Title: The CRH Family Coding for Cell Wall Glycosylphosphatidylinositol Proteins with a Predicted Transglycosidase Domain Affects Cell Wall Organization and Virulence of Candida albicans
Abstract: In Candida albicans UTR2 (CSF4), CRH11, and CRH12 are members of a gene family (the CRH family) that encode glycosylphosphatidylinositol-dependent cell wall proteins with putative transglycosidase activity. Deletion of genes of this family resulted in additive sensitivity to compounds interfering with normal cell wall formation (Congo red, calcofluor white, SDS, and high Ca2+ concentrations), suggesting that these genes contribute to cell wall organization. A triple mutant lacking UTR2, CRH11, and CRH12 produced a defective cell wall, as inferred from increased sensitivity to cell wall-degrading enzymes, decreased ability of protoplasts to regenerate a new wall, constitutive activation of Mkc1p, the mitogen-activated protein kinase of the cell wall integrity pathway, and an increased chitin content of the cell wall. Importantly, this was accompanied by a decrease in alkali-insoluble 1,3-β-glucan but not total glucan content, suggesting that formation of the linkage between 1,3-β-glucan and chitin might be affected. In support of this idea, localization of a Utr2p-GFP fusion protein largely coincided with areas of chitin incorporation in C. albicans.As UTR2 and CRH11 expression is regulated by calcineurin, a serine/threonine protein phosphatase involved in tolerance to antifungal drugs, cell wall morphogenesis, and virulence, this points to a possible relationship between calcineurin and the CRH family. Deletion of UTR2, CRH11, and CRH12 resulted in only a partial overlap with calcineurin-dependent phenotypes, suggesting that calcineurin has additional targets. Interestingly, cells deleted for UTR2, CRH11, and CRH12 were, like a calcineurin mutant, avirulent in a mouse model of systemic infection but retained the capacity to colonize target organs (kidneys) as the wild type. In conclusion, this work establishes the role of UTR2, CRH11, and CRH12 in cell wall organization and integrity. In Candida albicans UTR2 (CSF4), CRH11, and CRH12 are members of a gene family (the CRH family) that encode glycosylphosphatidylinositol-dependent cell wall proteins with putative transglycosidase activity. Deletion of genes of this family resulted in additive sensitivity to compounds interfering with normal cell wall formation (Congo red, calcofluor white, SDS, and high Ca2+ concentrations), suggesting that these genes contribute to cell wall organization. A triple mutant lacking UTR2, CRH11, and CRH12 produced a defective cell wall, as inferred from increased sensitivity to cell wall-degrading enzymes, decreased ability of protoplasts to regenerate a new wall, constitutive activation of Mkc1p, the mitogen-activated protein kinase of the cell wall integrity pathway, and an increased chitin content of the cell wall. Importantly, this was accompanied by a decrease in alkali-insoluble 1,3-β-glucan but not total glucan content, suggesting that formation of the linkage between 1,3-β-glucan and chitin might be affected. In support of this idea, localization of a Utr2p-GFP fusion protein largely coincided with areas of chitin incorporation in C. albicans.As UTR2 and CRH11 expression is regulated by calcineurin, a serine/threonine protein phosphatase involved in tolerance to antifungal drugs, cell wall morphogenesis, and virulence, this