The MP65 gene is required for cell wall integrity, adherence to epithelial cells and biofilm formation in Candida albicans
© Sandini et al; licensee BioMed Central Ltd. 2011
Received: 28 December 2010
Accepted: 16 May 2011
Published: 16 May 2011
The MP65 gene of Candida albicans (orf19.1779) encodes a putative β-glucanase mannoprotein of 65 kDa, which plays a main role in a host-fungus relationship, morphogenesis and pathogenicity. In this study, we performed an extensive analysis of a mp65Δ mutant to assess the role of this protein in cell wall integrity, adherence to epithelial cells and biofilm formation.
The mp65Δ mutant showed a high sensitivity to a range of cell wall-perturbing and degrading agents, especially Congo red, which induced morphological changes such as swelling, clumping and formation of hyphae. The mp65Δ mutant showed an activation of two MAPKs (Mkc1p and Cek1p), a high level of expression of two stress-related genes (DDR48 and SOD5), and a modulated expression of β-glucan epitopes, but no gross changes in cell wall polysaccharide composition. Interestingly, the mp65Δ mutant displayed a marked reduction in adhesion to BEC and Caco-2 cells and severe defects in biofilm formation when compared to the wild type. All of the mentioned properties were totally or partially recovered in a revertant strain, demonstrating the specificity of gene deletion.
We demonstrate that the MP65 gene of Candida albicans plays a significant role in maintaining cell wall integrity, as well as in adherence to epithelia and biofilm formation, which are major virulence attributes of this fungus.
Candida albicans is both a commensal and a pathogenic yeast, which is responsible for severe infections in humans, particularly in immunocompromised persons, such as AIDS and cancer patients, diabetics, newborns and the elderly [1, 2]. Although several anti-Candida agents are currently available, such as amphotericin B, azoles and echinocandins, there is clearly a need for new specific anti-fungal agents and drug-targets . The cell wall of C. albicans is an essential organelle that helps to withstand osmotic pressure and determines the shape of the cell. The cell wall is a plastic and dynamic structure, whose macromolecular composition, molecular organization and thickness can greatly vary depending on environmental conditions. The cell wall construction is also tightly controlled in space and time by many genes . Within a host-parasite relationship, the cell wall of C. albicans lies at the crossroads of pathogenicity and therapeutics. It contributes to pathogenicity through adherence and invasion, and it is the target of both pharmacological and immunological antifungal therapy [5, 6]. The cell wall comprises two main layers. The inner layer consists of a network of β1,3-glucan molecules, accounting for approximately 40% of the cell-wall mass, to which β1,6-glucan (about 20%) and chitin (2-4%) are covalently attached . The outer layer is composed of a dense layer of mannoproteins, termed "cell wall proteins" (CWP), which account for 35-40% of the cell-wall mass. Based on their linkage to other cell wall polysaccharides, two classes of CWPs can be distinguished. One class, which constitutes the majority of the CWPs, consists of CWPs that are covalently linked to β1,6-glucan via a remnant of a GPI anchor [8, 9]. The other class consists of the so-called "alkali sensitive linkage" (ASL)-CWPs, which are covalently linked to the β1,3-glucan network (without an interconnecting β1,6-glucan molecule) through an unknown linkage that is sensitive to mild alkaline conditions . The best-described ASL-CWPs are the family of Pir-proteins (proteins with internal repeats). Pir-proteins are thought to be pre-proteins that are processed at Kex2 endoprotease recognition sites ; the N-terminal part of mature proteins contains conserved internal tandem repeats, and the C-terminal half shares a high sequence similarity including four conserved cysteines.
The MP65 gene encodes a cell wall mannoprotein (Mp65p) of C. albicans. In a previous study [12–14], our research group identified, generated, and intensely studied native and recombinant forms of Mp65p and found that it is a major target of immune response in humans and mice [15–17]; we also found that Mp65p is a critical determinant of pathogenicity in experimental models of systemic infection in mice and vaginal infection in rats [18–21]. Mp65p is a putative β-glucanase adhesin with one N- and multiple potential O-glycosylation sites, homologous to Scw10p of S. cerevisiae, a member of the GH17 glycosyl-hydrolase family [14, 21, 22]. Moreover, it contains a putative Kex2 peptidase (KR) site , where the protein is cleaved for secretion and an RGD motif that characterizes various proteins of eukaryotic organisms involved in adhesion mechanisms, as both adhesins and adhesin receptors [24, 25]. Furthermore, we found that the MP65 gene can be used as a diagnostic marker for systemic C. albicans and non-albicans infections . In another study , we described the construction of the mp65Δ mutants and some of their genetic traits and biological properties, demonstrating that Mp65p is required for hyphal morphogenesis and experimental pathogenicity. In the present study, we explored the role of Mp65p in depth, examining whether it is required for cell wall integrity, adhesion to host tissues and biofilm formation.
Microorganisms, media and growth conditions
Strains used in this study
Sensitivity testing by microdilution method
To evaluate the sensitivity to cell wall-stressing agents, each C. albicans strain was initially grown for 24 h in YEPD; the cells were then washed with water, resuspended at OD600 nm = 1, and inoculated in YEPD at OD600 nm = 0.01; 95-ml volumes were then pipetted into microdilution plate wells. To these wells were added 5 ml of doubling dilutions of cell wall-stressing agents. The plates were incubated for 16 h at 30°C, and absorbance was read at 540 nm. All strains were tested in duplicate. The agents tested were: Congo red (Sigma, Milan, Italy; 100 mg/ml), calcofluor white (Sigma; 1000 mg/ml), SDS (Bio-Rad, Milan, Italy; 0.25%), caffeine (Sigma; 50 mM), and tunicamycin (Sigma; 100 mg/ml). The mentioned concentrations were the highest used to test each agent.
