Molecular Mechanisms of Fluconazole Resistance in Candida Dubliniensis Isolates from Human Immunodeficiency Virus-infected Patients with Oropharyngeal Candidiasis

Antimicrobial Agents and Chemotherapy. Jun, 2002  |  Pubmed ID: 12019078

Candida dubliniensis is a newly identified species of Candida that is phenotypically similar to but genetically distinct from C. albicans. This organism has been recovered with increasing frequency from the oral cavities of human immunodeficiency virus (HIV)-infected and AIDS patients and has been implicated as a causative agent of oral candidiasis and systemic disease. In the present study we characterized the molecular mechanisms of resistance to fluconazole (FLC) in C. dubliniensis clinical isolates from two different HIV-infected patients with oropharyngeal candidiasis. Isolates were identified to the species level by phenotypic and genotypic tests. DNA-typing techniques were used to assess strain identity. Antifungal susceptibility testing was performed by NCCLS techniques. Northern blotting analysis was used to monitor the expression of genes encoding lanosterol demethylase (ERG11) and efflux transporters (CDR and MDR1) in matched sets of C. dubliniensis-susceptible and -resistant isolates by using probes generated from their homologous C. albicans sequences. In addition, ERG11 genes were amplified by PCR, and their nucleotide sequences were determined in order to detect point mutations with a possible effect in the affinity for azoles. Decreasing susceptibilities to FLC were detected in C. dubliniensis isolates recovered from both patients during the course of treatment. FLC-resistant C. dubliniensis isolates from one patient demonstrated combined upregulation of the MDR1, CDR1, and ERG11 genes. Among the isolates from the second patient, all isolates showing decreased susceptibility to FLC demonstrated upregulation of MDR1, whereas the levels of mRNA for the ERG11 genes remained constant and the expression of CDR genes was negligible. Fourteen point mutations were found in the ERG11 genes of the isolates with decreased susceptibility to FLC. These data demonstrate that the development of azole resistance in C. dublinensis clinical isolates from HIV-infected patients treated with FLC is mediated by multiple molecular mechanisms of resistance, similar to the observations found in the case of C. albicans.

In Vitro Activity of Caspofungin (MK-0991) Against Candida Albicans Clinical Isolates Displaying Different Mechanisms of Azole Resistance

Journal of Clinical Microbiology. Jun, 2002  |  Pubmed ID: 12037093

Caspofungin inhibits the synthesis of 1,3-beta-D-glucan, a key step in fungal cell wall biosynthesis. Here we report on its potent in vitro activity (MIC at which 90% of the isolates tested are inhibited = 1 microg per ml of RPMI medium) against 32 Candida albicans fluconazole-susceptible and -resistant clinical isolates irrespective of the underlying resistance mechanism (alterations in ERG11 and/or upregulation of MDR and CDR genes encoding efflux pumps) and provide further evidence that caspofungin is not a substrate for multidrug transporters.

Investigation of Multidrug Efflux Pumps in Relation to Fluconazole Resistance in Candida Albicans Biofilms

The Journal of Antimicrobial Chemotherapy. Jun, 2002  |  Pubmed ID: 12039889

A main characteristic associated with microbial biofilms is their increased resistance to antimicrobial chemotherapies. However, at present very little is known about the phenotypic changes that occur during the transition from the planktonic to the biofilm mode of growth. Candida albicans biofilms displayed an organized three-dimensional structure, and consisted of a dense network of yeasts and filamentous cells deeply embedded in exopolymeric matrix. These biofilms were intrinsically resistant to fluconazole. Moreover, the resistance phenotype was maintained by sessile cells when resuspended as free-floating cells, thus demonstrating that biofilm integrity and the presence of exopolymeric material are not the sole determinants of biofilm resistance. Under planktonic conditions, one of the main mechanisms of azole resistance in C. albicans is through active efflux of these drugs mediated by ATP-binding cassette (ABC) transporters and major facilitators. In this study we used northern hybridization to monitor expression of genes belonging to two different types of efflux pump, the ABC transporters and major facilitators (encoded by CDR and MDR genes, respectively), in C. albicans populations under both planktonic and biofilm growth. It was demonstrated that expression of genes encoding both types of efflux pump were up-regulated during the course of biofilm formation and development. Moreover, antifungal susceptibilities of biofilms formed by a set of C. albicans mutant strains deficient in efflux pumps were investigated to determine their contribution to biofilm resistance. Remarkably, mutants carrying single and double deletion mutations in Delta(cdr)1, Delta(cdr)2, Delta(mdr)1, Delta(cdr)1/Delta(cdr)2 and Delta(mdr)1/Delta(cdr)1 were hypersusceptible to fluconazole when planktonic, but still retained the resistant phenotype during biofilm growth. These analyses demonstrate that C. albicans biofilm resistance is a complex phenomenon that cannot be explained by one mechanism alone, instead it is multifactorial and may involve different molecular mechanisms of resistance compared with those displayed by planktonic cells.

Replacement of Candida Albicans with C. Dubliniensis in Human Immunodeficiency Virus-infected Patients with Oropharyngeal Candidiasis Treated with Fluconazole

Journal of Clinical Microbiology. Sep, 2002  |  Pubmed ID: 12202543

Candida dubliniensis is an opportunistic yeast that has been increasingly implicated in oropharyngeal candidiasis (OPC) in human immunodeficiency virus (HIV)-infected patients but may be underreported due to its similarity with Candida albicans. Although most C. dubliniensis isolates are susceptible to fluconazole, the inducibility of azole resistance in vitro has been reported. Thus, the use of fluconazole prophylaxis in the treatment of these patients may have contributed to the increasing rates of isolation of C. dubliniensis. In this study, yeast strains were collected from the oral cavities of HIV-infected patients enrolled in a longitudinal study of OPC. Patients received fluconazole for the suppression or treatment of OPC, and isolates collected at both study entry and end of study were chosen for analysis. Samples were plated on CHROMagar Candida medium for initial isolation and further identified by Southern blot analysis with the species-specific probes Ca3 (for C. albicans) and Cd25 (for C. dubliniensis). Fluconazole MICs were determined by using NCCLS methods. At study entry, susceptible C. albicans isolates were recovered from oral samples in 42 patients who were followed longitudinally (1 to 36 months). C. albicans strains from 12 of these patients developed fluconazole resistance (fluconazole MIC, >/=64 micro g/ml). C. dubliniensis was not detected at end of study in any of these patients. Of the remaining 30 patients, eight (27%) demonstrated a replacement of C. albicans by C. dubliniensis when a comparison of isolates obtained at baseline and those from the last culture was done. For the 22 of these 30 patients in whom no switch in species was detected, the fluconazole MICs for initial and end-of-study C. albicans isolates ranged from 0.125 to 2.0 micro g/ml. For the eight patients in whom a switch to C. dubliniensis was detected, the fluconazole MICs for C. dubliniensis isolates at end of study ranged from 0.25 to 64 micro g/ml: the fluconazole MICs for isolates from six patients were 0.25 to 2.0 micro g/ml and those for the other two were 32 and 64 micro g/ml, respectively. In conclusion, a considerable number of patients initially infected with C. albicans strains that failed to develop fluconazole resistance demonstrated a switch to C. dubliniensis. C. dubliniensis in this setting may be underestimated due to lack of identification and may occur due to the impact of fluconazole on the ecology of oral yeast species.

