Human Pathogenic Yeast Candida Albicans Research Articles

Use of the Fluorescent Dye Thioflavin T to Track Amyloid Structures in the Pathogenic Yeast Candida albicans.

The human pathogenic yeast Candida albicans can attach to epithelial cells or indwelling medical devices to form biofilms. These microbial communities are highly problematic in the clinic as they reduce both sensitivity to antifungal drugs and detection of fungi by the immune system. Amyloid structures are highly organized quaternary structures that play a critical role in biofilm establishment by allowing fungal cells to adhere to each other. Thus, fungal amyloids are exciting targets to develop new antifungal strategies. Thioflavin T is a specific fluorescent dye widely used to study amyloid properties of target proteins in vitro (spectrophotometry) and in vivo (epifluorescence/confocal microscopy). Notably, thioflavin T has been used to demonstrate the ability of Als5, a C. albicans adhesin, to form an amyloid fiber upon adhesion. We have developed a pipeline that allows us to study amyloid properties of target proteins using thioflavin T staining in vitro and in vivo, as well as in intact fungal biofilms. In brief, we used thioflavin T to sequentially stain (i) amyloid peptides, (ii) recombinant proteins, (iii) fungal cells treated or not with amyloid peptides, (iv) fungal amyloids enriched by cell fractionation, and (v) intact biofilms of C. albicans. Contrary to other methods, our pipeline gives a complete picture of the amyloid behavior of target proteins, from in vitro analysis to intact fungal biofilms. Using this pipeline will allow an assessment of the relevance of the in vitro results in cells and the impact of amyloids on the development and/or maintenance of fungal biofilm. Key features • Study of amyloid properties of fungal proteins. • Visualization of the subcellular localization of fungal amyloid material using epifluorescence or confocal microscopy. • Unraveling of the amyloid properties of target proteins and their physiological meaning for biofilm formation. • Observation of the presence of amyloid structures with live-cell imaging on intact fungal biofilm using confocal microscopy.

In vitro Antioxidant, Enzyme Inhibitory and Antimicrobial Evaluation of Sideritis caesarea H. Duman, Aytaç & Başer Extracts

Sideritis caesarea H. Duman, Aytaç & Başer of the family Lamiaceae is an endemic plant with different ethnobotanical uses in Anatolia. In this present study, the antioxidant and biological activity potential of different S caesarea extracts and purified compounds were evaluated in vitro using DPPH•, CUPRAC, and ABTS• methods. In addition, their urease and chymotrypsin enzyme inhibitory activities, antimicrobial potential, and total phenol and flavonoid contents were determined. The experimental results showed that the two isolated flavonoids {4′- O-methylhypolaetin-7- O-[6″′- O-acetyl- β-D-allopyranosyl-(1→2)]-6″- O-acetyl- β-D-glucopyranoside, and isoscutellarein-7- O-[6″′- O-acetyl- β-D-allopyranosyl-(1→2)]-6″- O-acetyl- β-D-glucopyranoside}, as well as their fractions, except the n-hexane and water residue, showed remarkable antioxidant activity. Interestingly, the ethyl acetate and water residue fractions inhibited the urease enzyme activity by 49.6% and 49.1%, respectively; while the n-hexane and dichloromethane fractions attracted attention with their relative high chymotrypsin inhibitory activity of 85.8%, and 82.4%, respectively. When evaluated for their in vitro antimicrobial potential, including Helicobacter pylori, only Bacillus subtilis was susceptible to the n-butanol fraction at 1 mg/mL, while all of the studied samples showed activity against the human pathogenic yeast Candida albicans with the range of 0.25 to 1 mg/mL minimum inhibitory concentrations. The initial antioxidant activity, enzyme inhibitions, and antimicrobial results of S caesarea extracts and compounds suggest the further potential for in vivo evaluations.