points to a possible relationship between calcineurin and the CRH family. Deletion of UTR2, CRH11, and CRH12 resulted in only a partial overlap with calcineurin-dependent phenotypes, suggesting that calcineurin has additional targets. Interestingly, cells deleted for UTR2, CRH11, and CRH12 were, like a calcineurin mutant, avirulent in a mouse model of systemic infection but retained the capacity to colonize target organs (kidneys) as the wild type. In conclusion, this work establishes the role of UTR2, CRH11, and CRH12 in cell wall organization and integrity. The cell wall in yeast is an essential structure that maintains cell morphology and helps to stabilize internal osmotic conditions (1Klis F.M. Mol P. Hellingwerf K. Brul S. FEMS Microbiol. Rev. 2002; 26: 239-256Crossref PubMed Google Scholar). It is also involved in yeast adherence and filamentation, two major factors contributing to the virulence of Candida albicans (2Sundstrom P. Cell. Microbiol. 2002; 4: 461-469Crossref PubMed Scopus (263) Google Scholar). The cell wall shows a bilayered structure with external and internal parts having specific functions. The outer layer enriched in mannoproteins determines cell surface properties (2Sundstrom P. Cell. Microbiol. 2002; 4: 461-469Crossref PubMed Scopus (263) Google Scholar, 3Chauhan N. Li D. Singh P. Calderone R. Kruppa M. Calderone R.A. Candida and Candidiasis. American Society for Microbiology, Washington, D. C.2002: 159-175Google Scholar). In contrast, the inner layer forms the cell wall skeleton with 1,3-β-glucan chains as major constituents (4De Groot P.W.J. De Boer A.D. Cunningham J. Dekker H.L. De Jong L. Hellingwerf K.J. De Koster C. Klis F.M. Eukaryot. Cell. 2004; 3: 955-965Crossref PubMed Scopus (217) Google Scholar). These 1,3-β-glucan chains are linked by covalent bonds to 1,6-β-glucans and chitin to form a three-dimensional matrix that surrounds cells (5Lipke P.N. Ovalle R. J. Bacteriol. 1998; 180: 3735-3740Crossref PubMed Google Scholar). Recent studies about cell wall assembly have indicated that glycosylphosphatidylinositol (GPI) 3The abbreviations used are: GPI, glycosylphosphatidylinositol; MAP, mitogen-activated protein; CFW, calcofluor white; GFP, green fluorescent protein; ORF, open reading frame; CFU, colony-forming unit; PBS, phosphate-buffered saline; HPLC, high pressure liquid chromatography; CR, congo red; GH, glycoside hydrolase; CBM, carbohydrate-binding module. -anchored proteins may play an important role in cell wall synthesis and in modifying or cross-linking polymers (6Mouyna I. Fontaine T. Vai M. Monod M. Fonzi W.A. Diaquin M. Popolo L. Hartland R.P. Latge J.P. J. Biol. Chem. 2000; 275: 14882-14889Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar). Some GPI proteins such as the members of the Gas family have a 1,3-β-transglucosidase activity that is responsible for in vitro 1,3-β-glucan chain elongation (6Mouyna I. Fontaine T. Vai M. Monod M. Fonzi W.A. Diaquin M. Popolo L. Hartland R.P. Latge J.P. J. Biol. Chem. 2000; 275: 14882-14889Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar); others such as the Ecm33 family have an important, yet unresolved, role in cell wall biosynthesis and morphogenesis (7Martinez-Lopez R. Monteoliva L. Diez-Orejas R. Nombela C. Gil C. Microbiology. 2004; 150: 3341-3354Crossref PubMed Scopus (92) Google Scholar). Moreover, GPI proteins such as Hwp1p and members of the Als protein family are involved in C. albicans adhesion and virulence (8Hoyer L.L. Fundyga R. Hecht J.E. Kapteyn J.C. Klis F.M. Arnold J. Genetics. 