The Filamentation Pathway Controlled by the Efg1 Regulator Protein is Required for Normal Biofilm Formation and Development in Candida Albicans

FEMS Microbiology Letters. Aug, 2002  |  Pubmed ID: 12204378

Candida albicans biofilms are structured microbial communities composed of a mixture of yeast cells and hyphal elements, suggesting a pivotal role for the dimorphic switch in the development of biofilms. We have used C. albicans mutants defective in genes involved in filamentation (Deltacph1, Deltaefg1, Deltahst7, and Deltacst20) and compared these mutants to wild-type strains to determine whether filamentation is an integral factor for biofilm formation. Scanning electron microscopy revealed that Deltacph1, Deltahst7 and Deltacst20 mutants were able to filament and form structured biofilms displaying three-dimensional architecture similar to those formed by wild-type strains. However, Deltaefg1 and Deltacph1/Deltaefg1 mutants were unable to filament and did not form biofilms, but rather sparse monolayers of loosely attached elongated, rod-like, cells. Antimicrobial susceptibility testing showed intrinsic resistance of all mutant strains to fluconazole and amphotericin B when attached to the surface of biomaterials. These results suggest that hyphal formation is pivotal for biofilm development in C. albicans. However, the sessile lifestyle associated with adherent cells confers antifungal resistance, regardless of coherent biofilm formation.

In Vitro Activity of Caspofungin Against Candida Albicans Biofilms

Antimicrobial Agents and Chemotherapy. Nov, 2002  |  Pubmed ID: 12384370

Most manifestations of candidiasis are associated with biofilm formation on biological or inanimate surfaces. Candida albicans biofilms are recalcitrant to treatment with conventional antifungal therapies. Here we report on the activity of caspofungin, a new semisynthetic echinocandin, against C. albicans biofilms. Caspofungin displayed potent in vitro activity against sessile C. albicans cells within biofilms, with MICs at which 50% of the sessile cells were inhibited well within the drug's therapeutic range. Scanning electron microscopy and confocal scanning laser microscopy were used to visualize the effects of caspofungin on preformed C. albicans biofilms, and the results indicated that caspofungin affected the cellular morphology and the metabolic status of cells within the biofilms. The coating of biomaterials with caspofungin had an inhibitory effect on subsequent biofilm development by C. albicans. Together these findings indicate that caspofungin displays potent activity against C. albicans biofilms in vitro and merits further investigation for the treatment of biofilm-associated infections.

In Vitro Pharmacodynamic Properties of Three Antifungal Agents Against Preformed Candida Albicans Biofilms Determined by Time-kill Studies

Antimicrobial Agents and Chemotherapy. Nov, 2002  |  Pubmed ID: 12384379

We have examined the in vitro activities of fluconazole, amphotericin B, and caspofungin against Candida albicans biofilms by time-kill methodology. Fluconazole was ineffective against biofilms. Killing of biofilm cells was suboptimal at therapeutic concentrations of amphotericin B. Caspofungin displayed the most effective pharmacokinetic properties, with > or =99% killing at physiological concentrations.

Inhibition of Candida Albicans Biofilm Formation by Farnesol, a Quorum-sensing Molecule

Applied and Environmental Microbiology. Nov, 2002  |  Pubmed ID: 12406738

Farnesol is a quorum-sensing molecule that inhibits filamentation in Candida albicans. Both filamentation and quorum sensing are deemed to be important factors in C. albicans biofilm development. Here we examined the effect of farnesol on C. albicans biofilm formation. C. albicans adherent cell populations (after 0, 1, 2, and 4 h of adherence) and preformed biofilms (24 h) were treated with various concentrations of farnesol (0, 3, 30, and 300 micro M) and incubated at 37 degrees C for 24 h. The extent and characteristics of biofilm formation were then assessed microscopically and with a semiquantitative colorimetric technique based on the use of 2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide. The results indicated that the effect of farnesol was dependent on the concentration of this compound and the initial adherence time, and preincubation with 300 micro M farnesol completely inhibited biofilm formation. Supernatant media recovered from mature biofilms inhibited the ability of planktonic C. albicans to form filaments, indicating that a morphogenetic autoregulatory compound is produced in situ in biofilms. Northern blot analysis of RNA extracted from cells in biofilms indicated that the levels of expression of HWP1, encoding a hypha-specific wall protein, were decreased in farnesol-treated biofilms compared to the levels in controls. Our results indicate that farnesol acts as a naturally occurring quorum-sensing molecule which inhibits biofilm formation, and we discuss its potential for further development and use as a novel therapeutic agent.

Engineered Control of Cell Morphology in Vivo Reveals Distinct Roles for Yeast and Filamentous Forms of Candida Albicans During Infection

Eukaryotic Cell. Oct, 2003  |  Pubmed ID: 14555488

It is widely assumed that the ability of Candida albicans to switch between different morphologies is required for pathogenesis. However, most virulence studies have used mutants that are permanently locked into either the yeast or filamentous forms which are avirulent but unsuitable for discerning the role of morphogenetic conversions at the various stages of the infectious process. We have constructed a strain in which this developmental transition can be externally modulated both in vitro and in vivo. This was achieved by placing one copy of the NRG1 gene (a negative regulator of filamentation) under the control of a tetracycline-regulatable promoter. This modified strain was then tested in an animal model of hematogenously disseminated candidiasis. Mice injected with this strain under conditions permitting hyphal development succumbed to the infection, whereas all of the animals injected under conditions that inhibited this transition survived. Importantly, fungal burdens were almost identical in both sets of animals, indicating that, whereas filament formation appears to be required for the mortality resulting from a deep-seated infection, yeast cells play an important role early in the infectious process by extravasating and disseminating to the target organs. Moreover, these infecting Candida yeast cells still retained their pathogenic potential, as demonstrated by allowing this developmental transition to occur at various time points postinfection. We demonstrate here the importance of morphogenetic conversions in C. albicans pathogenesis. This engineered strain should provide a useful tool in unraveling the individual contributions of the yeast and filamentous forms at various stages of the infectious process.

Antifungal Combinations Against Candida Albicans Biofilms in Vitro

Antimicrobial Agents and Chemotherapy. Nov, 2003  |  Pubmed ID: 14576141

Candida biofilms display increased resistance to most antifungal agents. We have evaluated the efficacy of combinations of fluconazole (FLC), amphotericin B, and caspofungin (CSP) against Candida albicans biofilms in vitro. Indifference was observed for all the combinations of paired antifungal agents when a checkerboard titration method was used. Time-kill experiments revealed an antagonistic effect of high FLC doses with CSP.

Changes in Susceptibility to Posaconazole in Clinical Isolates of Candida Albicans

The Journal of Antimicrobial Chemotherapy. Jan, 2004  |  Pubmed ID: 14657086

To characterize the molecular mechanisms responsible for reduced susceptibility to azoles in Candida albicans clinical isolates.