Potential of an endophytic fungus Alternaria tenuissima PE2 isolated from Psidium guajava L. for the production of bioactive compounds

Searching of novel bioactive compounds from natural sources is increasing day by day for overcoming multidrug resistant microorganisms and endophytic fungi are considered as important sources in this regard. In the present study, one potent endophytic fungal isolate PE2 was isolated from the leaves of Psidium guajava L. which was identified as Alternaria tenuissima by 28S rDNA sequence homology. The isolate was found to produce thermostable, non-proteinacious antimicrobial substances. Ethyl acetate (EA) extract of the cell free supernatant (CFS) of the isolate was found effective against both Gram-positive and Gram- negative bacteria with MIC values of ∼500 µg/mL and ∼800 µg/mL, respectively. Degradations of bacterial cells upon treatment were supported by the release of intracellular materials as well as scanning electron microscopic (SEM) studies. EA extract also exhibited an adverse effect on bacterial central carbohydrate metabolic pathway enzymes viz. phospho fructokinase, isocitrate dehydrogenase and fructose-1,6-bisphosphatase. Production of inhibition zones against pathogenic fungi including human pathogenic yeast Candida albicans, inhibition of fungal radial growth and conidial germination upon treatment suggested antifungal potential of the EA extract of the isolate. SEM study revealed massive deformation of cellular integrity of C. albicans, indicated the probable effect of antimicrobial compounds. Thin layer chromatographic (TLC) separation and GC-MS analysis revealed the production of numbers of bioactive compounds by the isolate. Based on the present study, isolate Alternaria tenuissima PE2 could be considered as a promising source of natural bioactive metabolites.

Luffa operculata seed proteins: Identification by LC-ESI-MS/MS and biotechnological potential against Candida albicans and C. krusei

Loperculata is a plant commonly found in the North and Northeast of Brazil. Although the regional population knows its medicinal potential, there are few scientific studies about its antimicrobial potential. Thus, this study aimed to characterize the proteins from L. operculata seeds extracted using different solutions and evaluate their antimicrobial potentials. The protein extracts obtained with NaCl and sodium acetate buffer presented the best inhibitory activities against Candida albicans and C. krusei. The study of the mechanism of action revealed proteins from L. operculata seeds induced pore formation on the membrane and ROS overaccumulation. Scanning Electron Microscopy images also showed severe morphological changes in Candida albicans and C. krusei. Proteins from L.operculata seeds did not show antibacterial activity. The enzymatic assays revealed the presence of proteolytic enzymes, serine and cysteine protease inhibitors, and chitinases in both protein extracts. Proteomic analysis by LC-ESI-MS/MS identified 57 proteins related to many biological processes, such as defense to (a)biotic stress, energetic metabolism, protein folding, and nucleotide metabolism. In conclusion, the L. operculata seed proteins have biotechnological potential against the human pathogenic yeasts Candida albicans and C. krusei.

Small, Cationic Antifungal Proteins from Filamentous Fungi Inhibit Candida albicans Growth in 3D Skin Infection Models.

