida albicans strains lacking Hwp1 were unable to form stable attachments to human buccal epithelial cells and had a reduced capacity to cause systemic candidiasis in mice. This represents a paradigm for microbial adhesion that implicates essential host enzymes.

In Vivo Expression and Localization of Candida albicans Secreted Aspartyl Proteinases during Oral Candidiasis in HIV-Infected Patients

Isoforms of aspartyl proteinase (Sap), which are encoded by at least nine related SAP genes, have been implicated to be a major virulence factor of the opportunistic yeast Candida albicans in experimental infections. Although it is generally assumed that proteinases are important for infections, detailed information on the pathogenetic role of Saps is still lacking. The same applies to the question whether the genes and corresponding isoforms of the enzyme are expressed during oral infection. For in vivo investigations, parts of the lesional oral epithelium were collected from three HIV-infected patients with oropharyngeal candidiasis. Immunoelectron microscopy was performed (pre- and post-embedding gold labeling with silver enhancement) using an anti-Sap murine monoclonal antibody directed against the gene products Sap1-3. It was possible to demonstrate expression of Sap antigens in each of the three samples of human oral candidiasis. This suggests that at least one of the genes SAP1-3 was expressed at the time of sample collection. Furthermore, a possible role of the enzymes during the interaction of yeast cells and mucosal cells is suggested: the majority of Sap antigens is secreted by those C. albicans cells that adhere directly to the epithelial surface. Sap immunoreactivity can be detected in particular at the site of close contact between C. albicans and epithelial cells, suggesting a pathogenetic role of the Saps in host-fungal interaction. Thus, inhibition of the enzyme might prove to be an important alternative in the prevention and treatment of candidiasis.

Effectiveness of a vaccine composed of heat-killed Candida albicans and a novel mucosal adjuvant, LT(R192G), against systemic candidiasis.

The incidence of fungal infections caused by the opportunistic yeast Candida albicans has increased significantly in recent years. The ability to vaccinate selected patients against the organism would be advantageous. In this paper we describe a potential anti-C. albicans vaccine consisting of heat-killed C. albicans (HK-CA) in combination with the novel mucosal adjuvant LT(R192G), a genetically detoxified form of the heat-labile toxin of enterotoxigenic Escherichia coli. Groups of male CBA/J mice were immunized intranasally on three occasions at weekly intervals with 2 x 10(7) HK-CA per dose, alone or in conjunction with 10 micrograms of LT(R192G) per dose. Two weeks following the last application of antigen, some animals were challenged intravenously (i.v.) with 10(4), 10(5), or 10(6) viable C. albicans to assess protection as measured by survival and/or culture. Some groups of animals were footpad tested with C. albicans mannan to assess delayed-type hypersensitivity (DTH), and all the animals were bled for antibody assays. In two independent studies, all the animals immunized with HK-CA plus LT(R192G) were able to eradicate 10(4) C. albicans completely, as determined by kidney culture 4 weeks after challenge. Animals immunized with HK-CA only had reduced levels of C. albicans compared to the adjuvant or saline-only control. Greatly enhanced survival was observed when mice immunized with HK-CA plus LT(R192G) were challenged with 10(5) live C. albicans as well. Animals immunized with HK-CA plus LT(R192G) developed a significant DH response, while those given HK-CA alone developed only marginal DH responses. High immunoglobulin G (IgG) levels to cytoplasmic antigens developed in mice immunized with HK-CA plus LT(R192G), but they were found only after i.v. challenge. Addition of adjuvant shifted the antibody isotype production in i.v.-challenged animals to a response dominated by IgG2a. Clearly, intranasal immunization with killed C. albicans in conjunction with LT(R192G) afforded significant levels of protection. This novel approach offers new possibilities for the development of an effective vaccine against candidiasis for use in humans.

Regulation of Chemokine Gene Expression in Human Peripheral Blood Neutrophils Phagocytosing Microbial Pathogens

Production of chemokines (chemotactic cytokines) by neutrophils is likely to be important in the regulation of inflammation and the control of infection. In this study we show that exposure of human neutrophils to various microbial pathogens leads to the production of both macrophage inflammatory protein 1alpha (MIP-1alpha) and IL-8. The bacterial microbes, Salmonella typhimurium and Pseudomonas aeruginosa, and Staphylococcus aureus all strongly induced both IL-8 and MIP-1alpha secretion, whereas Streptococcus pneumoniae, Staphylococcus epidermidis, and the opportunistic yeast Candida albicans were less potent. Saccharomyces cerevisiae and zymosan both induced IL-8 secretion but failed to stimulate that of MIP-1alpha. Coincubation of neutrophils with the proinflammatory cytokine TNF-alpha and the micro-organisms also led to differential expression of MIP-1alpha and IL-8. Significant enhancement of the induction of both MIP-1alpha and IL-8 by S. typhimurium, P. aeruginosa, and S. pneumoniae as well as by C. albicans was observed. In contrast, while IL-8 production in response to S. cerevisiae and zymosan was enhanced in the presence of TNF-alpha, no MIP-1alpha was produced. These combined results indicate that while neutrophils exposed to some micro-organisms alone or in the presence of inflammatory cytokines such as TNF-alpha will produce both MIP-1alpha and IL-8, resulting in generation of signals for the recruitment of mononuclear leukocytes and neutrophils, respectively, certain types of microorganisms can skew this response toward synthesis of IL-8.

Increased mRNA levels of ERG16, CDR, and MDR1 correlate with increases in azole resistance in Candida albicans isolates from a patient infected with human immunodeficiency virus.