2001; 157: 1555-1567PubMed Google Scholar, 9Hoyer L.L. Trends Microbiol. 2001; 9: 176-180Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar, 10Hoyer L.L. Payne T.L. Bell M. Myers A.M. Scherer S. Curr. Genet. 1998; 33: 451-459Crossref PubMed Scopus (187) Google Scholar, 11Sundstrom P. Cutler J.E. Staab J.F. Infect. Immun. 2002; 70: 3281-3283Crossref PubMed Scopus (94) Google Scholar). In C. albicans, 104 ORFs coding for putative GPI proteins have been described by a genome-wide approach (12De Groot P.W.J. Hellingwerf K.J. Klis F.M. Yeast. 2003; 20: 781-796Crossref PubMed Scopus (219) Google Scholar). Of these, UTR2/CSF4 has been shown to be one of the genes that is most strongly regulated by calcineurin (13Karababa M. Valentino E. Pardini G. Coste A.T. Bille J. Sanglard D. Mol. Microbiol. 2006; 9: 1429-1451Crossref Scopus (180) Google Scholar), a Ca2+-calmodulin-dependent serine/threonine phosphatase playing a crucial role in the response of yeast to stress conditions. Calcineurin acts on the transcription factor Crz1p (for calcineurin-regulated zinc finger) (13Karababa M. Valentino E. Pardini G. Coste A.T. Bille J. Sanglard D. Mol. Microbiol. 2006; 9: 1429-1451Crossref Scopus (180) Google Scholar). A recent study reported the existence of a cluster of 60 genes dependent on calcineurin and Crz1p activation. These genes are involved in cell wall organization, cellular organization, cellular transport and homeostasis, cell metabolism, and protein fate (13Karababa M. Valentino E. Pardini G. Coste A.T. Bille J. Sanglard D. Mol. Microbiol. 2006; 9: 1429-1451Crossref Scopus (180) Google Scholar). Loss of calcineurin in C. albicans results in phenotypes comparable with those observed in strains with altered cell wall composition. This is consistent with the involvement of calcineurin in the regulation of genes, like UTR2, that are important for cell wall biogenesis. In Saccharomyces cerevisiae, the CRH family also consists of three GPI proteins, named CRH1, UTR2/CRH2, and CRR1, that are involved in cell wall construction (14Rodriguez-Pena J.M. Cid V.J. Arroyo J. Nombela C. Mol. Cell. Biol. 2000; 20: 3245-3255Crossref PubMed Scopus (111) Google Scholar). Deletion of CRH1 and CRH2 results in additive sensitivity to compounds (Congo red and calcofluor white) that interfere with cell wall assembly. Moreover, the double deletion mutant showed a 2-fold increase in the amount of alkali-soluble cell wall glucan in comparison to wild type, indicating that less glucan is bound to chitin (14Rodriguez-Pena J.M. Cid V.J. Arroyo J. Nombela C. Mol. Cell. Biol. 2000; 20: 3245-3255Crossref PubMed Scopus (111) Google Scholar). Studies with GFP fusion proteins indicated that Crh1p and Crh2p are located at the cell surface, particularly in chitin-rich areas, and that their temporal and spatial localization is specifically controlled during the cell cycle (14Rodriguez-Pena J.M. Cid V.J. Arroyo J. Nombela C. Mol. Cell. Biol. 2000; 20: 3245-3255Crossref PubMed Scopus (111) Google Scholar, 15Rodriguez-Pena J.M. Rodriguez C. Alvarez A. Nombela C. Arroyo J. J. Cell Sci. 2002; 115: 2549-2558PubMed Google Scholar). In addition, both proteins are shown to be covalently bound to the cell wall network (16Yin Q.Y. De Groot P.W.J. Dekker H.L. De Jong L. Klis F.M. De Koster C.G. J. Biol. Chem. 2005; 280: 20894-20901Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). Recently, two Crh homologues of Yarrowia lipolytica were found to be important for cell wall construction and were shown to have endo-1,3-β-glucosidase activity in vitro (17Hwang J.S. Seo D.H. Kim J.Y. Yeast. 