The C-terminal Antibody Binding Domain of Candida Albicans Mp58 Represents a Protective Epitope During Candidiasis

FEMS Microbiology Letters. Mar, 2004  |  Pubmed ID: 15033231

The 58-kDa surface mannoprotein of Candida albicans (mp58) elicits strong antibody responses during infection. Epitope mapping with sera from patients with candidiasis and control individuals indicated the presence of multiple IgG-reactive continuous epitopes on the protein, expanding both the amino- and carboxy-terminal domains and several internal regions. These immunoreactive regions were similar to the ones previously identified using sera from immunized animals. Two of the epitopic regions (including the C-terminal domain) showed increased reactivity with antibodies present in sera from patients with candidiasis as compared to control individuals. Patients who survived the infection displayed increased antibody reactivity towards the C-terminal epitope as compared to those succumbing to candidiasis. A monoclonal antibody directed towards this epitopic region conferred protection in serum therapy experiments in a murine model of hematogenously disseminated candidiasis. Together, these observations indicate the carboxy-terminal antibody binding domain of C. albicans mp58 represents a protective epitope during candidiasis.

Caspofungin Resistance in Candida Albicans: Correlating Clinical Outcome with Laboratory Susceptibility Testing of Three Isogenic Isolates Serially Obtained from a Patient with Progressive Candida Esophagitis

Antimicrobial Agents and Chemotherapy. Apr, 2004  |  Pubmed ID: 15047549

A patient with azole-refractory thrush-esophagitis responded initially to caspofungin, but the treatment eventually failed. In a murine model, caspofungin was effective against two early isolates for which the MICs of caspofungin were low, but it was less effective against a late isolate for which the MIC of caspofungin was greater. We concluded that there is a correlation between in vivo failure and rising in vitro caspofungin MICs.

Inducible Defense Mechanism Against Nitric Oxide in Candida Albicans

Eukaryotic Cell. Jun, 2004  |  Pubmed ID: 15189992

The yeast Candida albicans is an opportunistic pathogen that threatens patients with compromised immune systems. Immune cell defenses against C. albicans are complex but typically involve the production of reactive oxygen species and nitrogen radicals such as nitric oxide (NO) that damage the yeast or inhibit its growth. Whether Candida defends itself against NO and the molecules responsible for this defense have yet to be determined. The defense against NO in various bacteria and the yeast Saccharomyces cerevisiae involves an NO-scavenging flavohemoglobin. The C. albicans genome contains three genes encoding flavohemoglobin-related proteins, CaYHB1, CaYHB4, and CaYHB5. To assess their roles in NO metabolism, we constructed strains lacking each of these genes and demonstrated that just one, CaYHB1, is responsible for NO consumption and detoxification. In C. albicans, NO metabolic activity and CaYHB1 mRNA levels are rapidly induced by NO and NO-generating agents. Loss of CaYHB1 increases the sensitivity of C. albicans to NO-mediated growth inhibition. In mice, infections with Candida strains lacking CaYHB1 still resulted in lethality, but virulence was decreased compared to that in wild-type strains. Thus, C. albicans possesses a rapid, specific, and highly inducible NO defense mechanism involving one of three putative flavohemoglobin genes.

Antibody Response to Candida Albicans Cell Wall Antigens

FEMS Immunology and Medical Microbiology. Jul, 2004  |  Pubmed ID: 15196567

The cell wall of Candida albicans is not only the structure where many essential biological functions reside but is also a significant source of candidal antigens. The major cell wall components that elicit a response from the host immune system are proteins and glycoproteins, the latter being predominantly mannoproteins. Both carbohydrate and protein moieties are able to trigger immune responses. Proteins and glycoproteins exposed at the most external layers of the wall structure are involved in several types of interactions of fungal cells with the exocellular environment. Thus, coating of fungal cells with host antibodies has the potential to profoundly influence the host-parasite interaction by affecting antibody-mediated functions such as opsonin-enhanced phagocytosis and blocking the binding activity of fungal adhesins to host ligands. In this review we examine various members of the protein and glycoprotein fraction of the C. albicans cell wall that elicit an antibody response in vivo. Some of the studies demonstrate that certain cell wall antigens and anti-cell wall antibodies may be the basis for developing specific and sensitive serologic tests for the diagnosis of candidiasis, particularly the disseminated form. In addition, recent studies have focused on the potential of antibodies against the cell wall protein determinants in protecting the host against infection. Hence, a better understanding of the humoral response triggered by the cell wall antigens of C. albicans may provide the basis for the development of (i) effective procedures for the serodiagnosis of disseminated candidiasis, and (ii) novel prophylactic (vaccination) and therapeutic strategies to control this type of infections.

Denture Stomatitis: a Role for Candida Biofilms

Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontics. Jul, 2004  |  Pubmed ID: 15243471

To assess the contribution of Candida biofilms to the etiology of denture stomatitis.

Candida Biofilms: an Update

Eukaryotic Cell. Apr, 2005  |  Pubmed ID: 15821123

Candida Albicans Biofilms: More Than Filamentation

Current Biology : CB. Jun, 2005  |  Pubmed ID: 15964263

Candida albicans is the fungal species most commonly associated with biofilm formation in immunosuppressed patients. Recent work offers a fresh new look at the role of filamentation in C. albicans biofilm formation, and describes the application of a powerful tool for the molecular dissection of these important developmental processes.

Use of Genome Information for the Study of the Pathogenesis of Fungal Infections and the Development of Diagnostic Tools

Revista Iberoamericana De Micología : órgano De La Asociación Española De Especialistas En Micología. Dec, 2005  |  Pubmed ID: 16499417

One of the most exciting advances in Mycology is the application of genomic approaches. The advent of genomics, together with post-genomic studies, promises to revolutionize the studies on the pathogenesis of fungal infections. Approaches include comparative genomics to identify sequences that contribute to infection and disease and functional genomics and proteomics to analyze global patterns of gene and protein expression involved in fungal pathogenesis.

Drosophila Melanogaster As a Facile Model for Large-scale Studies of Virulence Mechanisms and Antifungal Drug Efficacy in Candida Species

The Journal of Infectious Diseases. Apr, 2006  |  Pubmed ID: 16518764

Candida species are the predominant fungal pathogens in humans and an important cause of mortality in immunocompromised patients. We developed a model of candidiasis in Toll (Tl)-deficient Drosophila melanogaster. Similar to the situation in humans, C. parapsilosis was less virulent than C. albicans when injected into Tl mutant flies. In agreement with findings in the mouse model of invasive candidiasis, cph1/cph1 and efg1/efg1 C. albicans mutants had attenuated virulence, and the efg1/efg1 cph1/cph1 double mutant was almost avirulent in Tl mutant flies. Furthermore, the conditional tet-NRG1 C. albicans strain displayed significantly attenuated virulence in flies fed food without doxycycline; virulence was restored to wild-type levels when the strain was injected into Tl mutant flies fed doxycycline-containing food. Fluconazole (FLC) mixed into food significantly protected Tl mutant flies injected with FLC-susceptible C. albicans strains, but FLC had no activity in flies injected with FLC-resistant C. krusei strains. The D. melanogaster model is a promising minihost model for large-scale studies of virulence mechanisms and antifungal drug activity in candidiasis.