Sensitivity testing by spotting in solid medium
To assess the susceptibility to specific cell wall-stressing agents, yeast cells were grown in YEPD, in agitation overnight (o.n.) at 28°C and then harvested, washed and re-suspended in sterile water. A sample containing 1.6 × 107 cells/ml and a series of 5-fold dilutions from the sample were prepared. Three μl of each dilution were spotted onto YEPD or YEPD buffered plates (buffered with 50 mM HEPES-NaOH pH 7.0, ), containing no additional chemicals (as control), Congo red (100 mg/ml in YEPD buffered plates), calcofluor white (100 mg/ml in YEPD buffered plates), SDS (0.025%), caffeine (10 mM), and tunicamycin (1.25 μg/ml). The plates were incubated for 24 h at 28°C.
Sensitivity to Zymolyase
Sensitivity to Zymolyase was assayed as described previously . Exponentially growing cells were adjusted to an OD600 nm value of 0.5 (approximately 2 × 107 cells/ml) in 10 mM Tris/HCl, pH 7.5, containing 25 μg/ml of Zymolyase 100T; the optical density decrease was monitored over a 140 min period.
For morphological observations (inclusive of flocculation), the cells were grown at 28°C in YEPD in the absence or presence of Congo red and observed under a light microscope at 2, 6 and 24 h. The images were captured with Nikon Microphot-Fx and Arkon software and imported to Adobe Photoshop 7 (Adobe System Incorporated, San Jose, CA). Finally, the cropped images were assembled into figures using Canvas 9 (Deneba, Miami, FL). For the flocculation studies, following o.n. growth, the cultures were transferred to test tubes and incubated for 10 min. For scanning electron microscopy (SEM) observations, C. albicans cells were grown in YEPD in the absence or presence of Congo red (50 μg/ml) at 28°C for 2, 6 and 24 h. After centrifuging, the cells were washed twice in distilled water and fixed with 2.5% (v/v) glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) containing 2% (w/v) sucrose, for 20 min at room temperature (r.t.). After 3 washes in the same buffer, the cells were postfixed with 1% (w/v) OsO4 for 1 h, dehydrated through graded ethanol concentrations, critical point-dried in CO2 (CPD 030 Balzers device, Bal-Tec, Balzers) and gold coated by sputtering (SCD 040 Balzers device, Bal-Tec). The samples were examined with a Cambridge Stereoscan 360 scanning electron microscope (Cambridge Instruments, Cambridge, United Kingdom). For transmission electron microscopy (TEM), cells were prefixed with glutaraldehyde, as previously mentioned, then post-fixed with the OsO4 solution o.n., at 4°C. The cells were then dehydrated in acetone gradient and embedded in epoxy resin (Agar 100 resin, Agar Scientific Ltd, Stansted, UK), as per routine procedures. Ultrathin sections, obtained with an LKB ultramicrotome (LKB, Bromma, Sweden), were stained with uranyl acetate and lead citrate. These were examined with a Philips 208 transmission electron microscope (FEI Company, Eindhoven, Netherlands).
Immuno-labelling studies in Electron Microscopy (EM)
For β-glucan localization in the post-embedding procedure, the ultrathin sections, obtained as described above, and collected on gold grids, were treated for 3 min with 0.5 mg of sodium borohydride per ml of ice-cold distilled water. After being washed in ice-cold distilled water (3 times, for 5 min) and in PBS containing 0.5% (w/v) bovine serum albumin, 0.05% Tween 20, and 5% fetal serum (3 times, 5 min each time), the sections were incubated with mAb 1E12 (diluted 1:10) o.n. at 4°C. After being washed at r.t. for 2 h by floating the grids on drops of PBS, the samples were labeled with rabbit anti-mouse immunoglobulin M (IgM) gold conjugate 10 nm (diluted 1:10; Sigma) and then washed in PBS buffer at r.t for 3 h. For negative control, the sections were incubated with IgM monoclonal antibody or with goat anti-mouse IgG-gold alone.
Adhesion to buccal ephitelial cells (BEC)
Adhesion to buccal epithelial cells (BEC) was assayed as described previously . Yeast cells were grown for 24 h at 28°C in Winge (0.3% yeast extract, 0.2% glucose), washed twice with PBS (0.02 M NaH2PO4 H2O, 0.02 M Na2HPO4 12H2O, 0.15 M NaCl, pH 7.4) and resuspended at 2 × 107 cells/ml in the same buffer by using a Bürker hemocytometer. One ml of yeast suspension was added to 105 BEC and incubated for 1 h at 37°C. The non-adhering fungal cells were washed off with 50 ml of PBS through a 12 μm polycarbonate filter. The filters were then gently smeared on glass slides, which were air-dried at r.t. o.n. stained with crystal violet (CV) and observed under a light microscope. The images were captured with Nikon Microphot-Fx and Arkon software at different magnifications, and
imported to Adobe Photoshop 7 (Adobe System incorporated, San Jose, CA) and then assembled into figures using Canvas 9 (Deneba, Miami, FL). Adherence was expressed as yeast cells adhering to 100 epithelial cells + standard error.