2006; 23: 803-812Crossref PubMed Scopus (6) Google Scholar). Much less is known about the CRH family in C. albicans. In an earlier study, Utr2p/Csf4p was selected as a potential cell surface factor using a bio-informatics approach (18Alberti-Segui C. Morales A.J. Xing H. Kessler M.M. Willins D.A. Weinstock K.G. Cottarel G. Fechtel K. Rogers B. Yeast. 2004; 21: 285-302Crossref PubMed Scopus (64) Google Scholar). Deletion of UTR2 in C. albicans resulted in altered colony morphogenesis, attenuated cell adhesion, and reduced virulence. However, neither the role in cell wall assembly nor the functionality of other members of the CRH gene family in C. albicans was investigated (18Alberti-Segui C. Morales A.J. Xing H. Kessler M.M. Willins D.A. Weinstock K.G. Cottarel G. Fechtel K. Rogers B. Yeast. 2004; 21: 285-302Crossref PubMed Scopus (64) Google Scholar). In this study, we have addressed the role of the putative transglycosidases encoded by UTR2, CRH11, and CRH12 in cell wall biology and pathogenesis of C. albicans. First, we show that strains lacking UTR2, CRH11, and CRH12 exhibit phenotypes typical for cell wall defects. Second, alterations in cell wall polysaccharide composition in mutants lacking UTR2 suggest that Utr2p is involved in formation of the linkage between 1,3-β-glucan and chitin. Third, we show that the CRH family is critical for virulence in an animal model of systemic infection. Furthermore, comparative phenotypic analysis of the CRH family mutants with a calcineurin mutant indicates a partial overlap and thus suggests the existence of other important calcineurin-regulated targets. Strains and Media—The C. albicans strains used in this study are listed in Table 1. C. albicans strains were grown either in complete medium YEPD containing 0.5% (w/v) yeast extract (Difco), 1% (w/v) bactopeptone (Difco), and 2% (w/v) glucose (Sigma) or in minimal medium YNB (yeast nitrogen base; Difco) plus 2% (w/v) glucose (Fluka). For growth on solid media, 2% (w/v) agar (Difco) was added to the media.TABLE 1Strains used in this studyStrainGenotypeParent strainRef. or sourceCAF2-1ura3Δ::imm434/URA3SC531421CAF4-2ura3Δ::imm434/ura3Δ::imm434CAF2-121DSY2091cnaΔ::hisG/cnaΔ:: hisG-URA3-hisGCAF4-244GPY01utr2Δ::hisG-URA3-hisG/UTR2CAF4-2This studyGPY02utr2Δ::hisG/UTR2GPY01This studyGPY03utr2Δ::hisG/utr2Δ::hisG-URA3-hisGGPY02This studyGPY04utr2Δ::hisG/utr2Δ::hisGGPY03This studyGPY07utr2Δ::hisG/utr2Δ::hisG; LEU2::UTR2GPY04This studyGPY37crh11Δ::hisG-URA3-hisG/CRH11CAF4-2This studyGPY74crh11Δ::hisG/CRH11GPY37This studyGPY80crh11Δ::hisG/crh11::hisG-URA3-hisGGPY74This studyGPY83crh11Δ::hisG/crh11Δ::hisGGPY80This studyGPY89crh11Δ::hisG/crh11Δ::hisG; LEU2::CRH11GPY83This studyGPY16crh12Δ::hisG-URA3-hisG/CRH12CAF4-2This studyGPY19crh12Δ::hisG/CRH12GPY16This studyGPY88crh12Δ::hisG/crh12Δ::hisG-URA3-hisGGPY19This studyGPY91crh12Δ::hisG/crh12Δ::hisGGPY88This studyGPY97crh12Δ::hisG/crh12Δ::hisG; LEU2::CRH12GPY91This studyGPY17utr2Δ::hisG/utr2Δ::hisG; crh12Δ::hisG-URA3-hisG/CRH12GPY04This studyGPY20utr2Δ::hisG/utr2Δ::hisG; crh12Δ::hisG/CRH12GPY17This studyGPY23utr2Δ::hisG/utr2Δ::hisG; crh12Δ::hisG/crh12Δ::hisG-URA3-hisGGPY20This studyGPY26utr2Δ::hisG/utr2Δ::hisG; crh12Δ::hisG/crh12Δ::hisGGPY23This