Resistance to antifungal drugs, specifically azoles such as fluconazole, in the opportunistic yeast Candida albicans has become an increasing problem in human immunodeficiency virus (HIV)-infected individuals. The molecular mechanisms responsible for this resistance have only recently become apparent and can include alterations in the target enzyme of the azole drugs (lanosterol 14alpha demethylase [14DM]), or in various efflux pumps from both the ABC transporter and major facilitator gene families. To determine which of these possible mechanisms was associated with the development of drug resistance in a particular case, mRNA levels have been studied in a series of 17 clinical isolates taken from a single HIV-infected patient over 2 years, during which time the levels of fluconazole resistance of the strain increased over 200-fold. Using Northern blot analysis of steady-state levels of total RNA from these isolates, we observed increased mRNA levels of ERG16 (the 14DM-encoding gene), CDR1 (an ABC transporter), and MDR1 (a major facilitator) in this series. The timing of the increase in mRNA levels of each of these genes correlated with increases in fluconazole resistance of the isolates. Increased mRNA levels were not observed for three other ABC transporters, two other genes in the ergosterol biosynthetic pathway, or the NADPH-cytochrome P-450 oxidoreductase gene that transfers electrons from NADPH to 14DM. Increases in mRNA levels of ERG16 and CDR1 correlated with increased cross-resistance to ketoconazole and itraconazole but not to amphotericin B. A compilation of the genetic alterations identified in this series suggests that resistance develops gradually and is the sum of several different changes, all of which contribute to the final resistant phenotype.

Evidence of multiple extracellular phospholipase activities of Aspergillus fumigatus

Extracellular phospholipase activity has been implicated in the pathogenesis of several bacterial infections. Recently, extracellular phospholipase activity has been proposed as a virulence factor in the opportunistic yeast Candida albicans. Aspergillus fumigatus is the most pathogenic member of its genus, responsible for > 90% of infections. Previously, no specific virulence factors have been determined. We investigated the ability of A. fumigatus to produce extracellular phospholipases at 37 degrees C. Fast atom bombardment was used to compare lipid-containing media before and at 5-h intervals during shaking culture of A. fumigatus. Lipids were extracted and analyzed. Many anions corresponding to phospholipid breakdown products were identified. Specific anion species identified indicated phospholipase A, B, C (PLC), and D activities. PLC activity was further investigated by using the synthetic substrate p-nitrophenylphosphorylcholine. PLC activity was initially observed after 30 h of growth and accumulated in broth cultures up to 50 h. At 55 h, there was a sharp increase in PLC activity which coincided with cultures reaching the stationary phase. Activity of the PLC was measured at different temperatures, with greater activity occurring at 37 degrees C than at lower temperatures. Phospholipases could represent a virulence determinant in A. fumigatus.

Macrophage colony-stimulating factor in murine candidiasis: serum and tissue levels during infection and protective effect of exogenous administration

Serum and tissue concentrations of the macrophage-specific colony-stimulating factor (CSF-1) and the number of CSF-1-responsive cells in bone marrow were investigated in mice chronically infected with a low-virulence strain of the opportunistic zoopathogenic yeast Candida albicans. CSF-1 levels in serum, brain, kidney, liver, and lung were significantly increased shortly after infection and remained elevated during the 2 weeks preceding the onset of specific T cell-dependent immunity. The number of monocytic precursor cells was also increased in the bone marrow of infected mice. When macrophages from naive donors were exposed in vitro to purified murine CSF-1, their anticandidal activity in vitro appeared to be enhanced. CSF-1 was also administered in vivo to prospective recipients of a lethal C. albicans challenge. The results showed that the factor could effectively potentiate the animals' resistance to the yeast, as shown by increased survival times and reduced recovery of viable C. albicans from the organs of the CSF-1-treated mice. Therefore, the present data suggest that CSF-1 is likely to contribute to early resistance to fungal infection and could be successfully exploited in experimental models of antifungal immunotherapy.

Vergleich verschiedener Immun-Dekorationsmethoden für die Rasterelektronenmikroskopie am Beispiel eines Protein-Antigens auf der Oberfläche der Hefe Candida albicans/ A Comparison of Decoration Techniques for the Demonstration of Immunocomplexes by Scanning Electron Microscopy: Labeling of a Protein Antigen on the Surface of the Yeast Candida albicans

Abstract The labeling of immunocomplexes for scanning electron microscopy (SEM) is a fairly new technique, and the various procedures, that have been proposed, have not yet been compared. Such comparative evaluation was performed with Candida protease as a target antigen. This secretory enzyme of the opportunistic yeast Candida albicans can be localized on the surface of fungal blastopores and mycelia, both after growth in proteinaceous medium and upon infection of murine peritoneal macrophages. The presence of the protease antigen was confirmed by immunofluorescence and by immunoperoxidase-light microscopy. The decoration of protease - anti protease complexes for SEM was attempted with colloids derived from the immunoperoxidase reaction, by the immunogold technique, and by antibodies linked to beads of synthetic polymers (polystyrene, polymethacrylate, polyacrolein). In addition, inactivated Staphylococcus aureus was used, which binds to antibodies through its protein-A. The high resolution by SEM of surface structures was matched only by the colloid based decoration techniques. All conjugates with beads suffered from inconsistent binding, which did not correspond with the distribution of the surface antigen. The comparatively best result with beads was obtained with polystyrene (Latex). Colloid based techniques in addition allow for critical point drying, which cannot be applied to synthetic beads in the usual manner.