Adhesion to Caco-2
The adhesion assay was set up in 24-well polystyrene plates as described previously , with only one modification: 2 × 102 cells in PBS (Phosphate Buffered Saline, Sigma) were added to each well.
Biofilm formation and quantification
Cells were grown for 24 h at 28°C in YEPD broth. These were washed twice with sterile PBS (10 mM phosphate buffer, 2.7 mM potassium chloride, 137 mM sodium chloride, pH 7.4, Sigma), and resuspended in RPMI 1640 supplemented with morpholinepropanesulfonic acid (MOPS) at 1 × 106 cells/ml. The cell suspension (250 μl) was seeded in presterilized, polystyrene flat-bottom 24-well microtiter plates (Falcon, Becton Dickinson, NY, USA) and incubated for 48 h at 37°C. After biofilm formation, the medium was aspirated, and non-adherent cells were removed by washing the biofilms 3 times with 250 μl of sterile PBS [3, 30]. The yeasts were quantified by the 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) reduction assay. The XTT (Sigma-Aldrich: 1 mg/ml in PBS) and menadione (Sigma: 0.1 M in acetone) solutions were prepared immediately before each assay. XTT solution was mixed with the menadione solution at a ratio of 1000:1 by volume; 250 μl of the XTT-menadione solution was then added to each well. The microtiter plates were then incubated in the dark for 1 h at 37°C. Following incubation, 250 μl of the XTT-menadione solution was recovered and centrifuged (to eliminate interference of cells with colorimetric readings); 100 μl of the solution was transferred to new wells, and the color change resulting from XTT reduction was measured at 490 nm with a microtiter plate reader (SpectraMax Plus microplate spectrophotometer; Molecular Devices, Ltd., Sunnyvale, CA). The absorbance values of the controls were then subtracted from the values of the test wells to eliminate spurious results due to background interference. Biofilm cultures were grown in triplicate, and each assay was performed 3 times. For the photographs, the biofilms were stained with CV  and the images captured with a Nikon Eclipse TE300 inverted microscope. For dry weight determinations, the biofilms were grown as described above and dried o.n. in a laminar flow hood. Three 24-well microtiter plates, for each C. albicans strain were used. The dry weight was given by the difference between the weight of dried plate containing biofilm and the same clean and sterile pre-weighed plate. The dry weight was expressed as the mean + S. D. of 3 plates.
Quantitative Real-Time RT-PCR
Oligonucleotides used in this study
5' to 3' sequence
Protein Extract and Western Analysis
To investigate if the cell wall integrity pathway was activated by the presence of Congo red, C. albicans cells were grown in YPD medium at 28°C, to mid-exponential phase, then treated with Congo red (50 μg/ml), 1.5 h before collection. The cells were then washed and resuspended in extraction buffer [100 mM Tris- HCl pH 7.5, 0.01% (w/v) SDS, 1 mM dithiothreitol, 10% (w/v) glycerol, protease inhibitor mixture (Roche Applied Science, Lewes, UK)] and then disrupted with glass beads. The lysate was centrifuged at 12 000 rpm for 10 min. The protein extracts were quantified using the Comassie protein assay reagent (Bio-Rad). One hundred and fifty μg of protein was separated on a 10% SDS-PAGE linear gel and then blotted to the nitrocellulose membrane. Before blocking, the equal loading was verified by MemCode ™ Reversible Protein Stain Kit (Pierce) together with the intensity of nonspecific bands. The membrane was then blocked in TBS plus 0.1% Tween 20 and 5 mg/ml dry milk (Carnation) at r.t. for 2 h. The anti-phospho-p44/42 MAPK (Thr202/Tyr204) antibody (New England Biolabs Inc., Hertfordshire, UK) was used to detect phosphorylated forms of Mkc1p and Cek1p MAPKs. The anti-MAPK antibody was used to reveal the total amount of Mkc1p. The anti-Kss1p polyclonal antibody (Santa Cruz Biotechnology), raised in rabbit against Kss1p of S. cerevisiae, was used to detect the total amount of Cek1p. The Act1p signal, obtained using the anti-Act1p antibody (SIGMA), was used as the loading control.
To detect antigen expression, a suspension of 106-107 yeast cells was fixed with 2% paraformaldehyde at r.t. for 30 min. After washing with ice-cold PBS, samples were incubated at 4°C for 30 min with mAb 1E12 diluted 1:100 and then with a goat anti-mouse IgM-fluorescein-conjugated antibody (Sigma) diluted 1:25. After washing, cells were immediately analyzed. Fluorescence was analyzed with FACScan flow cytometer (Becton Dickinson, Mountain View, CA) equipped with a 15 mW, 488 nm, air-cooled argon ion laser. FITC fluorescence was measured through a 530 nm band-pass filter and acquired in log mode. Negative controls were obtained by incubating samples with mouse IgM lambda (Sigma). The β-glucan content was expressed in arbitrary units (A.U.) and was calculated as the ratio of the labeled samples on the mean fluorescence channel (mfc) of the corresponding negative controls. The mfc was calculated by Cell Quest software (Becton Dickinson, Mountain View, CA).
Cell wall components
The determination of the sugar monomers, after cell wall polysaccharides extraction with acid hydrolysis, was performed using HPLC with a Dionex Bio-LC system as previously described .
Differences in mean values of analytical determinations were assessed by the Student's t test, and significance was set at P < 0.05.
Cell wall integrity
To further assess the importance of Mp65p for cell wall assembly and integrity, we performed a cell wall digestion assay with a cell wall-corrupting β1,3-glucanase enzyme (Zymolyase 100 T) by measuring the half-life (the time required for a 50% decrease in the OD) of spheroplast lysis.