studyGPY87crh11Δ::hisG/crh11Δ::hisG; crh12Δ::hisG-URA3-hisG/CRH12GPY83This studyGPY99crh11Δ::hisG/crh11Δ::hisG; crh12Δ::hisG/CRH12GPY87This studyGPY100crh11Δ::hisG/crh11Δ::hisG; crh12Δ::hisG/crh12Δ::hisG-URA3-hisGGPY99This studyGPY36utr2Δ::hisG/utr2Δ::hisG; crh11Δ::hisG-URA3-hisG/CRH11GPY04This studyGPY75utr2Δ::hisG/utr2Δ::hisG; crh11Δ::hisG/CRH11GPY36This studyGPY109utr2Δ::hisG/utr2Δ::hisG; crh11Δ::hisG/crh11Δ::hisG-URA3-hisGGPY75This studyGPY27utr2Δ::hisG/utr2Δ::hisG; crh12Δ::hisG/crh12Δ::hisG; crh11Δ::hisG-URA3-hisG/CRH11GPY26This studyGPY84utr2Δ::hisG/utr2Δ::hisG; crh12Δ::hisG/crh12Δ::hisG; crh11Δ::hisG/CRH11GPY27This studyGPY101utr2Δ::hisG/utr2Δ::hisG; RPS/RPS::pGP52GPY04This studyGPY102utr2Δ::hisG/utr2Δ::hisG; crh12Δ::hisG/crh12Δ::hisG; crh11Δ::hisG/crh11Δ::hisG-URA3-hisGGPY84This studyGPY103utr2Δ::hisG/utr2Δ::hisG; crh12Δ::hisG/crh12Δ::hisG; crh11Δ::hisG/crh11Δ::hisGGPY102This studyMKY378RPS10/RPS10::Clp10CAF4-213MKY379cnaΔ::hisG/cnaΔ::hisG; RPS10/RPS10::Clp10DSY210113GPY120utr2Δ::hisG/utr2Δ::hisG; crh12Δ::hisG/crh12Δ::hisG; crh11Δ::hisG crh11Δ::hisG; RPS10/RPS10::Clp10GPY103This studyGPY121utr2Δ::hisG/utr2Δ::hisG; RPS10/RPS10::Clp10GPY04This studyGPY122crh11Δ::hisG/crh11Δ::hisG; RPS10/RPS10::Clp10GPY83This studyGPY123crh12Δ::hisG/crh12Δ::hisG; RPS10/RPS10::Clp10GPY91This studyGPY127utr2Δ::hisG/UTR2::caSAT1; RPS10/RPS10::Clp10GPY121This studyGPY128crh11Δ::hisG/CRH11::caSAT1; RPS10/RPS10::Clp10GPY122This studyGPY129crh12Δ::hisG/CRH12::caSAT1; RPS10/RPS10::Clp10GPY123This studyGPY132utr2Δ::hisG/UTR2::caSAT1; crh12Δ::hisG/crh12Δ::hisG; crh11Δ::hisG/crh11Δ::hisG; RPS10/RPS10::Clp10GPY120This studyGPY133utr2Δ::hisG/utr2Δ::hisG; crh12Δ::hisG/crh12Δ::hisG; crh11Δ::hisG/CRH11::caSAT1; RPS10/RPS10::Clp10GPY120This studyGPY134utr2Δ::hisG/utr2Δ::hisG; crh12Δ::hisG/CRH12::caSAT1; crh11Δ::hisG/crh11Δ::hisG RPS10/RPS10::Clp10GPY120This studyDSY2548mkc1Δ::hisG/mkc1Δ::hisG-URA3-hisGCAF4-259 Open table in a new tab Escherichia coli DH5α was used as host for plasmid constructions and propagation (19Hanahan D. Glover D.M. DNA Cloning. A Practical Approach. IRL Press at Oxford University Press, Oxford1985: 109-135Google Scholar). DH5α was grown in LB (Luria-Bertani broth; Difco) or LB plates, supplemented with ampicillin (0.1 mg/ml) when required. Transformation Procedures—C. albicans strains were transformed by a one-step transformation method of yeast in stationary phase as reported previously (20Coste A.T. Karababa M. Ischer F. Bille J. Sanglard D. Eukaryot. Cell. 2004; 3: 1639-1652Crossref PubMed Scopus (303) Google Scholar). Disruption of UTR2, CRH11, and CRH12 in C. albicans—Two different regions of UTR2 (orf19.1671 available on line) were amplified from C. albicans genomic DNA with the following pairs of primers (supplemental Table I): UTR2-1-XbaI/UTR2-1-XhoI and UTR2-2-XbaI/UTR2-2-XhoI in a PCR, and cloned into compatible restriction sites of pBluescriptKS+ to yield pGP01 and pGP03 (supplemental Table II), respectively. Deletions in the cloned UTR2 regions were created by PCR using outward directed primer pairs UTR2-1-PstI/UTR2-1-BglII and UTR2-2-PstI/UTR2-2-BglII (supplemental Table I) with pGP01 and pGP03 as templates. The products of these PCRs were digested with PstI and BglII and ligated to a PstI-BglII 3.7-kb fragment from pMB7 containing the hisG-URA3-hisG“Ura-blaster” cassette to yield pGP02 and pGP04 (supplemental Table II), respectively. The construction of two different UTR2 cassettes was aimed to enhance the recovery of targeted sequential genome deletions, because one cassette (pGP04) is internal to the first one (pGP02). Finally, disruption cassettes were linearized by digestion with ApaI and SacI and used for sequential disruption of both UTR2 alleles with intermediate marker regeneration as described by Fonzi and Irwin (21Fonzi W.A. Irwin M.Y. Genetics. 