Proteomics for the Analysis of the Candida Albicans Biofilm Lifestyle

Proteomics. Nov, 2006  |  Pubmed ID: 17001605

Candida albicans is an opportunistic pathogenic fungus capable of causing infections in immunocompromised patients. Candidiasis is often associated with the formation of biofilms on the surface of inert or biological materials. Biofilms are structured microbial communities attached to a surface and encased within a matrix of exopolymeric substance (EPS). At present, very little is known about the changes in protein profiles that occur during the transition from the planktonic to the biofilm mode of growth. Here, we report the use of proteomics for the comparative analysis of subcellular fractions obtained from C. albicans biofilm and planktonic cultures, including cell surface-associated proteins and secreted components present in liquid culture supernatants (for planktonic cultures) and EPS (for biofilms). The analysis revealed a high degree of similarity between the protein profiles associated with the planktonic and biofilm extracts, and led to the identification of several differentially expressed protein spots. Among the differentially expressed proteins, there was a preponderance of metabolic enzymes that have been described as cell surface proteins and immunodominant antigens. Proteins found in the biofilm matrix included a few predicted to form part of the secretome, and also many secretion-signal-less proteins. These observations contribute to our understanding of the C. albicans biofilm lifestyle.

Inhibition of Filamentation Can Be Used to Treat Disseminated Candidiasis

Antimicrobial Agents and Chemotherapy. Oct, 2006  |  Pubmed ID: 17005810

Candida albicans remains the leading causative agent of invasive fungal infection. Although the importance of filamentation in C. albicans pathogenesis has been extensively investigated, in vivo studies to date have been unable to dissect the role of this developmental process in the establishment of infection versus the development of active disease as characterized by damage to the host leading to mortality. To address this issue, we genetically engineered a C. albicans tet-NRG1 strain in which filamentation and virulence can be modulated both in vitro and in vivo simply by the presence or absence of doxycycline (DOX): this strain enabled us, in a prior study, to demonstrate that yeast-form cells were able to infect the deep organs but caused no disease unless filamentation (induced by the addition of DOX) was allowed to occur. In the present study, we examined whether inhibiting filamentation (by withdrawing the DOX) at 24 or 48 h postinfection could serve as an effective therapeutic intervention against candidiasis. The results obtained indicate that DOX removal led to an alteration in the morphology of the infecting fungal cells and a dramatic increase in survival, but as with conventional antifungal drug therapy regimens, mortality rates increased markedly the longer this intervention was delayed. These observations reinforce the importance of invasive filamentous growth in causing the damage to the host and the lethality associated with active disease and suggest this process could be fruitfully targeted for the development of new antifungal agents.

Biofilm Formation by Candida Albicans Mutants for Genes Coding Fungal Proteins Exhibiting the Eight-cysteine-containing CFEM Domain

FEMS Yeast Research. Nov, 2006  |  Pubmed ID: 17042757

Several features and functions of a Candida albicans gene, PGA10 (also designated as RBT51), coding for a putative polypeptide species belonging to a subset of fungal proteins containing an eight-cysteine domain referred as CFEM (Common in several Fungal Extracellular Membrane proteins), are described. The ORF of the gene (ORF19.5674) encoded a protein of 250 amino acids, with a predicted molecular mass of 25.17 kDa. The product of the PGA10 gene also exhibited some features reminiscent of a class II-type hydrophobin. Deletion of PGA10 resulted in a cascade of pleiotropic effects, mostly affecting cell-surface-related properties. Thus, the null pga10Delta mutant displayed an increased sensitivity to cell-wall-perturbing agents and formed fragile biofilms that appeared partially split and weakly attached to the substratum. The biofilm-forming ability of several C. albicans mutants with single, double and triple deletions of genes encoding other protein species also containing the CFEM domain (RBT5 and WAP1/CSA1) was determined. These mutants also exhibited an abnormal ability to form biofilms. Overall, the evidence presented here suggests that fungal proteins containing the CFEM domain (Pga10p/Rbt51p, Rbt5p and Wap1p/Csa1p) may play a key role in the formation, development and/or maintenance of the biofilm structure in C. albicans.

A Proteomic-based Approach for the Identification of Candida Albicans Protein Components Present in a Subunit Vaccine That Protects Against Disseminated Candidiasis

Proteomics. Nov, 2006  |  Pubmed ID: 17051645

Candidiasis has become a prevalent infection in different types of immunocompromised patients. The cell wall of Candida albicans plays important functions during the host-fungus interactions. Cell wall (surface) proteins of C. albicans are major elicitors of host immune responses during candidiasis, and represent candidates for vaccine development. Groups of mice were vaccinated subcutaneously with a beta-mercaptoethanol (beta-ME) extract from C. albicans containing cell wall proteins. Vaccinated mice were then infected with a lethal dose of C. albicans. Increased survival and decreased fungal burden were observed in vaccinated mice as compared to a control group, and 75% of vaccinated mice with the beta-ME extract survived this otherwise lethal infection. We used a proteomic approach (2-DE followed by immunoblotting) to demonstrate a complex polypeptidic pattern associated with the beta-ME extract used in the vaccine formulation and to detect immunogenic components recognized by antibodies in immune sera from vaccinated animals. Reactive protein spots were identified by MALDI-TOF-MS and searches in genomic databases. As a conclusion, vaccination strategies using C. albicans cell wall proteins induce protective responses. These antigens can be identified by proteomic approaches and may be used as components of subcellular vaccines against candidiasis.

Proteomics to Study Candida Albicans Biology and Pathogenicity

Infectious Disorders Drug Targets. Dec, 2006  |  Pubmed ID: 17168799

Candida albicans is an opportunistic pathogenic fungus capable of causing infections in an expanding population of immunosuppressed patients. The implementation of proteomics in the post-genomic era of this organism can provide vital information about its biological complexity and pathogenic traits. C. albicans proteomic analyses to date have focused on the understanding of the cell wall, virulence, dimorphism, antifungal drug effects and resistance, and serological response, among others. This exciting and rapid growing discipline should become an indispensable tool in C. albicans research, particularly to address problems that cannot be solved by genomic studies. Furthermore, in the near future it is expected that results from proteomic experiments will lead to novel techniques for the management of candidiasis.

A Role for Efg1p in Candida Albicans Interactions with Extracellular Matrices

FEMS Microbiology Letters. Mar, 2006  |  Pubmed ID: 16487333

Candida albicans interactions with extracellular matrices extracellular matrices represent hallmarks of invasive candidiasis. We have assessed the role of Efg1p, a key transcriptional regulator, in the interactions between C. albicans and extracellular matrices. Wild-type C. albicans cells were able to bind and subsequently penetrate the extracellular matrices layer, whereas the Deltaefg1/Deltaefg1 mutant strain showed a drastically reduced ability to interact with extracellular matrices. Results indicate that, besides its control of morphogenesis, Efg1p also regulates C. albicans interactions with extracellular matrices. Proteomic analyses indicated that expression of 30 surface-associated proteins is affected in the Deltaefg1/Deltaefg1 mutant, some of which may be involved in adherence to extracellular matrices.