The mp65Δ mutant proved to be more sensitive to β-1,3-glucanase activity than the wild type and the revertant strains (30-min spheroplast half-life versus 60 and 37 min, respectively), indicating marked changes in the cell wall composition, organization or both, which could only in part be recovered by reintroduction of one copy of the MP65 gene (Figure 1C).
The above results suggest that the mp65Δ mutant may express cell wall damage response genes in the absence of exogenous cell wall-perturbing agents. We assayed the expression of the following five cell wall damage response genes: DDR48, PHR1, STP4, CHT2 and SOD5 [6, 44–46]. Figure 2B shows that of the five genes mentioned only DDR48 and SOD5 had an altered expression in the mp65Δ mutant when compared to wild type and revertant strains. These findings suggest that the MP65 gene was required for the cell wall integrity and that DDR48 and SOD5 may be involved in the recovery of cell wall function when the MP65 gene is deleted. Overall, the MP65 mutation may have had a direct effect on the cell wall, given that Mp65p is a cell wall-located putative β1-3 glucanase enzyme , in addition to the indirect effects due to the altered expression of cell wall damage response genes.
Morphological and biochemical properties of the mp65Δ mutant strain
We also investigated the possible chemical changes in the cell wall composition. As previously demonstrated in Saccharomyces cerevisiae (fks1, mnn9, gas1, kre6, knr4, and chs3 strains)  and C. albicans mutants (kre5, crh) [43, 48, 49], the defective expression in the genes implicated in cell wall biogenesis and regulation may also result in dramatic changes in the chemical composition of the cell wall. Hence, we measured the amount of main cell wall polysaccharide components (i.e., mannan, glucan and chitin). The comparison of the mp65Δ mutant with wild type indicated no statistically significant differences in any of these components (Figure 4C). However, there was a trend of an increase in chitin content in the mp65Δ mutant compared to the wild type cells (2.56 ± 0.57 vs. 1.75 ± 0.45: these values are the mean percentage distribution of chitin of 3 independent experiments expressed as mean + S.D.).
Adherence and biofilm formation
The cell wall is a dynamic structure that is remodeled when fungal cells are exposed to severe stress conditions, including hyphal growth, mutations of genes coding for cell wall components, and host immune responses . This remodeling leads to a reorganization of the cell wall architecture following the activation of different cell-wall compensatory mechanisms . The 65-kDa mannoprotein (Mp65p) of C. albicans was previously shown to be a major target of anti-Candida immune responses in humans [15–17] and, more recently, a putative β-glucanase adhesin which plays a critical role in hyphal formation and virulence of this fungus [18–21].
In light of these findings, we have now specifically addressed the role of Mp65p in cell wall biogenesis and integrity, as well as the adherence to epithelial cells and biofilm formation.
Also based on previous work performed with scw4scw10 mutants of S. cerevisiae  and pkc1, mkc1, hog1 , pmr1 , och1 , sun41  and crh mutants of C. albicans , we first examined the sensitivity of the mp65Δ mutant to a range of cell wall-perturbing agents to determine the effects of the MP65 gene deletion on the integrity of the cell wall.
Our data show that Mp65p plays an important role in membrane/cell wall stability. This was evident from: i) the increased sensitivity of the mp65Δ mutant to a number of agents whose effects have been associated with altered cell wall; ii) the constitutive activation of the cell wall integrity pathway in the mutant; iii) the increased expression in the mutant, in the absence of stressing agents, of DDR48 and SOD5, two cell wall damage response genes which code for, respectively, a cell-wall protein and an antioxidant enzyme [44–46].
Interestingly, the cell wall defects consequential to the MP65 gene deletion did not bring about gross detectable changes in the cell wall chemistry, as seen in other mutants of β-glucanase enzyme families [50, 52]. While further investigations are needed to detect small chemical changes, which are likely to occur in the mutant cell wall, we believe that the MP65 gene deletion may mostly affect cell wall organization, with associated remodeling of its main polymeric constituents. This interpretation is supported by the comparable contents of all the 3 cell wall polysaccharides (mannan, glucan and chitin), which overall accounted for more than 95% of the cell wall dry weight, and by the rather marked differences in β-glucan expression, zymolyase sensitivity and morphological changes on the other. In particular, the disposition of β-glucan appears to be affected in the mp65Δ mutant, which displays a much lower reactivity than the wild type cell, as detected by an antibody which recognizes both β-1,3 and β-1,6 glucan configurations. This would suggest that β glucan is much less accessible to the antibody in the mp65Δ mutant than in the wild type strain. This lower antibody accessibility to the target may modulate immune responses to the pathogen, in view of the critical role exerted by β-glucan polysaccharide in fungal recognition by the immune system .
Notably, the re-integration of one MP65 gene copy in the revertant strain did not induce a full recovery of the lost or decreased function of the mp65Δ mutant. This is in line with the repeatedly observed gene dosage effects in C. albicans .