ABSTRACTThe emerging resistance of human-pathogenic fungi to antifungal drugs urges the development of alternative therapeutic strategies. The small, cationic antifungal proteins (AFPs) from filamentous ascomycetes represent promising candidates for next-generation antifungals. These bio-molecules need to be tested for tolerance in the host and efficacy against fungal pathogens before they can be safely applied in humans. Testing of the efficacy and possible adverse effects of new drug candidates in three-dimensional (3D) human-cell based models represents an advantageous alternative to animal experiments. In, this study, as a proof-of-principle, we demonstrate the usefulness of 3D skin infection models for screening new antifungal drug candidates for topical application. We established a cutaneous infection with the opportunistic human-pathogenic yeast Candida albicans in a commercially available 3D full-thickness (FT) skin model to test the curative potential of distinct AFPs from Penicillium chrysogenum (PAFopt, PAFB, and PAFC) and Neosartorya (Aspergillus) fischeri (NFAP2) in vitro. All tested AFPs were comparably well tolerated by the skin models. The infected 3D models exhibited reduced epidermal permeability barriers, allowing C. albicans to colonize the epidermal and dermal layers, and showed increased secretion of the pro-inflammatory cytokine IL-6 and the chemokine IL-8. AFP treatment diminished the fungal burden and penetration depth of C. albicans in the infected models. The epidermal permeability barrier was restored and the secretion of IL-8 was decreased following AFP treatment. In summary, our study proves that the tested AFPs exhibit antifungal potential against cutaneous C. albicans infection in a 3D FT skin model.IMPORTANCE Candida albicans represents one of the most prevalent opportunistic fungal pathogens, causing superficial skin and mucosal infections in humans with certain predisposing health conditions and life-threatening systemic infections in immunosuppressed patients. The emerging drug resistance of this human-pathogenic yeast and the limited number of antifungal drugs for prevention and treatment of infections urgently demands the identification of new antifungal compounds with novel mechanisms of action. Small, cationic antifungal proteins (AFPs) from filamentous fungi represent promising candidates for next-generation antifungals for topical application. These bio-molecules need to be tested for tolerance by the host and efficacy in pathogen clearance prior to being involved in clinical trials. In a proof-of-principle study, we provide evidence for the suitability of 3D human-cell based models as advantageous alternatives to animal experiments. We document the tolerance of specific AFPs and their curative efficacy against cutaneous C. albicans infection in a 3D skin model.

ORF19.2286 Gene: Isolation and Purification of Deoxyhypusine Hydroxylase from the Human Pathogenic Yeast Candida albicans

ORF19.2286 Gene: Isolation and Purification of Deoxyhypusine Hydroxylase from the Human Pathogenic Yeast Candida albicans

ORF19.2286 Gene: Isolation and Purification of Deoxyhypusine Hydroxylase from the Human Pathogenic Yeast Candida albicans

Candida albicans (C. albicans) is a fungal pathogen that causes infections of the wet body surfaces and the blood in immunocompromised patients or individuals with imbalanced microflora. Since the cases of clinically meaningful candidosis are on the rise, efficient С. albicans therapy is in a high demand. Informed drug design requires well-characterized С. albicans targets, including these aimed at disrupting its post-translational modifications. C. albicans ORF19.2286 gene encodes an ortholog of human deoxyhypusine hydroxylase (DOHH). Here, this ORF was cloned from the SC5314 strain and re-expressed in Escherichia coli as sGB1 - CaDOHH construct with 6xHis tag on the N-terminus of the fusion protein, then purified, and GB1-tag was removed with Tobacco etch virus (TEV) protease. Several amino acid sequence differences between C. albicans and animal DOHHs were noted, and are useful for a selection of the binding sites for antimicrobials in CaDOHH. We present the protocol for the heterologous expression and purification of C. albicans DOHH, which is suitable for further crystallization.

Investigation of the phytochemical and antimicrobial activity of Primula veris L. extracts

Abstract Methanol, ethanol (50%) and aqueous extracts were prepared from the roots, leaves and flowers of Primula veris collected and dried under natural conditions in meadows near Marosludas in April 2018. The polyphenol contents of the plant parts, as well as the antioxidant capacity were measured by ABTS spectrophotometric methods. For the microbiology section, aqueous and ethanolic extracts (100 mg/ml) were prepared from the roots, leaves and flowers of Primula veris followed by testing of their antimicrobial activity against human pathogenic yeasts (Candida albicans, Candida parapsilosis, Candida glabrata) and bacteria (Gram negatives:, Escherichia coli, Acinetobacter sp., Pseudomonas aeruginosa, Enterobacter cloacae; Gram-positive: Enterococcus faecalis, Listeria monocytogenes, Staphylococcus aureus) by agar well diffusion method. Ethanol proved to be the most effective solvent for all three drugs to determine the polyphenol content. The flower drug contains the highest amounts of polyphenols. With the ABTS method of antioxidant measurement, ethanolic extracts are more effective than aqueous extracts. Agar well diffusion studies showed that all extracts caused complete inhibition within the inhibition zone in the yeasts tested. The inhibitory effect of aqueous extracts was stronger than that of ethanolic extracts in the case of all three plant parts. The greatest inhibition was caused by extracts of the roots with ethanol. The three yeasts tested showed similarly high sensitivity to roots extracts, while the inhibitory effect was weaker for leaf extracts. Candida parapsilosis was resistant to leaf and flower extracts. Pseudomonas aeruginosa, Enterococcus faecalis and Staphylococcus aureus, like fungi, showed good sensitivity to extracts, while other bacteria showed little or no inhibitory effect.

