Andida Albicans Research Articles

A family of secreted pathogenesis‐related proteins in Candida albicans

Analysing culture supernatants of yeast and hyphal cells of Candida albicans, we found two close homologues of pathogenesis-related (PR-) 1 proteins, Rbe1p and Rbt4p, in the secretome. Due to sequence homology, three additional, yet not characterized open reading frames, ORF19.6200, ORF19.2787 and ORF19.2336, together with RBE1 and RBT4 were assigned to a novel family of CaPRY proteins. In a Δrbe1/Δrbt4 deletion strain, genome-wide transcriptional analysis revealed differential transcription of only a limited set of genes implicated in virulence and oxidative stress response. Single deletion of RBE1 or RBT4 in a clinical C. albicans isolate resulted in a moderate but significant attenuation in virulence in a mouse model for disseminated candidiasis. However, a synergistic effect was observed in a Δrbe1/Δrbt4 double deletion strain, where virulence was strongly affected. Remarkably, transcription of RBT4 and RBE1 was each upregulated in blastospores of Δrbe1 or hyphae of Δrbt4 deletion strains respectively, indicating functional complementation thereby compensating a potential virulence defect in the single deletion strains. Furthermore, the double deletion strain showed increased sensitivity to attack by polymorphonuclear leucocytes. Therefore, the crucial contribution of both C. albicans pathogenesis-related proteins to virulence might be vested in protection against phagocyte attack.

Association of subgingival colonization of Candida albicans and other yeasts with severity of chronic periodontitis

The aim of this study is to analyze the relationship between the subgingival colonization by Candida albicans and other yeasts with the severity of chronic periodontitis (CP). After sample size calculation, 40 patients with CP and 20 healthy subjects (HS) were included in the study. Cases of slight-moderate (MCP, n = 23) and severe CP (SCP, n = 17) were defined according to the Centers for Disease Control/American Association of Periodontology classification. Subgingival samples were acquired using sterile paper-points from the sulcus or the deepest periodontal pocket of each healthy and subject with CP, respectively, and were cultured aerobically on three selective media. Yeast colonies that grew on the surface of plates were later identified by biochemical reactions. Statistical tests were used to analyze the association between subgingival yeast colonization (number of yeast-positive individuals and colony forming units (CFU) per subject) and periodontal disease status, considering statistical significance when P < 0.05. Although several yeast species were found (C. parapisilosis, Rhodotorula sp., C. dubliniensis and C. tropicalis), only C. albicans was present in all the patients with yeast-positive CP. Twelve patients (30%) with CP presented yeasts in the subgingival biofilm while only three patients (15%) in the HS group were positive for these microorganisms. No statistical difference was found between the CP and HS groups (P = 0.084). However, when the CP group was divided on the basis of severity, statistical differences were observed between the SCP and MCP groups (47% vs. 17%, P = 0.043), and between the SCP and HS groups (47% vs. 15%, P = 0.033). No statistical difference was observed between the MCP and HS groups (17% vs. 15%, P = 0.832). High densities of yeasts were found only in patients with MCP and SCP (mean and range 61.25 (0-100) CFU/plate and mean and range 51 (0-101) CFU/plate, respectively). In this group of patients, subgingival colonization of some yeasts, especially C. albicans, was associated with the severity of CP.

Pseudomonas aeruginosa lipopolysaccharide inhibits Candida albicans hyphae formation and alters gene expression during biofilm development

Elucidation of bacterial and fungal interactions in multispecies biofilms will have major impacts on understanding the pathophysiology of infections. The objectives of this study were to (i) evaluate the effect of Pseudomonas aeruginosa lipopolysaccharide (LPS) on Candida albicans hyphal development and transcriptional regulation, (ii) investigate protein expression during biofilm formation, and (iii) propose likely molecular mechanisms for these interactions. The effect of LPS on C.albicans biofilms was assessed by XTT-reduction and growth curve assays, light microscopy, scanning electron microscopy (SEM), and confocal laser scanning microscopy (CLSM). Changes in candidal hypha-specific genes (HSGs) and transcription factor EFG1 expression were assessed by real-time polymerase chain reaction and two-dimensional gel electrophoresis, respectively. Proteome changes were examined by mass spectrometry. Both metabolic activities and growth rates of LPS-treated C.albicans biofilms were significantly lower (P<0.05). There were higher proportions of budding yeasts in test biofilms compared with the controls. SEM and CLSM further confirmed these data. Significantly upregulated HSGs (at 48h) and EFG1 (up to 48h) were noted in the test biofilms (P<0.05) but cAMP levels remained unaffected. Proteomic analysis showed suppression of candidal septicolysin-like protein, potential reductase-flavodoxin fragment, serine hydroxymethyltransferase, hypothetical proteins Cao19.10301(ATP7), CaO19.4716(GDH1), CaO19.11135(PGK1), CaO19.9877(HNT1) by P.aeruginosa LPS. Our data imply that bacterial LPS inhibit C.albicans biofilm formation and hyphal development. The P.aeruginosa LPS likely target glycolysis-associated mechanisms during candidal filamentation.