Some β-glucanase mutants have been shown to be endowed with low pathogenicity potential which is not entirely attributable to their inability to make tissue invasive hyphae [22, 50]. The adherence to host tissues or to abiotic surfaces is an important attribute of Candida that is positively correlated with pathogenicity . In C. albicans and C. glabrata, but also in the less pathogenic yeast S. cerevisiae, multiple adhesion proteins (known as "adhesins", "flocculins" or "agglutinins") have been identified, such as Als family proteins, Hwp1, Eap1 in C. albicans and Epa proteins in C. glabrata. These proteins provide these organisms with a variety of adherence properties, such as their interactions with other cells (during mating) and with abiotic surfaces and host tissues. Mp65p is a putative β-glucanase adhesin, which is critical to C. albicans adherence to an abiotic surface . In this study, we explored whether the adherence to epithelial cells was also affected in the mp65Δ mutant. We thus compared the ability of the wild type and the mp65Δ mutant strains to adhere to BEC and Caco-2 cell monolayers by using two in vitro adhesion assays. In both assays, the mp65Δ mutant consistently displayed a significant decrease in adherence. These findings, together with the capacity of an anti-Mp65p serum to inhibit almost totally the adherence to the plastic by the wild type strain , highlights the more exstensive role of Mp65p as an adhesin, in that its adhesion is not limited to inert surfaces. Nevertheless, the decreased adherence of the mp65Δ mutant could also be indirectly due to the suggested alteration in cell wall organization, with a possible decreased cell surface expression of other C. albicans adhesins, such as those previously mentioned.
Biofilms are typically found on medical devices, such as catheter surfaces, and they have attracted attention because of their persistence and resistance to antifungals [3, 30]. Given that biofilm formation begins with surface adherence and that mp65Δ mutant loses adherence to the polystyrene plates, as demonstrated in our previous paper , we also investigated whether the ability of the mp65Δ mutant in forming biofilms had altered. As consistently shown by our data, the mp65Δ mutant displayed a strongly defective biofilm formation, in contrast to wild type that produced abundant biofilm.
The findings reported in the current paper significantly extend beyond the previously reported role of Mp65p in hyphal cell wall biogenesis and actually confirm that morphogenesis and cell wall remodeling are intimately related issues [22, 50, 55]. The knock-out of the MP65 gene affects biological properties that are of potential relevance for candidiasis. Together with the defective hyphal morphogenesis , these findings provide some further functional correlates to the previously demonstrated loss of invasive and mucosal pathogenicity by the mp65Δ null mutant. Overall, the MP65 gene appears to play a role in cell wall structure and stability which, by still unknown mechanisms, are translated into fungal virulence. For all of the discussed reasons, and with the previously reported evidence of Mp65p being a major target of host immune response to C. albicans , this protein remains an interesting potential target for therapeutic or immunotherapeutic interventions.
This work was supported in part by grants from the Istituto Superiore di Sanità (National AIDS Project, under contract No. 50/C). The authors are also grateful to Dr. Paola Chiani in providing the mAb 1E12, Laura Toccaceli and Dr Giuseppe Esposito for their skilful assistance with SEM photographs and statistics respectively. Special thanks to Dr. Andrea Savarino for his kind assistance in photographing the biofilm, and for his invaluable suggestions for our future project. Thanks Dr. G. Mandarino and Dr. Anna Marella for their help in manuscript preparation and to Prof. Antonio Cassone for critical reading of the manuscript and suggestions. We also wish to thank Maurice Di Santolo for the English revision of the manuscript.
- Cassone A: Fungal vaccines: real progress from real challenges. Lancet Infect Dis. 2008, 8: 114-124. 10.1016/S1473-3099(08)70016-1.PubMedView ArticleGoogle Scholar
- Angiolella L, Stringaro AR, De Bernardis F, Posteraro B, Bonito M, Toccacieli L, Torosantucci A, Colone M, Sanguinetti M, Cassone A, Palamara AT: Increase of virulence and its phenotypic traits in drug-resistant strains of Candida albicans. Antimicrob Agents Chemother. 2008, 52: 927-936. 10.1128/AAC.01223-07.PubMedPubMed CentralView ArticleGoogle Scholar
- Morgunova E, Saller S, Haase I, Cushman M, Bacher A, Fischer M, Ladenstein R: Lumazine synthase from Candida albicans as an anti-fungal target enzyme: structural and biochemical basis for drug design. J Biol Chem. 2007, 282: 17231-17241. 10.1074/jbc.M701724200.PubMedView ArticleGoogle Scholar