Sphaerostilbellins, New Antimicrobial Aminolipopeptide Peptaibiotics from Sphaerostilbella toxica.

Sphaerostilbella toxica is a mycoparasitic fungus that can be found parasitizing wood-decay basidiomycetes in the southern USA. Organic solvent extracts of fermented strains of S. toxica exhibited potent antimicrobial activity, including potent growth inhibition of human pathogenic yeasts Candida albicans and Cryptococcus neoformans, the respiratory pathogenic fungus Aspergillus fumigatus, and the Gram-positive bacterium Staphylococcus aureus. Bioassay-guided separations led to the purification and structure elucidation of new peptaibiotics designated as sphaerostilbellins A and B. Their structures were established mainly by analysis of NMR and HRMS data, verification of amino acid composition by Marfey’s method, and by comparison with published data of known compounds. They incorporate intriguing structural features, including an N-terminal 2-methyl-3-oxo-tetradecanoyl (MOTDA) residue and a C-terminal putrescine residue. The minimal inhibitory concentrations for sphaerostilbellins A and B were measured as 2 μM each for C. neoformans, 1 μM each for A. fumigatus, and 4 and 2 μM, respectively, for C. albicans. Murine macrophage cells were unaffected at these concentrations.

The Evolutionary Conserved γ-Core Motif Influences the Anti-Candida Activity of the Penicillium chrysogenum Antifungal Protein PAF.

Small, cysteine-rich and cationic antimicrobial proteins (AMPs) from filamentous ascomycetes represent ideal bio-molecules for the development of next-generation antifungal therapeutics. They are promising candidates to counteract resistance development and may complement or even replace current small molecule-based antibiotics in the future. In this study, we show that a 14 amino acid (aa) long peptide (Pγ) spanning the highly conserved γ-core motif of the Penicillium chrysogenum antifungal protein (PAF) has antifungal activity against the opportunistic human pathogenic yeast Candida albicans. By substituting specific aa we elevated the positive net charge and the hydrophilicity of Pγ and created the peptide variants Pγvar and Pγopt with 10-fold higher antifungal activity than Pγ. Similarly, the antifungal efficacy of the PAF protein could be significantly improved by exchanging the respective aa in the γ-core of the protein by creating the protein variants PAFγvar and PAFγopt. The designed peptides and proteins were investigated in detail for their physicochemical features and mode of action, and were tested for cytotoxicity on mammalian cells. This study proves for the first time the important role of the γ-core motif in the biological function of an AMP from ascomycetes. Furthermore, we provide a detailed phylogenetic analysis that proves the presence and conservation of the γ-core motif in all AMP classes from Eurotiomycetes. We emphasize the potential of this common protein motif for the design of short antifungal peptides and as a protein motif in which targeted aa substitutions enhance antimicrobial activity.

The Dietary Food Components Capric Acid and Caprylic Acid Inhibit Virulence Factors in Candida albicans Through Multitargeting.