The stepwise acquisition of fluconazole resistance mutations causes a gradual loss of fitness in Candida albicans

The pathogenic yeast Candida albicans can develop resistance to the widely used antifungal agent fluconazole, which inhibits ergosterol biosynthesis. Resistance is often caused by gain-of-function mutations in the transcription factors Mrr1, Tac1 and Upc2, which result in constitutive overexpression of multidrug efflux pumps and ergosterol biosynthesis genes respectively. It is not known how the permanently changed gene expression program in resistant strains affects their fitness in the absence of drug selection pressure. We have systematically investigated the effects of activating mutations in Mrr1, Tac1 and Upc2, individually and in all possible combinations, on the degree of fluconazole resistance and on the fitness of C. albicans in an isogenic strain background. All combinations of different resistance mechanisms resulted in a stepwise increase in drug resistance, culminating in 500-fold increased fluconazole resistance in strains possessing mutations in the three transcription factors and an additional resistance mutation in the drug target enzyme Erg11. The acquisition of resistance mutations was associated with reduced fitness under non-selective conditions in vitro as well as in vivo during colonization of a mammalian host. Therefore, without compensatory mutations, the inability to appropriately regulate gene expression results in a loss of competitive fitness of drug-resistant C. albicans strains.

Functional control of the Candida albicans cell wall by catalytic protein kinase A subunit Tpk1

The cyclic AMP protein kinase A pathway governs numerous biological features of the fungal pathogen Candida albicans. The catalytic protein kinase A subunits, Tpk1 (orf19.4892) and Tpk2 (orf19.2277), have divergent roles, and most studies indicate a more pronounced role for Tpk2. Here we dissect two Tpk1-responsive properties: adherence and cell wall integrity. Homozygous tpk1/tpk1 mutants are hyperadherent, and a Tpk1 defect enables biofilm formation in the absence of Bcr1, a transcriptional regulator of biofilm adhesins. A quantitative gene expression-based assay reveals that tpk1/tpk1 and bcr1/bcr1 genotypes show mixed epistasis, as expected if Tpk1 and Bcr1 act mainly in distinct pathways. Overexpression of individual Tpk1-repressed genes indicates that cell surface proteins Als1, Als2, Als4, Csh1 and Csp37 contribute to Tpk1-regulated adherence. Tpk1 is also required for cell wall integrity, but has no role in the gene expression response to cell wall inhibition by caspofungin. Interestingly, increased expression of the adhesin gene ALS2 confers a cell wall defect, as manifested in hypersensitivity to the cell wall inhibitor caspofungin and a shallow cell wall structure. Our findings indicate that Tpk1 governs C. albicans cell wall properties through repression of select cell surface protein genes.

Cell surface shaving of Candida albicans biofilms, hyphae, and yeast form cells

We used a brief trypsin treatment followed by peptide separation and identification using nano-LC followed by off-line MS/MS to identify the surface proteins on live Candida albicans organisms growing in biofilms and planktonic yeast cells and hyphae. One hundred thirty-one proteins were present in at least two of the three replicates of one condition and distributed in various combinations of the three growth conditions. Both previously reported and new surface proteins were identified and these were distributed between covalently attached proteins and noncovalently attached proteins of the cell wall.

Candida albicans adherence to an acrylic resin modified by experimental photopolymerised coatings: an in vitro study

This study evaluated whether photopolymerised coatings containing zwitterion or hydrophilic monomers would reduce the adhesion of Candida albicans to an acrylic resin. Disc-shaped samples (n=468) were fabricated with rough or smooth surfaces. The samples did not receive any surface treatment (control) or were coated with one of the following experimental coatings (2-hydroxyethyl methacrylate - HE; 3-hydroxypropyl methacrylate - HP; and 2-trimethylammonium ethyl methacrylate chloride - T; and sulfobetaine methacrylate - S). The concentrations of the constituent monomers were 25, 30 or 35%. The water contact angles of the samples were measured, and half of the samples were exposed to saliva. The adherent yeast cells were counted after crystal violet staining. For the smooth samples, the groups S35, HP35 and HE35 showed significantly lower number of adhered Candida than control, in the absence of saliva. There were no significant differences among the experimental and control groups for the rough samples, but the saliva decreased the cell numbers for groups S25, S30 and HP30. The photoelectron spectroscopy analysis confirmed the changes in the chemical compositions of the experimental samples. The experimental photopolymerised coatings changed the chemical composition and decreased C.albicans adhesion in the groups S35, HP35 and HE35, suggesting that they should be further investigated.