- Ram AF, Klis FM: Identification of fungal cell wall mutants using susceptibility assays based on Calcofluor white and Congo red. Nat Protoc. 2006, 1: 2253-2256. 10.1038/nprot.2006.397.PubMedView ArticleGoogle Scholar
- Norice CT, Smith FJ, Solis N, Filler SG, Mitchell AP: Requirement for Candida albicans Sun41 in biofilm formation and virulence. Eukaryot Cell. 2007, 6: 2046-2055. 10.1128/EC.00314-07.PubMedPubMed CentralView ArticleGoogle Scholar
- Torosantucci A, Chiani P, Bromuro C, De Bernardis F, Palma AS, Liu Y, Mignogna G, Maras B, Colone M, Stringaro A, Zamboni S, Feizi T, Cassone A: Protection by anti-beta-glucan antibodies is associated with restricted beta-1,3 glucan binding specificity and inhibition of fungal growth and adherence. PLoS One. 2009, 4: e5392-10.1371/journal.pone.0005392.PubMedPubMed CentralView ArticleGoogle Scholar
- Brown JA, Catley BJ: Monitoring polysaccharide synthesis in Candida albicans. Carbohydr Res. 1992, 227: 195-202.View ArticleGoogle Scholar
- de Groot PW, de Boer AD, Cunningham J, Dekker HL, de Jong L, Hellingwerf KJ, de Koster C, Klis FM: Proteomic analysis of Candida albicans cell walls reveals covalently bound carbohydrate-active enzymes and adhesins. Eukaryot Cell. 2004, 3: 955-965. 10.1128/EC.3.4.955-965.2004.PubMedPubMed CentralView ArticleGoogle Scholar
- de Groot PW, Ram AF, Klis FM: Features and functions of covalently linked proteins in fungal cell walls. Fungal Genet Biol. 2005, 42: 657-675. 10.1016/j.fgb.2005.04.002.PubMedView ArticleGoogle Scholar
- Ecker M, Deutzmann R, Lehle L, Mrsa V, Tanner W: Pir proteins of Saccharomyces cerevisiae are attached to beta-1,3-glucan by a new protein-carbohydrate linkage. J Biol Chem. 2006, 281: 11523-11529. 10.1074/jbc.M600314200.PubMedView ArticleGoogle Scholar
- Mrsa V, Seidl T, Gentzsch M, Tanner W: Specific labelling of cell wall proteins by biotinylation. Identification of four covalently linked O-mannosylated proteins of Saccharomyces cerevisiae. Yeast. 1997, 13: 1145-1154. 10.1002/(SICI)1097-0061(19970930)13:12<1145::AID-YEA163>3.0.CO;2-Y.PubMedView ArticleGoogle Scholar
- Gomez MJ, Torosantucci A, Arancia S, Maras B, Parisi L, Cassone A: Purification and biochemical characterization of a 65-kilodalton mannoprotein (MP65), a main target of anti-Candida cell-mediated immune responses in humans. Infect Immun. 1996, 64: 2577-2584.PubMedPubMed CentralGoogle Scholar
- Gomez MJ, Maras B, Barca A, La Valle R, Barra D, Cassone A: Biochemical and immunological characterization of MP65, a major mannoprotein antigen of the opportunistic human pathogen Candida albicans. Infect Immun. 2000, 68: 694-701. 10.1128/IAI.68.2.694-701.2000.PubMedPubMed CentralView ArticleGoogle Scholar
- La Valle R, Sandini S, Gomez MJ, Mondello F, Romagnoli G, Nisini R, Cassone A: Generation of a recombinant 65-kilodalton mannoprotein, a major antigen target of cell-mediated immune response to Candida albicans. Infect Immun. 2000, 68: 6777-6784. 10.1128/IAI.68.12.6777-6784.2000.PubMedPubMed CentralView ArticleGoogle Scholar
- Nisini R, Romagnoli G, Gomez MJ, La Valle R, Torosantucci A, Mariotti S, Teloni R, Cassone A: Antigenic properties and processing requirements of 65-kilodalton mannoprotein, a major antigen target of anti-Candida human T-cell response, as disclosed by specific human T-cell clones. Infect Immun. 2001, 69: 3728-3736. 10.1128/IAI.69.6.3728-3736.2001.PubMedPubMed CentralView ArticleGoogle Scholar
- Pietrella D, Bistoni G, Corbucci C, Perito S, Vecchiarelli A: Candida albicans mannoprotein influences the biological function of dendritic cells. Cell Microbiol. 2006, 8: 602-612. 10.1111/j.1462-5822.2005.00651.x.PubMedView ArticleGoogle Scholar
- Torosantucci A, Gomez MJ, Bromuro C, Casalinuovo I, Cassone A: Biochemical and antigenic characterization of mannoprotein constituents released from yeast and mycelial forms of Candida albicans. J Med Vet Mycol. 1991, 29: 361-372. 10.1080/02681219180000591.PubMedView ArticleGoogle Scholar
- Cassone A, De Bernardis F, Torososantucci A: An outline of the role of anti-Candida antibodies within the context of passive immunization and protection from candidiasis. Curr Mol Med. 2005, 5: 377-382. 10.2174/1566524054022549.PubMedView ArticleGoogle Scholar
- De Bernardis F, O'Mahony R, Liu H, La Valle R, Bartollino S, Sandini S, Grant S, Brewis N, Tomlinson I, Basset RC, Holton J, Roitt IM, Cassone A: Human domain antibodies against virulence traits of Candida albicans inhibit fungus adherence to vaginal epithelium and protect against experimental vaginal candidiasis. J Infect Dis. 2007, 195: 149-157. 10.1086/509891.PubMedView ArticleGoogle Scholar