Capric acid and caprylic acid are the dietary food components. They are found to inhibit the virulence factors like morphogenesis, adhesion, and biofilm formation in the human pathogenic yeast Candida albicans. Our study demonstrated that yeast-to-hyphal signal transduction pathways were affected by capric acid and caprylic acid. The expression profile of genes associated with serum-induced morphogenesis showed reduced expressions of Cdc35, Hwp1, Hst7, and Cph1 by the treatment with both the fatty acids. Cell elongation gene, Ece1, was surprisingly downregulated by 5208-fold by the treatment of caprylic acid. Nrg1 and Tup1, negative regulators of hyphal formation, were overexpressed in presence of capric or caprylic acid. Cell cycle studies revealed that capric and caprylic acids arrested cell cycle at G2/M and S phase. Targeting the virulence factors like yeast-to-hyphal transition is efficacious for treatment of opportunistic fungal infections. This research suggests that both capric and caprylic acid may be effective interventions for treating C. albicans yeast infections.

Antifungal activity and cytotoxicity of extracts and triterpenoid saponins obtained from the aerial parts of Anagallis arvensis L.

Ethnopharmacological relevanceAnagallis arvensis L. (Primulaceae) is used in argentinean northwestern traditional medicine to treat fungal infections. We are reporting the isolation and identification of compounds with antifungal activity against human pathogenic yeast Candida albicans, and toxicity evaluation. Aim of the studyto study the antifungal activity of extracts and purified compounds obtained form A. arvensis aerial parts, alone and in combinations with fluconazole (FLU), and to study the toxicity of the active compounds. Materials and methodsDisk diffusion assays were used to perform an activity-guided isolation of antifungal compounds from the aerial parts of A. arvensis. Broth dilution checkerboard and viable cell count assays were employed to determine the effects of samples and combinations of FLU + samples against Candida albicans. The chemical structures of active compounds were elucidated by spectroscopic analysis. Genotoxic and haemolytic effects of the isolated compounds were determined. ResultsFour triterpenoid saponins (1–4) were identified. Anagallisin C (AnC), exerted the highest inhibitory activity among the assayed compounds against C. albicans reference strain (ATCC 10231), with MIC-0 =1µg/mL. The Fractional Inhibitory Concentration Index (FICI=0.129) indicated a synergistic effect between AnC (0.125µg/mL) and FLU (0.031µg/mL) against C. albicans ATCC 10231. AnC inhibited C. albicans 12–99 FLU resistant strain (MIC-0 =1µg/mL), and the FICI=0.188 indicated a synergistic effect between AnC (0.125µg/mL) and fluconazole (16µg/mL). The combination AnC+ FLU exerted fungicidal activity against both C. albicans strains. AnC exerted inhibitory activity against C. albicans ATCC 10231 sessile cells (MIC50=0.5µg/mL and MIC80=1µg/mL) and against C. albicans 12–99 sessile cells (MIC50=0.75µg/mL and MIC80=1.25µg/mL). AnC exerted haemolytic effect against human red blood cells at 15µg/mL and did not exerted genotoxic effect on Bacillus subtilis rec strains. ConclusionsThe antifungal activity and lack of genotoxic effects of AnC give support to the traditional use of A. arvensis as antifungal and makes AnC a compound of interest to expand the available antifungal drugs.

Factor H Binds to Extracellular DNA Traps Released from Human Blood Monocytes in Response to Candida albicans.