Determinação da Concentração Inibitória Mínima do Clotrimazol e da Terbinafina sobre isolados de Candida albicans oriundas da mucosa bucal de pacientes HIV positivos e HIV negativos

Candidiasis is a fungi infection caused by yeasts from Candida genus, that live in the mucosas and just causes illness when there are favoring conditions to its growth, like immune compromised HIV infected patients. Resistance reports verified in C andida albicans isolates , especially in those immune compromised, and fast and efficient treatment need cause there to be a great interest in studies capable of using in vitro susceptibility tests to adequate therapy choose. The objective of this study was to determine the Minimum Inhibitory Concentration (MIC) of antifungal agents clotrimazole and terbinafine on 19 isolates of Candida albicans isolated from HIV positive patients

Complement plays a central role in Candida albicans‐induced cytokine production by human PBMCs

In experimental studies, the role of complement in antifungal host defense has been attributed to its opsonizing capability. In this study, we report that in humans an activated complement system mainly augments Candida albicans-induced host proinflammatory cytokine production via C5a-C5aR signaling, while phagocytosis and intracellular killing of Candida are not influenced. By blocking the C5a-C5aR signaling pathway, either with anti-C5a antagonist antibodies or with the C5aR antagonist W-54001, C. albicans-induced IL-6 and IL-1β levels were significantly reduced. Recombinant C5a augmented cytokine production. In addition, using serum from patients with various complement deficiencies, we demonstrated a crucial role of C5, but not C6 or the membrane attack complex, in C. albicans-induced IL-6 and IL-1β production in monocytes. These findings reveal a central role of anaphylatoxin C5a in augmenting host proinflammatory cytokine production upon contact with C. albicans, and define the role of the complement system in anti-Candida host defense in humans.

Dosage‐dependent roles of the Cwt1 transcription factor for cell wall architecture, morphogenesis, drug sensitivity and virulence in Candida albicans

The Cwt1 transcription factor is involved in cell wall architecture of the human fungal pathogen Candida albicans. We demonstrate here that deficiency of Cwt1 leads to decreased beta1,6-glucan in the cell wall, while mannoproteins are increased in the cell wall of exponentially growing cells and are released into the medium of stationary phase cells. Hyphal morphogenesis of cwt1 mutants is reduced on the surfaces of some inducing media. Unexpectedly, the CWT1/cwt1 heterozygous strains shows some stronger in vitro phenotypes compared to the homozygous mutant. The heterozygous but not the homozygous strain is also strongly impaired for its virulence in a mouse model of systemic infection. We suggest that an intermediate dosage of Cwt1 affects phenotypes profoundly, while its complete absence may elicit compensatory responses of C. albicans.

Efecto inhibitorio in vitro de ajoeno sobre aislamientos de Candida recuperados de secreciones vaginales

Aims The main purpose of this work was to evaluate the in vitro activity of ajoene of the Candida, obtained from vaginal discharges. Methods For this, 136 samples were analyzed. The yeasts were recovered and identified by conventional mycological methods. The susceptibility to ajoene (at 20, 15, 12.5, 10, 6.25 and 3.125 μg/ml) was performed according to the CLSI M27-A2 document with the EUCAST modifications. The ATCC reference strains 90028 (C andida albicans), 22019 ( Candida parapsilosis), and 6258 (Candida krusei) were included in this study. The minimal inhibitory concentration (MIC) was considered as the minimal concentration of ajoena able to inhibit 80% of the fungal growth. Results Fifty five yeasts were recovered, 36 (65.4%) of them were causing candidosis and 19 (34.5%) were colonizing. C. albicans was the most frequent (81.8%) of the six isolated species, prevailing on the patients with candidosis (54.5%). The non- albicans species were less frequently isolated (18.2%), and Candida glabrata was the prevailing agent (7.3%) followed by Candida tropicalis (3.6%), C. krusei, C. parapsilosis, Candida guilliermondii and Candida sp. (1.8% each of them). The susceptibility tests to ajoeno showed inhibition of fungal growth in 98.2% of the isolates, showing MIC values ⩽15 μg/ml, and in (one isolate of C. glabrata) (1.8%) this value was >20 μg/ml. The reference strains showed MIC values of 3.125 and 10 μg/ml. Conclusions The results here presented, obtained from a significant number of isolates, mainly C. albicans, demonstrate, once more, the potential of ajoeno as an antifungal agent.