- Pietrella D, Lupo P, Rachini A, Sandini S, Ciervo A, Perito S, Bistoni F, Vecchiarelli A: A Candida albicans mannoprotein deprived of its mannan moiety is efficiently taken up and processed by human dendritic cells and induces T-cell activation without stimulating proinflammatory cytokine production. Infect Immun. 2008, 76: 4359-4367. 10.1128/IAI.00669-08.PubMedPubMed CentralView ArticleGoogle Scholar
- Sandini S, La Valle R, De Bernardis F, Macri C, Cassone A: The 65 kDa mannoprotein gene of Candida albicans encodes a putative beta-glucanase adhesin required for hyphal morphogenesis and experimental pathogenicity. Cell Microbiol. 2007, 9: 1223-1238. 10.1111/j.1462-5822.2006.00862.x.PubMedView ArticleGoogle Scholar
- Klis FM, Boorsma A, De Groot PW: Cell wall construction in Saccharomyces cerevisiae. Yeast. 2006, 23: 185-202. 10.1002/yea.1349.PubMedView ArticleGoogle Scholar
- Newport G, Kuo A, Flattery A, Gill C, Blake JJ, Kurtz MB, Abruzzo GK, Agabian N: Inactivation of Kex2p diminishes the virulence of Candida albicans. J Biol Chem. 2003, 278: 1713-1720. 10.1074/jbc.M209713200.PubMedView ArticleGoogle Scholar
- Calderone R, Suzuki S, Cannon R, Cho T, Boyd D, Calera J, Chibana H, Herman D, Holmes A, Jeng HW, Kaminishi H, Matsumoto T, Mikami T, O'Sullivan JM, Sudoh M, Suzuki M, Nakashima Y, Tanaka T, Tompkins GR, Watanabe T: Candida albicans: adherence, signaling and virulence. Med Mycol. 2000, 38 (Suppl 1): 125-137.PubMedView ArticleGoogle Scholar
- Gale CA, Bendel CM, McClellan M, Hauser M, Becker JM, Berman J, Hostetter MK: Linkage of adhesion, filamentous growth, and virulence in Candida albicans to a single gene INT1. Science. 1998, 279: 1355-1358. 10.1126/science.279.5355.1355.PubMedView ArticleGoogle Scholar
- Arancia S, Sandini S, Cassone A, De Bernardis F: Use of 65 kDa mannoprotein gene primers in PCR methods for the identification of five medically important Candida species. Mol Cell Probes. 2009, 23: 218-226. 10.1016/j.mcp.2009.04.003.PubMedView ArticleGoogle Scholar
- van der Vaart JM, Caro LH, Chapman JW, Klis FM, Verrips CT: Identification of three mannoproteins in the cell wall of Saccharomyces cerevisiae. J Bacteriol. 1995, 177: 3104-3110.PubMedPubMed CentralGoogle Scholar
- De Bernardis F, Agatensi L, Ross IK, Emerson GW, Lorenzini R, Sullivan PA, Cassone A: Evidence for a role for secreted aspartate proteinase of Candida albicans in vulvovaginal candidiasis. J Infect Dis. 1990, 161: 1276-1283. 10.1093/infdis/161.6.1276.PubMedView ArticleGoogle Scholar
- Dieterich C, Schandar M, Noll M, Johannes FJ, Brunner H, Graeve T, Rupp S: In vitro reconstructed human epithelia reveal contributions of Candida albicans EFG1 and CPH1 to adhesion and invasion. Microbiol. 2002, 148: 497-506.View ArticleGoogle Scholar
- Hiller E, Heine S, Brunner H, Rupp S: Candida albicans Sun41p, a putative glycosidase, is involved in morphogenesis, cell wall biogenesis, and biofilm formation. Eukaryot Cell. 2007, 6: 2056-2065. 10.1128/EC.00285-07.PubMedPubMed CentralView ArticleGoogle Scholar
- Reynolds TB, Fink GR: Bakers' yeast, a model for fungal biofilm formation. Science. 2001, 291: 878-881. 10.1126/science.291.5505.878.PubMedView ArticleGoogle Scholar
- De Bernardis F, Cassone A, Sturtevant J, Calderone R: Expression of Candida albicans SAP1 and SAP2 in experimental vaginitis. Infect Immun. 1995, 63: 1887-1892.PubMedPubMed CentralGoogle Scholar
- Livak KJ, Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT. Methods. 2001, 25: 402-408. 10.1006/meth.2001.1262.PubMedView ArticleGoogle Scholar
- François JM: A simple method for quantitative determination of polysaccharides in fungal cell walls. Nat Protoc. 2006, 1: 2995-3000.PubMedView ArticleGoogle Scholar
- Bates S, MacCallum DM, Bertram G, Munro CA, Hughes HB, Buurman ET, Brown AJ, Odds FC, Gow NA: Candida albicans Pmr1p, a secretory pathway P-type Ca2+/Mn2+-ATPase, is required for glycosylation and virulence. J Biol Chem. 2005, 280: 23408-23415. 10.1074/jbc.M502162200.PubMedView ArticleGoogle Scholar
- Plaine A, Walker L, Da Costa G, Mora-Montes HM, McKinnon A, Gow NA, Gaillardin C, Munro CA, Richard ML: Functional analysis of Candida albicans GPI-anchored proteins: roles in cell wall integrity and caspofungin sensitivity. Fungal Genet Biol. 2008, 45: 1404-1414. 10.1016/j.fgb.2008.08.003.PubMedPubMed CentralView ArticleGoogle Scholar
- Kunze D, MacCallum D, Odds FC, Hube B: Multiple functions of DOA1 in Candida albicans. Microbiol. 2007, 153: 1026-1041. 10.1099/mic.0.2006/002741-0.View ArticleGoogle Scholar
- Bates S, Hughes HB, Munro CA, Thomas WP, MacCallum DM, Bertram G, Atrih A, Ferguson MA, Brown AJ, Odds FC, Gow NA: Outer chain N-glycans are required for cell wall integrity and virulence of Candida albicans. J Biol Chem. 2006, 281: 90-98.PubMedView ArticleGoogle Scholar