Inhibition on Candida Albicans Biofilm Formation Using Divalent Cation Chelators (EDTA)

Mycopathologia. Dec, 2007  |  Pubmed ID: 17909983

Candida albicans can readily form biofilms on both inanimate and biological surfaces. In this study we investigated a means of inhibiting biofilm formation using EDTA (Ethylenediaminetetra-acetic acid), a divalent cation chelating agent, which has been shown to affect C. albicans filamentation. Candida albicans biofilms were formed in 96-well microtitre plates. Cells were allowed to adhere for 1, 2, and 4 h at 37 degrees C, washed in PBS, and then treated with different concentrations of EDTA (0, 2.5, 25, and 250 mM). EDTA was also added to the standardized suspension prior to adding to the microtiter plate and to a preformed 24 h biofilm. All plates were then incubated at 37 degrees C for an additional 24 h to allow for biofilm formation. The extent and characteristics of biofilm formation were then microscopically assessed and with a semi-quantitative colorimetric technique based on the use of an XTT-reduction assay. Northern blot analysis of the hyphal wall protein (HWP1) expression was also monitored in planktonic and biofilm cells treated with EDTA. Microscopic analysis and colorimetric readings revealed that filamentation and biofilm formation were inhibited by EDTA in a concentration dependent manner. However, preformed biofilms were minimally affected by EDTA (maximum of 31% reduction at 250 mM). The HWP1 gene expression was reduced in EDTA-treated planktonic and biofilm samples. These results indicate that EDTA inhibits C. albicans biofilm formation are most likely through its inhibitory effect on filamentation and indicates the potential therapeutic effects of EDTA. This compound may serve a non-toxic means of preventing biofilm formation on infections with a C. albicans biofilm etiology.

Use of a Genetically Engineered Strain to Evaluate the Pathogenic Potential of Yeast Cell and Filamentous Forms During Candida Albicans Systemic Infection in Immunodeficient Mice

Infection and Immunity. Jan, 2008  |  Pubmed ID: 17967861

The pathogenesis of Candida albicans systemic infection is complex and results from the balance between its intrinsic virulence attributes and the host immune responses. Morphogenetic transitions between yeast cell and filamentous forms are considered one of the main virulence attributes in C. albicans. We have examined the pathogenesis of a genetically engineered C. albicans strain in which morphogenetic conversions can be externally manipulated in immunodeficient mice; these included B-cell deficient, nude (T cell deficient), SCID (lacking both functional T and B cells), and DBA/2N (C5 deficient with impaired neutrophil activity) mice. We also tested mice severely immunosuppressed by cyclophosphamide-cortisone acetate treatment. Mice with specific immune defects were able to survive an infection by yeast cells but not filamentous forms. However, yeast cells displayed a pathogenic effect leading to lethality in the severely immunosuppressed mice.

UME6, a Novel Filament-specific Regulator of Candida Albicans Hyphal Extension and Virulence

Molecular Biology of the Cell. Apr, 2008  |  Pubmed ID: 18216277

The specific ability of the major human fungal pathogen Candida albicans, as well as many other pathogenic fungi, to extend initial short filaments (germ tubes) into elongated hyphal filaments is important for a variety of virulence-related processes. However, the molecular mechanisms that control hyphal extension have remained poorly understood for many years. We report the identification of a novel C. albicans transcriptional regulator, UME6, which is induced in response to multiple host environmental cues and is specifically important for hyphal extension. Although capable of forming germ tubes, the ume6Delta/ume6Delta mutant exhibits a clear defect in hyphal extension both in vitro and during infection in vivo and is attenuated for virulence in a mouse model of systemic candidiasis. We also show that UME6 is an important downstream component of both the RFG1-TUP1 and NRG1-TUP1 filamentous growth regulatory pathways, and we provide evidence to suggest that Nrg1 and Ume6 function together by a negative feedback loop to control the level and duration of filament-specific gene expression in response to inducing conditions. Our results suggest that hyphal extension is controlled by a specific transcriptional regulatory mechanism and is correlated with the maintenance of high-level expression of genes in the C. albicans filamentous growth program.

A Seed and Feed Model for the Formation of Candida Albicans Biofilms Under Flow Conditions Using an Improved Modified Robbins Device

Revista Iberoamericana De Micología : órgano De La Asociación Española De Especialistas En Micología. Mar, 2008  |  Pubmed ID: 18338926

A variety of manifestations of Candida albicans infections are associated with the formation of biofilms on the surface of biomaterials. In order to maintain their niche these adherent populations need to withstand the continuous bathing action of physiological fluids (saliva, blood), which also provide water and nutrients to the fungal cells. Thus, it was the aim of this study to examine and further characterize the development of C. albicans biofilms under shear forces and a flow of replenishing nutrients, emulating the conditions that fungal cells would normally encounter within the host. An improved modified Robbins device (MRD) was designed to hold six poly methyl methacrylate (PMMA) plugs of 25 mm in diameter. A "seed and feed" model of biofilm formation was then implemented for which the apparatus was initially seeded with a C. albicans cell suspension to allow initial adhesion of fungal cells to the biomaterial. Following this initial step, sterile medium was then pumped through the MRD at a constant flow rate. Scanning electron microscopy (SEM) and confocal scanning laser microscopy (CSLM) demonstrated a high degree of heterogeneity associated with the structure of biofilms formed under flowing conditions using the MRD. In addition, these biofilms displayed a complex three dimensional architecture and increased production of exopolymeric material.

Candida Albicans-endothelial Cell Interactions: a Key Step in the Pathogenesis of Systemic Candidiasis

Infection and Immunity. Oct, 2008  |  Pubmed ID: 18573891

A Simple and Reproducible 96-well Plate-based Method for the Formation of Fungal Biofilms and Its Application to Antifungal Susceptibility Testing

Nature Protocols. 2008  |  Pubmed ID: 18772877

The incidence of fungal infections has increased significantly over the past decades. Very often these infections are associated with biofilm formation on implanted biomaterials and/or host surfaces. This has important clinical implications, as fungal biofilms display properties that are dramatically different from planktonic (free-living) populations, including increased resistance to antifungal agents. Here we describe a rapid and highly reproducible 96-well microtiter-based method for the formation of fungal biofilms, which is easily adaptable for antifungal susceptibility testing. This model is based on the ability of metabolically active sessile cells to reduce a tetrazolium salt (2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide) to water-soluble orange formazan compounds, the intensity of which can then be determined using a microtiter-plate reader. The entire procedure takes approximately 2 d to complete. This technique simplifies biofilm formation and quantification, making it more reliable and comparable among different laboratories, a necessary step toward the standardization of antifungal susceptibility testing of biofilms.