1993; 134: 717-728Crossref PubMed Google Scholar). The UTR2 revertant strain was obtained by reintroduction of UTR2 into the utr2Δ C. albicans mutant. A PCR was performed with the primers UTR2-5′-BamHI/UTR2-1-XhoI to yield a fragment containing the entire UTR2 ORF, including 3′- and 5′-flanking regions. The resulting fragment was cloned into pDS178 (22de Micheli M. Bille J. Schueller C. Sanglard D. Mol. Microbiol. 2002; 43: 1197-1214Crossref PubMed Scopus (146) Google Scholar) to obtain pGP05. This plasmid was linearized with SalI, which cuts within LEU2 to favor integration at the LEU2 genomic locus, and was transformed into C. albicans. CRH11 (orf19.2706) and CRH12 (orf19.3966) disruption cassettes were obtained using the same strategy as outlined above. PCRs were performed with the pairs of primers listed in supplemental Table I. Plasmids used for CRH11 and CRH12 disruptions are listed in supplemental Table II. Southern and Northern Blot Analysis—Genomic DNA was extracted from C. albicans and digested with appropriate restriction enzymes (supplemental Fig. 1). Northern blotting was performed as reported by Sanglard et al. (23Sanglard D. Kuchler K. Ischer F. Pagani J.L. Monod M. Bille J. Antimicrob. Agents Chemother. 1995; 39: 2378-2386Crossref PubMed Scopus (711) Google Scholar). The TEF3 probe was used as internal standard and originated from a 0.7-kb EcoRI-PstI fragment from pDC1 described in Sanglard et al. (23Sanglard D. Kuchler K. Ischer F. Pagani J.L. Monod M. Bille J. Antimicrob. Agents Chemother. 1995; 39: 2378-2386Crossref PubMed Scopus (711) Google Scholar). Phenotypic Analyses—C. albicans strains to be tested for their susceptibility to cell wall-perturbing agents and drugs were grown overnight in liquid YEPD medium. Cells were diluted to 1.5 × 107 cells per ml with serial 10-fold dilutions. Of each dilution 5 μl was spotted onto YEPD plates or plates containing 100 μg/ml congo red (CR; Sigma), 40 μg/ml calcofluor white (CFW; Sigma), 0.06% SDS (Fluka), or 500 mm CaCl2 (Fluka). Plates were incubated for 48 h at 34 °C. Protoplast Formation, Cell Wall Regeneration, and Cell Wall Degradation Assays—Cells grown overnight in YEPD were washed twice in TE, pH 8.0. Aliquots containing 1.5 × 107 cells were resuspended in PRO buffer (25 mm EDTA, 1 m sorbitol, 20 mm Tris-HCl, pH 7.5) with 50 μg/ml 100T Zymolyase (Seikagaku, Tokyo, Japan) and 1% (v/v) β-mercaptoethanol (Sigma) and incubated at 37 °C until cell wall lysis occurred. Protoplast formation was checked by microscopic observation. To regenerate cell walls, protoplasts were suspended in a soft-top agar (YEPD broth containing 0.7% agar and 1.2 m sorbitol, auto-claved and cooled to 45 °C). This suspension was poured evenly across the top of YEPD agar plates and allowed to solidify. To determine the number of intact cells present in the samples, the same amount of protoplasts was plated onto YEPD agar without top agar. Plates were incubated at 37 °C for 48 h. Numbers of cells grown in YEPD with or without top agar (nt and nnt, respectively), and the starting inoculum (ni) were