Upon systemic infection with human pathogenic yeast Candida albicans (C. albicans), human monocytes and polymorph nuclear neutrophilic granulocytes are the first immune cells to respond and come into contact with C. albicans. Monocytes exert immediate candidacidal activity and inhibit germination, mediate phagocytosis, and kill fungal cells. Here, we show that human monocytes spontaneously respond to C. albicans cells via phagocytosis, decondensation of nuclear DNA, and release of this decondensed DNA in the form of extracellular traps (called monocytic extracellular traps: MoETs). Both subtypes of monocytes (CD14++CD16−/CD14+CD16+) formed MoETs within the first hours upon contact with C. albicans. MoETs were characterized by the presence of citrullinated histone, myeloperoxidase, lactoferrin, and elastase. MoETs were also formed in response to Staphylococcus aureus and Escherichia coli, indicating a general reaction of monocytes to infectious microbes. MoET induction differs from extracellular trap formation in macrophages as MoETs are not triggered by simvastatin, an inhibitor of cholesterol synthesis and inducer of extracellular traps in macrophages. Extracellular traps from both monocytes and neutrophils activate complement and C3b is deposited. However, factor H (FH) binds via C3b to the extracellular DNA, mediates cofactor activity, and inhibits the induction of the inflammatory cytokine interleukin-1 beta in monocytes. Altogether, the results show that human monocytes release extracellular DNA traps in response to C. albicans and that these traps finally bind FH via C3b to presumably support clearance without further inflammation.

Opportunistic Sampling of Roadkill as an Entry Point to Accessing Natural Products Assembled by Bacteria Associated with Non-anthropoidal Mammalian Microbiomes.

Few secondary metabolites have been reported from mammalian microbiome bacteria despite the large numbers of diverse taxa that inhabit warm-blooded higher vertebrates. As a means to investigate natural products from these microorganisms, an opportunistic sampling protocol was developed, which focused on exploring bacteria isolated from roadkill mammals. This initiative was made possible through the establishment of a newly created discovery pipeline, which couples laser ablation electrospray ionization mass spectrometry (LAESIMS) with bioassay testing, to target biologically active metabolites from microbiome-associated bacteria. To illustrate this process, this report focuses on samples obtained from the ear of a roadkill opossum (Dideiphis virginiana) as the source of two bacterial isolates (Pseudomonas sp. and Serratia sp.) that produced several new and known cyclic lipodepsipeptides (viscosin and serrawettins, respectively). These natural products inhibited biofilm formation by the human pathogenic yeast Candida albicans at concentrations well below those required to inhibit yeast viability. Phylogenetic analysis of 16S rRNA gene sequence libraries revealed the presence of diverse microbial communities associated with different sites throughout the opossum carcass. A putative biosynthetic pathway responsible for the production of the new serrawettin analogues was identified by sequencing the genome of the Serratia sp. isolate. This study provides a functional roadmap to carrying out the systematic investigation of the genomic, microbiological, and chemical parameters related to the production of natural products made by bacteria associated with non-anthropoidal mammalian microbiomes. Discoveries emerging from these studies are anticipated to provide a working framework for efforts aimed at augmenting microbiomes to deliver beneficial natural products to a host.

Antifungal and Antibacterial Activity of Extracts and Alkaloids of Selected Amaryllidaceae Species

Alkaloidal extracts of six selected species of Amaryllidaceae were studied with respect to their antibacterial and anti-yeast activity and their alkaloidal fingerprint. Twenty-five alkaloids were determined by GC/MS, and sixteen of them identified from their mass spectra, retention times and retention indexes. In the antimicrobial assay, Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus were used, along with isolates of the human pathogenic yeasts Candida albicans, C. glabrata, C. dubliniensis and Lodderomyces elongiosporus. The six extracts, together with 19 Amaryllidaceae alkaloids isolated in our laboratory, showed almost no inhibitory activity against the bacteria tested. However, promising anti-yeast properties were detected; the most potent activity was shown by lycorine, which inhibited C. dubliniensis with a MIC of 32 µg/mL, C. albicans and L. elongiosporus, both with MICs of 64 µg/mL, followed by caranine inhibiting C. dubliniensis with a MIC of 128 µg/mL. Among the alkaloidal extracts, Narcissus jonquilla cv. Baby Moon showed the most potent anti-yeast activity, with minimal and average MIC values of 128 and 192 µg/mL, respectively, followed by Leucojum aestivum, Narcissus poeticus var. recurvus and N. canaliculatus (average MICs 256, 267 and 299 µg/mL, respectively). The lowest MIC value among extracts was obtained for N. canaliculatus against L. elongiosporus (MIC 64 µg/mL).