Effect of Tunicamycin on Candida Albicans Biofilm Formation and Maintenance

The Journal of Antimicrobial Chemotherapy. Mar, 2009  |  Pubmed ID: 19098294

Candida albicans is a common opportunistic pathogen of the human body and is the frequent causative agent of candidiasis. Typically, these infections are associated with the formation of biofilms on both host tissues and implanted biomaterials. As a result of the intrinsic resistance of C. albicans biofilms to most antifungal agents, new strategies are needed to combat these infections.

Expression Levels of a Filament-specific Transcriptional Regulator Are Sufficient to Determine Candida Albicans Morphology and Virulence

Proceedings of the National Academy of Sciences of the United States of America. Jan, 2009  |  Pubmed ID: 19116272

Candida albicans, the major human fungal pathogen, undergoes a reversible morphological transition from single yeast cells to pseudohyphal and hyphal filaments (elongated cells attached end-to-end). Because typical C. albicans infections contain a mixture of these morphologies it has, for many years, been difficult to assess the relative contribution of each form to virulence. In addition, the regulatory mechanisms that determine growth in pseudohyphal and hyphal morphologies are largely unknown. To address these questions we have generated a C. albicans strain that can be genetically manipulated to grow completely in the hyphal form under non-filament-inducing conditions in vitro. This was achieved by inducing high-level constitutive expression of UME6, a recently identified filament-specific transcriptional regulator of C. albicans hyphal extension. We show that high-level UME6 expression significantly increases hyphal formation and promotes virulence in a mouse model of systemic candidiasis. Our results strongly suggest that shifting the morphology of a C. albicans population toward the hyphal form, and/or increasing hyphal-specific gene expression, during the course of infection is sufficient to improve virulence potential. We also demonstrate that lower levels of UME6 expression specify growth largely in the pseudohyphal form and that increasing UME6 levels is sufficient to cause cells to gradually shift from pseudohyphal to hyphal morphology. In addition, we show that UME6 levels differentially induce the expression of several known filament-specific transcripts. These findings suggest that a common transcriptional regulatory mechanism functions to specify both pseudohyphal and hyphal morphologies in a dosage-dependent manner.

Efficacy of a Genetically Engineered Candida Albicans Tet-NRG1 Strain As an Experimental Live Attenuated Vaccine Against Hematogenously Disseminated Candidiasis

Clinical and Vaccine Immunology : CVI. Mar, 2009  |  Pubmed ID: 19144791

We report on the efficacy of the genetically engineered Candida albicans tet-NRG1 strain as an experimental live, attenuated vaccine against disseminated candidiasis in both immunocompetent and immunodeficient mice mostly dependent on T-cell immunity. This experimental vaccination model may represent an important tool to unravel the mechanisms of protective immunity during candidiasis.

Design of a Simple Model of Candida Albicans Biofilms Formed Under Conditions of Flow: Development, Architecture, and Drug Resistance

Mycopathologia. Sep, 2009  |  Pubmed ID: 19370400

Candida albicans biofilms on most medical devices are exposed to a flow of body fluids that provide water and nutrients to the fungal cells. While C. albicans biofilms grown in vitro under static conditions have been exhaustively studied, the same is not true for biofilms developed under continuous flow of replenishing nutrients. Here, we describe a simple flow biofilm (FB) model that can be built easily with materials commonly available in most microbiological laboratories. We demonstrate that C. albicans biofilms formed using this flow system show increased architectural complexity compared to biofilms grown under static conditions. C. albicans biofilms under continuous medium flow grow rapidly, and by 8 h show characteristics similar to 24 h statically grown biofilms. Biomass measurements and microscopic observations further revealed that after 24 h of incubation, FB was more than twofold thicker than biofilms grown under static conditions. Microscopic analyses revealed that the surface of these biofilms was extremely compact and wrinkled, unlike the open hyphal layer typically seen in 24 h static biofilms. Results of antifungal drug susceptibility tests showed that C. albicans cells in FB exhibited increased resistance to most clinically used antifungal agents.

Adhesion of Candida Albicans to Endothelial Cells Under Physiological Conditions of Flow

Infection and Immunity. Sep, 2009  |  Pubmed ID: 19581400

Candida albicans is a commensal organism that under certain circumstances can become pathogenic. During systemic infection C. albicans is disseminated via the circulation to distant organs, where it causes multiple organ failure. Despite the severity of systemic C. albicans infection, little is known about the mechanisms involved in the adhesion of this organism to the endothelium lining blood vessels. Previous studies have used static assays to examine adhesion. However, these do not realistically model blood vessels, where circulating C. albicans cells must adhere to the endothelium under conditions of flow and shear stress. Furthermore, there is conflicting evidence concerning the role played by yeast, pseudohyphal, and hyphal forms of C. albicans in adhesion to endothelium. To test the hypothesis that there may be differences in the abilities of these three morphogenic forms of C. albicans to adhere to endothelium under conditions of flow, we developed an in vitro flow adhesion assay. We found that all three forms of C. albicans rapidly bound to confluent endothelial cells under conditions of flow. Maximum adhesion was found at low shear stress levels similar to that found in postcapillary venules. Moreover, yeast forms bound in significantly greater numbers than did pseudohyphal and hyphal forms, respectively, contrasting with previous findings from static assays. These findings are consistent with recent in vivo data suggesting that yeast forms may be capable of adhering to the endothelium and migrating into the tissues before undergoing morphogenic change to cause tissue damage.

Treatment and Prevention of Candida Albicans Biofilms with Caspofungin in a Novel Central Venous Catheter Murine Model of Candidiasis

The Journal of Antimicrobial Chemotherapy. Sep, 2009  |  Pubmed ID: 19584104

We sought to develop a novel model of central venous catheter (CVC)-associated candidiasis in mice and to use this model to examine the efficacy of caspofungin to treat and prevent Candida albicans biofilms in vivo.

Characteristics of Candida Albicans Biofilms Grown in a Synthetic Urine Medium

Journal of Clinical Microbiology. Dec, 2009  |  Pubmed ID: 19794044

Urinary tract infections (UTIs) are the most common type of nosocomial infection, and Candida albicans is the most frequent organism causing fungal UTIs. Presence of an indwelling urinary catheter represents a significant risk factor for UTIs. Furthermore, these infections are frequently associated with the formation of biofilms on the surface of these catheters. Here, we describe the characterization of C. albicans biofilms formed in vitro using synthetic urine (SU) medium and the frequently used RPMI medium and compare the results. Biofilms of C. albicans strain SC5314 were formed in 96-well microtiter plates and on silicon elastomer pieces using both SU and RPMI media. Biofilm formation was monitored by microscopy and a colorimetric XTT [2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] reduction assay. As in biofilms grown in RPMI medium, time course studies revealed that biofilm formation using SU medium occurred after an initial adherence phase, followed by growth, proliferation, and maturation. However, microscopy techniques revealed that the architectural complexity of biofilms formed in SU medium was lower than that observed for those formed using RPMI medium. In particular, the level of filamentation of cells within the biofilms formed in SU medium was diminished compared to those in the biofilms grown in RPMI medium. This observation was also corroborated by expression profiling of five filamentation-associated genes using quantitative real-time reverse transcriptase PCR. Sessile C. albicans cells were resistant to fluconazole and amphotericin B, irrespective of the medium used to form the biofilms. However, caspofungin exhibited potent in vitro activity at therapeutic levels against C. albicans biofilms grown in both SU and RPMI media.