- Kuo SC, Lampen JO, Ruiz-Herrera J, Elorza MV, Valentín E, Sentandreu R: Tunicamycin-an inhibitor of yeast glycoprotein synthesis. Biochem Biophys Res Commun. 1974, 58: 287-95. 10.1016/0006-291X(74)90925-5.PubMedView ArticleGoogle Scholar
- Pierce CG, Thomas DP, López-Ribot JL: Effect of tunicamycin on Candida albicans biofilm formation and maintenance. J Antimicrob Chemother. 2009, 63: 473-9. 10.1093/jac/dkn515.PubMedPubMed CentralView ArticleGoogle Scholar
- Ruiz-Herrera J, Elorza MV, Valentín E, Sentandreu R: Molecular organization of the cell wall of Candida albicans and its relation to pathogenicity. FEMS Yeast Res. 2006, 6: 14-29. 10.1111/j.1567-1364.2005.00017.x.PubMedView ArticleGoogle Scholar
- Navarro-Garcia F, Eisman B, Fiuza SM, Nombela C, Pla J: The MAP kinase Mkc1p is activated under different stress conditions in Candida albicans. Microbiol. 2005, 151: 2737-2749. 10.1099/mic.0.28038-0.View ArticleGoogle Scholar
- Pardini G, De Groot PW, Coste AT, Karababa M, Klis FM, de Koster CG, Sanglard D: The CRH family coding for cell wall glycosylphosphatidylinositol proteins with a predicted transglycosidase domain affects cell wall organization and virulence of Candida albicans. J Biol Chem. 2006, 281: 40399-40411. 10.1074/jbc.M606361200.PubMedView ArticleGoogle Scholar
- Blankenship JR, Fanning S, Hamaker JJ, Mitchell AP: An extensive circuitry for cell wall regulation in Candida albicans. Plos Pathogens. 2010, 6: e1000752-10.1371/journal.ppat.1000752.PubMedPubMed CentralView ArticleGoogle Scholar
- Dib L, Hayek P, Sadek H, Beyrouthy B, Khalaf RA: The Candida albicans Ddr48 protein is essential for filamentation, stress response, and confers partial antifungal drug resistance. Med Sci Monit. 2008, 14: 113-121.Google Scholar
- Martchenko M, Alarco AM, Harcus D, Whiteway M: Superoxide dismutases in Candida albicans: transcriptional regulation and functional characterization of the hyphal induced SOD5 gene. Mol Biol Cell. 2004, 15: 456-467.PubMedPubMed CentralView ArticleGoogle Scholar
- Chiani P, Bromuro C, Cassone A, Torosantucci A: Anti-beta-glucan antibodies in healthy human subjects. Vaccine. 2009, 27: 513-519. 10.1016/j.vaccine.2008.11.030.PubMedView ArticleGoogle Scholar
- Herrero AB, Magnelli P, Mansour MK, Levitz SM, Bussey H, Abeijon C: KRE5 gene null mutant strains of Candida albicans are avirulent and have altered cell wall composition and hypha formation properties. Eukaryot Cell. 2004, 3: 1423-1432. 10.1128/EC.3.6.1423-1432.2004.PubMedPubMed CentralView ArticleGoogle Scholar
- Kapteyn JC, Hoyer LL, Hecht JE, Muller WH, Andel A, Verkleij AJ, Makarow M, Van Den Ende H, Klis FM: The cell wall architecture of Candida albicans wild type cells and cell wall-defective mutants. Mol Microbiol. 2000, 35: 601-611.PubMedView ArticleGoogle Scholar
- Calderon J, Zavrel M, Ragni E, Fonzi WA, Rupp S, Popolo L: PHR1, a pH-regulated gene of Candida albicans encoding a glucan-remodelling enzyme, is required for adhesion and invasion. Microbiol. 2010, 156: 2484-2494. 10.1099/mic.0.038000-0.View ArticleGoogle Scholar
- Sestak S, Hagen I, Tanner W, Strahl S: Scw10p, a cell-wall glucanase/transglucosidase important for cell-wall stability in Saccharomyces cerevisiae. Microbiol. 2004, 150: 3197-3208. 10.1099/mic.0.27293-0.View ArticleGoogle Scholar
- Fonzi WA: PHR1 and PHR2 of Candida albicans encode putative glycosidases required for proper cross-linking of beta-1,3- and beta-1,6-glucans. J Bacteriol. 1999, 181: 7070-7079.PubMedPubMed CentralGoogle Scholar
- Netea MG, Gow NA, Munro CA, Bates S, Collins C, Ferwerda G, Hobson RP, Bertram G, Hughes HB, Jansen T, Jacobs L, Buurman ET, Gijzen K, Williams DL, Torensma R, McKinnon A, MacCallum DM, Odds FC, Van der Meer JW, Brown AJ, Kullberg BJ: Immune sensing of Candida albicans requires cooperative recognition of mannans and glucans by lectin and Toll-like receptors. J Clin Invest. 2006, 116: 1642-1650. 10.1172/JCI27114.PubMedPubMed CentralView ArticleGoogle Scholar
- Calderone RA, Fonzi WA: Virulence factors of Candida albicans. Trends Microbiol. 2001, 9: 327-335. 10.1016/S0966-842X(01)02094-7.PubMedView ArticleGoogle Scholar
- Hope H, Schmauch C, Arkowitz RA, Bassilana M: The Candida albicans ELMO homologue functions together with Rac1 and Dck1, upstream of the MAP Kinase Cek1, in invasive filamentous growth. Mol Microbiol. 2010, 76: 1572-1590. 10.1111/j.1365-2958.2010.07186.x.PubMedView ArticleGoogle Scholar
- Murad AM, Lee PR, Broadbent ID, Barelle CJ: CIp10, an efficient and convenient integrating vector for Candida albicans. Yeast. 2000, 16: 325-327. 10.1002/1097-0061(20000315)16:4<325::AID-YEA538>3.0.CO;2-#.PubMedView ArticleGoogle Scholar