determined as colony-forming units (CFU) by counting colonies of serial dilutions on YEPD agar. Cell wall regeneration was expressed as a percentage (Pcw) and determined by the following equation: Pcw = (nn - nnt)/(ni - nnt). Cell wall degradation was expressed as the percentage of intact cells after lysis caused by Zymolyase activity and was monitored by taking aliquots at 10-min intervals and measuring the optical density at 540 nm. Cell Wall Isolation and Analysis of Cell Wall Composition—C. albicans strains were grown overnight in liquid YPD at 30 °C to an A600 of about 2. The detailed procedure for cell wall isolation is described by De Groot et al. (4De Groot P.W.J. De Boer A.D. Cunningham J. Dekker H.L. De Jong L. Hellingwerf K.J. De Koster C. Klis F.M. Eukaryot. Cell. 2004; 3: 955-965Crossref PubMed Scopus (217) Google Scholar). To determine alkali-resistant 1,6-β- and 1,3-β-glucans, cell walls (about 4 mg dry weight) were extracted by incubation (three times) in 1 ml of 3% (w/v) NaOH at 75 °C for 1 h (24Magnelli P. Cipollo J.F. Abeijon C. Anal. Biochem. 2002; 301: 136-150Crossref PubMed Scopus (102) Google Scholar). Extracted walls were washed three times with 1 ml of H2O and freeze-dried. To release alkali-resistant 1,6-β-glucan, alkali-extracted walls were incubated with recombinant endo-1,6-β-glucanase (Prozyme, San Leandro, CA) as described (25Kapteyn J.C. Hoyer L.L. Hecht J.E. Muller W.H. Andel A. Verkleij A.J. Makarow M. Van Den Ende H. Klis F.M. Mol. Microbiol. 2000; 35: 601-611Crossref PubMed Scopus (267) Google Scholar). The supernatant after centrifugation represents the alkali-resistant 1,6-β-glucan fraction. The remaining cell wall pellet was washed two times with 1 ml of H2O and freeze-dried. Alkali-resistant 1,3-β-glucan was solubilized by incubating with the recombinant endo-1,3-β-glucanase Quantazyme (Qbiogene Morgan Irvine, CA) as described (25Kapteyn J.C. Hoyer L.L. Hecht J.E. Muller W.H. Andel A. Verkleij A.J. Makarow M. Van Den Ende H. Klis F.M. Mol. Microbiol. 2000; 35: 601-611Crossref PubMed Scopus (267) Google Scholar). Supernatants containing either 1,6-β-glucan or 1,3-β-glucan were analyzed with the phenol-sulfuric acid assay using glucose as a reference (26Dubois M. Gilles K. Hamilton J.K. Rebers P.A. Smith F. Nature. 1951; 168: 167Crossref PubMed Scopus (1338) Google Scholar). For total cell wall glucan and mannan determination, cell wall carbohydrates were hydrolyzed to monomers using the sulfuric acid hydrolysis method (27Dallies N. Francois J. Paquet V. Yeast. 1998; 14: 1297-1306Crossref PubMed Scopus (202) Google Scholar). About 4 mg of freeze-dried walls were incubated in 100 μl of 72% (v/v) H2SO4 for 3 h at room temperature. The samples were then diluted with 575 μl of distilled H2O to obtain a 2 m H2SO4 solution and incubated for 4 h at 100 °C. Amounts of mannose and glucose in the samples were determined by HPLC (GE Healthcare) on a REZEX organic acid analysis column (Phenomenex, Torrance, CA) at 40 °C with 7.2 mm H2SO4 as eluent, using an RI1530 refractive index detector (Jasco, Great Dunmow, UK). The chromatograms were analyzed using AZUR chromatography software and compared with chromatograms of known amounts of mannose, glucose, and glucosamine. Chitin was determined following the protocol described by Kapteyn et al. (28Kapteyn J.C. Van Den Ende H. Klis F.M. Biochim. Biophys. Acta. 1999; 1426: 373-383Crossref PubMed Scopus (319) Google Scholar). Protein