Presence of Extracellular DNA in the Candida Albicans Biofilm Matrix and Its Contribution to Biofilms

Mycopathologia. May, 2010  |  Pubmed ID: 20012895

DNA has been described as a structural component of the extracellular matrix (ECM) in bacterial biofilms. In Candida albicans, there is a scarce knowledge concerning the contribution of extracellular DNA (eDNA) to biofilm matrix and overall structure. This work examined the presence and quantified the amount of eDNA in C. albicans biofilm ECM and the effect of DNase treatment and the addition of exogenous DNA on C. albicans biofilm development as indicators of a role for eDNA in biofilm development. We were able to detect the accumulation of eDNA in biofilm ECM extracted from C. albicans biofilms formed under conditions of flow, although the quantity of eDNA detected differed according to growth conditions, in particular with regards to the medium used to grow the biofilms. Experiments with C. albicans biofilms formed statically using a microtiter plate model indicated that the addition of exogenous DNA (>160 ng/ml) increases biofilm biomass and, conversely, DNase treatment (>0.03 mg/ml) decreases biofilm biomass at later time points of biofilm development. We present evidence for the role of eDNA in C. albicans biofilm structure and formation, consistent with eDNA being a key element of the ECM in mature C. albicans biofilms and playing a predominant role in biofilm structural integrity and maintenance.

Dispersion As an Important Step in the Candida Albicans Biofilm Developmental Cycle

PLoS Pathogens. Mar, 2010  |  Pubmed ID: 20360962

Biofilms are dynamic microbial communities in which transitions between planktonic and sessile modes of growth occur interchangeably in response to different environmental cues. In the last decade, early events associated with C. albicans biofilm formation have received considerable attention. However, very little is known about C. albicans biofilm dispersion or the mechanisms and signals that trigger it. This is important because it is precisely C. albicans cells dispersed from biofilms that are the main culprits associated with candidemia and establishment of disseminated invasive disease, two of the gravest forms of candidiasis. Using a simple flow biofilm model recently developed by our group, we have performed initial investigations into the phenomenon of C. albicans biofilm dispersion, as well as the phenotypic characteristics associated with dispersed cells. Our results indicate that C. albicans biofilm dispersion is dependent on growing conditions, including carbon source and pH of the media used for biofilm development. C. albicans dispersed cells are mostly in the yeast form and display distinct phenotypic properties compared to their planktonic counterparts, including enhanced adherence, filamentation, biofilm formation and, perhaps most importantly, increased pathogenicity in a murine model of hematogenously disseminated candidiasis, thus indicating that dispersed cells are armed with a complete arsenal of "virulence factors" important for seeding and establishing new foci of infection. In addition, utilizing genetically engineered strains of C. albicans (tetO-UME6 and tetO-PES1) we demonstrate that C. albicans biofilm dispersion can be regulated by manipulating levels of expression of these key genes, further supporting the evidence for a strong link between biofilms and morphogenetic conversions at different stages of the C. albicans biofilm developmental cycle. Overall, our results offer novel and important insight into the phenomenon of C. albicans biofilm dispersion, a key part of the biofilm developmental cycle, and provide the basis for its more detailed analysis.

The Transcriptional Regulator Nrg1p Controls Candida Albicans Biofilm Formation and Dispersion

Eukaryotic Cell. Oct, 2010  |  Pubmed ID: 20709787

The ability of Candida albicans to reversibly switch morphologies is important for biofilm formation and dispersion. In this pathogen, Nrg1p functions as a key negative regulator of the yeast-to-hypha morphogenetic transition. We have previously described a genetically engineered C. albicans tet-NRG1 strain in which NRG1 expression levels can be manipulated by the presence or absence of doxycycline (DOX). Here, we have used this strain to ascertain the role of Nrg1p in regulating the different stages of the C. albicans biofilm developmental cycle. In an in vitro model of biofilm formation, the C. albicans tet-NRG1 strain was able to form mature biofilms only when DOX was present in the medium, but not in the absence of DOX, when high levels of NRG1 expression blocked the yeast-to-hypha transition. However, in a biofilm cell retention assay in which biofilms were developed with mixtures of C. albicans tet-NRG1 and SC5314 strains, tet-NRG1 yeast cells were still incorporated into the mixed biofilms, in which an intricate network of hyphae of the wild-type strain provided for biofilm structural integrity and adhesive interactions. Also, utilizing an in vitro biofilm model under conditions of flow, we demonstrated that C. albicans Nrg1p exerts an exquisite control of the dispersal process, as overexpression of NRG1 leads to increases in dispersion of yeast cells from the biofilms. Our results demonstrate that manipulation of NRG1 gene expression has a profound influence on biofilm formation and biofilm dispersal, thus identifying Nrg1p as a key regulator of the C. albicans biofilm life cycle.

An Easy and Economical in Vitro Method for the Formation of Candida Albicans Biofilms Under Continuous Conditions of Flow

Virulence. Nov-Dec, 2010  |  Pubmed ID: 21178492

Candida albicans can develop biofilms on medical devices and these biofilms are most often nourished by a continuous flow of body fluids and subjected to shear stress forces. While many C. albicans biofilm studies have been carried out using in vitro static models, more limited information is available for biofilms developed under conditions of flow. We have previously described a simple flow biofilm model (SFB) for the development of C. albicans biofilms under conditions of continuous media flow. Here, we recount in detail from a methodological perspective, this model that can be assembled easily using materials commonly available in most microbiological laboratories. The entire procedure takes approximately two days to complete. Biofilms developed using this system are robust, and particularly suitable for studies requiring large amounts of biofilm cells for downstream analyses. This methodology simplifies biofilm formation under continuous replenishment of nutrients. Moreover, this technique mimics in vivo flow conditions, thereby making it physiologically more relevant than the currently dominant static models.

Effects of Fluconazole, Amphotericin B, and Caspofungin on Candida Albicans Biofilms Under Conditions of Flow and on Biofilm Dispersion

Antimicrobial Agents and Chemotherapy. Jul, 2011  |  Pubmed ID: 21518839

We have examined the effect of continuous perfusion with antifungals on Candida albicans biofilms under conditions of flow, closely mimicking physiological conditions encountered within patients. Biofilms displayed high levels of resistance to fluconazole, and this antifungal exerted minor effects on dispersion levels. Amphotericin B proved effective in reducing viability of cells within the biofilms and dispersion, but only at high concentrations. Under flow conditions, caspofungin exhibited potent activity against biofilms and drastically reduced biofilm dispersion.