The Aspergillus fumigatus septins play pleiotropic roles in septation, conidiation, and cell wall stress, but are dispensable for virulence

Septins are a conserved family of GTPases that regulate important cellular processes such as cell wall integrity, and septation in fungi. The requirement of septins for virulence has been demonstrated in the human pathogenic yeasts Candida albicans and Cryptococcus neoformans, as well as the plant pathogen Magnaporthe oryzae. Aspergillus spp. contains five genes encoding for septins (aspA–E). While the importance of septins AspA, AspB, AspC, and AspE for growth and conidiation has been elucidated in the filamentous fungal model Aspergillus nidulans, nothing is known on the role of septins in growth and virulence in the human pathogen Aspergillus fumigatus. Here we deleted all five A. fumigatus septins, and generated certain double and triple septin deletion strains. Phenotypic analyses revealed that while all the septins are dispensable in normal growth conditions, AspA, AspB, AspC and AspE are required for regular septation. Furthermore, deletion of only the core septin genes significantly reduced conidiation. Concomitant with the absence of an electron-dense outer conidial wall, the ΔaspB strain was also sensitive to anti-cell wall agents. Infection with the ΔaspB strain in a Galleria mellonella model of invasive aspergillosis showed hypervirulence, but no virulence difference was noted when compared to the wild-type strain in a murine model of invasive aspergillosis. Although the deletion of aspB resulted in increased release of TNF-α from the macrophages, no significant inflammation differences in lung histology was noted between the ΔaspB strain and the wild-type strain. Taken together, these results point to the importance of septins in A. fumigatus growth, but not virulence in a murine model.

Telomeric ORFS in Candida albicans: does mediator tail wag the yeast?

Recent studies of fungal genomes have shown that subtelomeric regions of chromosomes are areas of rapid evolution that facilitate adaptation to novel niches [1]. Several years ago, analysis of the genome of the human pathogenic yeast Candida albicans revealed the presence of a large family of telomeric orfs (TLO genes) [2]. The function of this gene family remained an enigma in C. albicans genetics for many years; however, recent studies have revealed that the TLO genes encode a subunit of the Mediator complex with roles in transcriptional regulation [3,4]. This gene family expansion is unique to C. albicans, the species responsible for the majority of human yeast infections and the species that is most commonly recovered as a human commensal. If selective pressures in the host have driven this expansion, it is likely that this gene family somehow contributes to the success of C. albicans as a commensal and opportunistic pathogen. To support this hypothesis, it was first necessary to determine the exact function of Tlo proteins in C. albicans. Armed with this knowledge, investigators are now beginning to understand how possession of multiple copies of TLO could contribute to the virulence properties of C. albicans.

Cold-active antibacterial and antifungal activities and antibiotic resistance of bacteria isolated from an alpine hydrocarbon-contaminated industrial site

Selection pressure in hydrocarbon-contaminated soils may lead not only to increased microbial resistance to antibiotics, but also to increased capacity of the soil indigenous population to produce antimicrobial compounds. Therefore, we studied the antibiotic resistance pattern and antibacterial and/or antifungal activities of 47 bacterial strains isolated from an industrial alpine site heavily polluted with petroleum hydrocarbons. Resistance to penicillin was more widespread (49%) than resistance to chloramphenicol or rifampicin (28%) or streptomycin (26%). Only 9% of the strains were resistant to tetracycline. The ability to produce cold-active (10 °C) antimicrobial compounds was tested by using human pathogenic bacteria (Escherichia coli, Shigella flexneri, Salmonella enterica, Pseudomonas aeruginosa, Staphylococcus aureus) and yeasts (Candida albicans, Cryptococcus neoformans) as indicator microorganisms. About two-thirds of the 47 tested strains produced compounds that inhibited growth of at least one indicator microorganism. Six strains inhibited growth of both bacteria and yeast indicators; 12 and 16 strains showed either antibacterial or antifungal activity, respectively. The most versatile bacteria with regard to multiple antibiotic resistance and antimicrobial activity belonged to Actinobacteria or Gammaproteobacteria. The antimicrobial compounds produced by three Pseudomonas spp. and two Serratia spp. strains were characterized in more detail by TLC and HPLC. Depending on the sensitivity of growth inhibition to enzymes, the compounds produced by the three pseudomonads contained a proteinaceous component.