Epidemiology of C andida albicans isolates from heroin addicts analysed by DNA typing

Candida albicans is a ubiquitous commensal organism of humans. Several studies have examined outbreaks of candidiasis in heroin addicts utilizing a variety of methods to assess the epidemiological relatedness of the isolates and suggested the association of certain subtypes with disease in this patient population. The aim of the present study was to assess a separate group of isolates of C. albicans from heroin addicts in Spain using a DNA typing method. Results showed that, of the 34 isolates from heroin addicts, 20 were in subgroup IA, 10 were in subgroup IB and no isolates were of the subtype IA2. In addition, four isolates were in a recently described subgroup IC. Control isolates from the same geographical region (Spain) showed a distribution similar to the Spanish heroin addict isolates (12 subgroup IA, three subgroup IB, two subgroup IC and no isolates of the subtype IA2). In this study isolates from the same locality appeared similar irrespective of the patient population from which they were isolated. These results indicated that there may be differing geographical diversity of C. albicans than has previously been reported and that the newly described genotypic subgroup (IC) of C. albicans may be more widespread than previously shown.

Exogenous Farnesol Interferes with the Normal Progression of Cytokine Expression during Candidiasis in a Mouse Model

Candida albicans, a dimorphic fungus composed of yeast and mycelial forms, is the most common human fungal pathogen. Th1 cytokines such as interleukin-2 (IL-2), gamma interferon (IFN-gamma), and tumor necrosis factor alpha (TNF-alpha), which are induced by macrophage IL-12, are critical to resistance against systemic candidiasis, while Th2 cytokines such as IL-4 and IL-5 are less critical. Farnesol is a quorum-sensing molecule produced by C. albicans that controls the formation of mycelia but is also a virulence factor. To determine whether farnesol enhances the virulence of C. albicans by modulating the production of Th1 and Th2 cytokines, mice were pretreated with farnesol prior to intravenous infection with a sublethal dose of farnesol-producing C. albicans. Production of IL-2, IL-4, IL-5, TNF-alpha, IFN-gamma, and IL-12 was evaluated by bead-array flow cytometry and enzyme-linked immunosorbent assay. Mice exhibited an elevation in serum TNF-alpha levels at 48 h and an elevation in IFN-gamma and IL-12 levels at 6 to 12 h after infection with C. albicans. Pretreatment with farnesol significantly reduced the elevation of both IFN-gamma and IL-12 but not TNF-alpha. In contrast, mice pretreated with farnesol exhibited an unexpected elevation in IL-5 levels. To determine whether farnesol has a direct effect on macrophage production of IL-12, peritoneal macrophages were pretreated with farnesol prior to stimulation with IFN-gamma plus lipopolysaccharide (LPS). Farnesol inhibited production of both IL-12 p40 and p70 from IFN-gamma/LPS-stimulated macrophages. Therefore, the role of farnesol in systemic candidiasis is likely due to its ability to inhibit the critical Th1 cytokines IFN-gamma and IL-12 and perhaps to enhance a Th2 cytokine, IL-5.

Enhanced Killing ofCandida albicansby Human Macrophages Adherent to Type 1 Collagen Matrices via Induction of Phagolysosomal Fusion

Candida albicans, a component of the normal flora of the alimentary tract and mucocutaneous membranes, is the leading cause of invasive fungal disease in premature infants, diabetics, and surgical patients and of oropharyngeal disease in AIDS patients. As little is known about the regulation of monocyte/macrophage anti-Candida activity, we sought to determine if fungicidal activity might be regulated by extracellular matrix proteins to which monocytes/macrophages are adherent in vivo. Compared to monocyte/macrophages that adhered to plastic, human monocytes and monocyte-derived macrophages that adhered to type 1 collagen matrices, but not to fibronectin, vitronectin, or laminin, demonstrated a significant increase in candidacidal activity. The enhancement of monocyte fungicidal activity was maintained over a 4-h period, whereas macrophage fungicidal activity was maximum at 1 h. Although adherence of monocytes and macrophages to collagen matrices concomitantly enhanced the production of superoxide anion, only the fungicidal activity of collagen-adherent monocytes was partially blocked by superoxide dismutase and catalase. Remarkably, we found that only 10% of the phagosomes in C. albicans-infected macrophages that adhered to plastic fused with lysosomes. In contrast, 80% of yeast-containing phagosomes of collagen-adherent macrophages fused with lysosomes. These data suggest that nonoxidative mechanisms are critical for human macrophage anti-Candida activity and that C. albicans pathogenicity is mediated, in part, by its ability to inhibit phagolysosomal fusion in macrophages.