Validation of the Tetracycline Regulatable Gene Expression System for the Study of the Pathogenesis of Infectious Disease

PloS One. 2011  |  Pubmed ID: 21633704

Understanding the pathogenesis of infectious disease requires the examination and successful integration of parameters related to both microbial virulence and host responses. As a practical and powerful method to control microbial gene expression, including in vivo, the tetracycline-regulatable system has recently gained the favor of many investigative groups. However, some immunomodulatory effects of the tetracyclines, including doxycycline, could potentially limit its use to evaluate host responses during infection. Here we have used a well-established murine model of disseminated candidiasis, which is highly dependent on both the virulence displayed by the fungal cells and on the host immune status, to validate the use of this system. We demonstrate that the pathogenesis of the wild type C. albicans CAF2-1 strain, which does not contain any tet-regulatable element, is not affected by the presence of doxycycline. Moreover levels of key cytokines, chemokines and many other biomarkers, as determined by multi-analyte profiling, remain essentially unaltered by the presence of the antibiotic during infection. Our results indicate that the levels of doxycycline needed to control the tetracycline regulatable promoter gene expression system have no detectable effect on global host responses during candidiasis. Because tet-regulatable systems are now being increasingly used in a variety of pathogenic microorganisms, these observations have wide implications in the field of infectious diseases.

Hsp90 Governs Dispersion and Drug Resistance of Fungal Biofilms

PLoS Pathogens. Sep, 2011  |  Pubmed ID: 21931556

Fungal biofilms are a major cause of human mortality and are recalcitrant to most treatments due to intrinsic drug resistance. These complex communities of multiple cell types form on indwelling medical devices and their eradication often requires surgical removal of infected devices. Here we implicate the molecular chaperone Hsp90 as a key regulator of biofilm dispersion and drug resistance. We previously established that in the leading human fungal pathogen, Candida albicans, Hsp90 enables the emergence and maintenance of drug resistance in planktonic conditions by stabilizing the protein phosphatase calcineurin and MAPK Mkc1. Hsp90 also regulates temperature-dependent C. albicans morphogenesis through repression of cAMP-PKA signalling. Here we demonstrate that genetic depletion of Hsp90 reduced C. albicans biofilm growth and maturation in vitro and impaired dispersal of biofilm cells. Further, compromising Hsp90 function in vitro abrogated resistance of C. albicans biofilms to the most widely deployed class of antifungal drugs, the azoles. Depletion of Hsp90 led to reduction of calcineurin and Mkc1 in planktonic but not biofilm conditions, suggesting that Hsp90 regulates drug resistance through different mechanisms in these distinct cellular states. Reduction of Hsp90 levels led to a marked decrease in matrix glucan levels, providing a compelling mechanism through which Hsp90 might regulate biofilm azole resistance. Impairment of Hsp90 function genetically or pharmacologically transformed fluconazole from ineffectual to highly effective in eradicating biofilms in a rat venous catheter infection model. Finally, inhibition of Hsp90 reduced resistance of biofilms of the most lethal mould, Aspergillus fumigatus, to the newest class of antifungals to reach the clinic, the echinocandins. Thus, we establish a novel mechanism regulating biofilm drug resistance and dispersion and that targeting Hsp90 provides a much-needed strategy for improving clinical outcome in the treatment of biofilm infections.

Effect of Exogenous Administration of Candida Albicans Autoregulatory Alcohols in a Murine Model of Hematogenously Disseminated Candidiasis

Journal of Basic Microbiology. Nov, 2011  |  Pubmed ID: 22052380

Candida albicans supernatants contain a mixture of autoregulatory alcohols. In vitro, when added individually or in combination, these alcohols inhibit the yeast to filamentous form conversion. Here we evaluate the in vivo effect of the exogenous administration of a Cocktail solution simulating the composition of alcohols present in a C. albicans culture supernatant (1 ml; 94 μmol l(-1) isoamyl alcohol, 70 μmol l(-1) 2-phenylethanol, 3.2 n mol l(-1) E -nerolidol, and 18 n mol l(-1) E,E -farnesol) using the well established murine model of hematogenously disseminated candidiasis. Mice injected intraperitoneally with the Cocktail solution demonstrated increased survival and decreased organ fungal burden compared to control mice. Histological observations suggest that the Cocktail, to some extent, has an inhibitory effect on cell filamentation within the kidney. These findings suggest that the exogenous administration of C. albicans autoregulatory alcohols displays a protective effect during disseminated candidiasis. (© 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim).

Sodium Hypochlorite, Chlorhexidine Gluconate, and Commercial Denture Cleansers As Disinfecting Agents Against Candida Albicans: An in Vitro Comparison Study

General Dentistry. Nov, 2011  |  Pubmed ID: 22313927

When treating patients who have candidiasis, removable dental appliances in active use should be treated as well. The authors aimed to determine, in vitro, the lowest concentration of sodium hypochlorite that would eliminate Candida albicans biofilm, as well as the effectiveness of additional products against C. albicans. Strains of C. albicans formed biofilms on microtiter plates. Sodium hypochlorite was added in dilutions (1:1 to 1:512) and Peridex was added in concentrations of 25%, 50%, and 100%. The plates were incubated for 30 minutes. One tablet each of Efferdent, Polident for Partials, and Polident for Dentures was dissolved in 200 mL of sterile water and added to additional groups of plates. One group was incubated for 30 minutes; the other was incubated for 18 hours. An XTT spectrophotometric reduction assay measured biofilm metabolic activity. Biofilm activity decreased 100% for all strains exposed to sodium hypochlorite for 30 minutes in concentrations of 1:32 or stronger. Biofilm activity decreased 100% for most strains when treated with 50% or 100% Peridex for 30 minutes and Polident for Dentures for 18 hours. From these results, it appears appropriate for providers to recommend a solution of two teaspoons of sodium hypochlorite in one cup of water (1:25) for 30 minutes to treat dentures contaminated with C. albicans.

Addition of DNase Improves the in Vitro Activity of Antifungal Drugs Against Candida Albicans Biofilms

Mycoses. Jan, 2012  |  Pubmed ID: 21668524

Cells within Candida albicans biofilms display decreased susceptibility to most clinically used antifungal agents. We recently demonstrated that extracellular DNA (eDNA) plays an important role in biofilm integrity, as a component of the biofilm matrix. This study aimed at gaining insights into the contributions of eDNA to C. albicans biofilms antifungal susceptibility by the investigation of the impact of the combined use of deoxyribonuclease I (DNase) and antifungals to treat biofilms. Candida albicans biofilms were formed using a simple and reproducible 96-well plate-based method. The activity of the combined use of 0.13 mg l(-1) DNase and antifungals was estimated using the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT) reduction assay and total viable counts. Herein, we report the improved efficacy of amphotericin B when in combination with DNase against C. albicans biofilms, as assessed using XTT readings and viable counts. Furthermore, although DNase increased the efficacy of caspofungin in the reduction of mitochondrial activity, no changes were observed in terms of culturable cells. Deoxyribonuclease I did not affect biofilm cells susceptibility to fluconazole. This work suggests that agents that target processes affecting the biofilm structural integrity may have potential use as adjuvants of a catheter-lock therapy.