Quantitative Expression of the Candida albicans Aspartyl Proteinase Genes SAP7, SAP8, SAP9, SAP10 in Human Serum in vitro

The different members of the secreted aspartyl proteinase (Sap) family of the human pathogenic yeast Candida albicans are proposed to play different roles during infection and are differentially expressed at various body sites. In recent reports, expression analysis has focused on the genes SAP1-6, while the expression pattern of SAP7-10 was less well studied. We analyzed the SAP7-SAP10 expression profile of C. albicans under human serum influence that may be elucidated in the course of blood infection in humans and how this in vitro expression profile is associated with hyphal formation. The phenotypes of strains were examined under scanning electron microscopy. Quantitative RT-PCR (2-(deltadeltaC)T) was used to monitor SAP expression of C. albicans wild type cells and mutants lacking SAP9 and/or SAP10. Of the four analyzed SAP genes, only SAP7 was detectably induced in the double mutant and in the wild type strains in the model that mimics bloodstream infections. On the other hand, in the wild types (isolate 83 and CAF2-1), SAP7 was expressed 0.8- or 0.4-fold less than SAP10, respectively. Our findings suggest that Sap7 may respond to the challenge of the human blood environment. Furthermore, the results support the notion that compensatory upregulation of SAP7 and SAP8 in the deltasap9/deltasap10 mutant occurs in these conditions. SAP7-10 expression was strain-specific. Our findings point to a link between morphogenesis and expression of SAP9 in serum, where these conditions induce both hyphae and SAP9, but temporal gene expression patterns might be controlled by other factors.

A Coordinated Interdependent Protein Circuitry Stabilizes the Kinetochore Ensemble to Protect CENP-A in the Human Pathogenic Yeast Candida albicans

Unlike most eukaryotes, a kinetochore is fully assembled early in the cell cycle in budding yeasts Saccharomyces cerevisiae and Candida albicans. These kinetochores are clustered together throughout the cell cycle. Kinetochore assembly on point centromeres of S. cerevisiae is considered to be a step-wise process that initiates with binding of inner kinetochore proteins on specific centromere DNA sequence motifs. In contrast, kinetochore formation in C. albicans, that carries regional centromeres of 3–5 kb long, has been shown to be a sequence independent but an epigenetically regulated event. In this study, we investigated the process of kinetochore assembly/disassembly in C. albicans. Localization dependence of various kinetochore proteins studied by confocal microscopy and chromatin immunoprecipitation (ChIP) assays revealed that assembly of a kinetochore is a highly coordinated and interdependent event. Partial depletion of an essential kinetochore protein affects integrity of the kinetochore cluster. Further protein depletion results in complete collapse of the kinetochore architecture. In addition, GFP-tagged kinetochore proteins confirmed similar time-dependent disintegration upon gradual depletion of an outer kinetochore protein (Dam1). The loss of integrity of a kinetochore formed on centromeric chromatin was demonstrated by reduced binding of CENP-A and CENP-C at the centromeres. Most strikingly, Western blot analysis revealed that gradual depletion of any of these essential kinetochore proteins results in concomitant reduction in cellular protein levels of CENP-A. We further demonstrated that centromere bound CENP-A is protected from the proteosomal mediated degradation. Based on these results, we propose that a coordinated interdependent circuitry of several evolutionarily conserved essential kinetochore proteins ensures integrity of a kinetochore formed on the foundation of CENP-A containing centromeric chromatin.