The Hyphal and Yeast Forms ofCandida albicansBind the Complement Regulator C4b-Binding Protein

Candida albicans, an important pathogenic yeast, activates all three pathways of the complement system. To understand how this yeast evades the effects of the activated system, we have analyzed the binding of the classical pathway inhibitor C4b-binding protein (C4BP) by C. albicans. Purified native as well as recombinant C4BP bound dose dependently to the yeast and hyphal forms, as shown by multiple methods, such as confocal microscopy, flow cytometry, a novel enzyme-linked immunosorbent assay, absorption from human serum, and direct binding assays with purified proteins. A prominent binding site was identified at the tip of the germ tube, a structure that is considered important for tissue penetration and pathogenesis. The binding site in C4BP was localized to the two N-terminal complement control protein domains by using recombinant deletion constructs and site-specific monoclonal antibodies. As the alternative pathway inhibitors factor H and FHL-1 also bind to C. albicans, the binding of all three plasma proteins was compared. Simultaneous binding of the classical regulator C4BP and the alternative pathway regulator factor H was demonstrated by confocal microscopy. In addition, FHL-1 competed for binding with C4BP, suggesting that these two related complement regulators bind to the same structures on the yeast surface. The surface-attached C4BP maintains its complement regulatory activities and inactivates C4b. The surface-attached human C4BP serves multiple functions relevant for immune evasion and likely pathogenicity. It inhibits complement activation at the yeast surface and, in addition, mediates adhesion of C. albicans to host endothelial cells.

Co-regulation of pathogenesis with dimorphism and phenotypic switching in Candida albicans, a commensal and a pathogen

AbstractCandida albicans, a common fungal pathogen of humans, can colonize in many diverse environments of the host and convert between a harmless commensal and a pathogen. Recent advances indicate that C. albicans uses a common set of conserved pathways to regulate dimorphism, mating and phenotypic switching. Major pathways known to regulate dimorphism include a mitogen-activated protein (MAP) kinase pathway through Cph1, the cAMP-dependent protein kinase pathway via Efg1, and Tup1-mediated repression through Rfg1 and Nrg1. The Cph1-mediated MAP kinase pathway is critical for the mating process, while all three pathways are implicated in the regulation of white-opaque switching. All these developmental pathways regulate the expression of hypha-specific and/or phase-specific genes. A high proportion of hypha-specific genes and phase-specific genes encode proteins that contribute directly or indirectly to pathogenesis and virulence of C. albicans. Therefore, virulence genes are co-regulated with cell morphogenesis. This supports a previous notion that the unique aspects of C. albicans commensalism and pathogenesis may lie in the developmental programs of dimorphism and phenotypic switching.

Carotid body resection: Approved by the executive committee of the American academy of allergy and immunology

Executive Committee: John A. Anderson Hvman Chai Henry N. C[alqlall Elliot F. Ellis Jordan N. Fink A[len P. Kap/an Philip L. Lieberman William E. Pierson John E. Salw~ggio Albert L. Sheff~,r Rawnond G. SMvin REFERENCES I. Tru,s CO: Ti.,,sue injury induced by ('amhda Albicans: mental and neurohgic manifestations. J ()rlhomolecular Psychiatry 7:l 7, 1978 2 Tru:,s CO: Restoration of immunologic competence to ('andida albicans. J Orthomolecular Psychialr? 9:287. 191'10 3 Tru!,s CO: The role of Candi, hl albicans m human dlncss. J Orthomolecular Psychiatry I 0:228. J 981 4. Crook W G The yeast connection: a medical breakthrough, ed 2. JJckson. fenn., 1984. PlofL'ssional Book~,

Candida albicans antibodies in candidiasis.

AbstractCandida albicans agglutinins and precipitins were examined in sera from 250 blood donors, 51 patients with malignant diseases without candidiasis, and 54 patients with malignant diseases and candidiasis. Agglutinins were encountered in 46.5% of persons without candidiasis, in 58.2% of patients with candidiasis in the oral cavity and/or pharynx and larynx, and in 54.5% of patients with severe candida infections. Agglutinins in titres of ≥8 were found in 34.9% of patients with candidiasis in the oral cavity and/or pharynx and larynx and in 15.0% of persons without candidiasis (0.005>P>0.001). Agglutinins in titres ≥128 were found only in patients with candidiasis, and an increase or a decrease in titre was demonstrated in some patients during the course of the disease. Precipitins were found in 1% of persons without candidiasis and in 20.4% of patients with candidiasis, and most often in sera with high agglutinin titres.