Structure Analysis—Functional domains of Utr2p, Crh11p, and Crh12p, glycoside hydrolase (GH) domains and a carbohydrate-binding module (CBM), were identified using the SMART tool analysis software and the CAZy Carbohydrate-Active enZymes data base. Most likely, GPI addition of amino acid (ω)-sites (Ser440, Asn430, and Gly478) were predicted using big-PI Fungal Predictor software. The three-dimensional structure of the putative transglycosidase domain of Utr2p was obtained by homology modeling. The GH16 domain boundaries of Utr2p were predicted by alignment of multiprotein families using the Pfam protein families data base. The secondary structure of the Utr2p GH16 domain was predicted and aligned on template structures of a fold library using threading to identify a Protein Data Bank template for homology modeling. At the level of secondary structure, Utr2p favorably aligns with Bacillus lichenoformis 1,3-1,4-β-d-glucan 4-glucanohydrolase (Protein Data Bank code 1GBG). This template also belongs to the CAZy GH16 family and cleaves 1,4-β-glycosidic bonds that are adjacent to 1,3-β-glycosidic bonds in mixed-linked glucans. Multiple sequence alignment with hierarchical clustering was used to align the Utr2p GH16 target and 1GBG template sequence for homology modeling. Optimization of this alignment was carried out manually. Detection of Mkc1p by Immunoblotting—C. albicans cell extracts for immunoblotting were prepared from cells grown to mid-log phase as described by Navarro-Garcia et al. (29Navarro-Garcia F. Eisman B. Fiuza S.M. Nombela C. Pla J. Microbiology. 2005; 151: 2737-2749Crossref PubMed Scopus (99) Google Scholar). Briefly, yeasts incubated for 2 h in the presence or absence of CR (20 μg/ml) were resuspended in lysis buffer and broken using glass beads in a Mini-Bead Beater-8 (Biospec Products, Bartlesville, OK) applying three 30-s rounds with intermediate ice cooling (1 min). Equal amount of proteins (150 μg), verified by the Bradford assay (30Bradford D.S. Cooper K.M. Oegema Jr., T.R. J. Bone Jt. Surg. Am. 1983; 65: 1220-1231Crossref PubMed Scopus (85) Google Scholar) and Ponceau S staining, were separated by SDS-PAGE and blotted onto a nitrocellulose membrane. Dually phosphorylated Mkc1p was detected using 1,000-fold diluted anti-phospho-p44/42 MAP kinase (Thr202/Tyr204) anti-serum (New England Biolabs, Ipswich, MA). Western blots were developed using an ECL kit following the manufacturer's instructions (Amersham Biosciences). Construction of GFP Fusion Proteins—To create a fusion protein of Utr2p with the GFP, a fragment of the UTR2 ORF (from +1to +1263), lacking the ω-site and C-terminal GPI signal sequence, was amplified with the primer pair UTR2-5′-GFP-BamHI/GFP-3′-UTR2. GFP was amplified from yEGFP (31Cormack B.P. Bertram G. Egerton M. Gow N.A. Falkow S. Brown A.J. Microbiology. 1997; 143: 303-311Crossref PubMed Scopus (502) Google Scholar) with primers UTR2-5′-GFP and GFP-3′-ClaI. These two fragments share a 30-bp overlap and were used as templates in a second PCR amplification step with external primers UTR2-5′-BamHI and GFP-ClaI. The resulting UTR2-GFP fragment was cloned into pBluescriptSK+ digested with BamHI and ClaI to yield pGP50. To obtain a functional Utr2p-GFP protein, a UTR2 fragment from +1263 to +2861 containing the ω-site and C-terminal sequence was amplified using primers UTR2-5′-ClaI/UTR2-1-XhoI and cloned into a compatible site of pGP50 to yield pGP51. The final UTR2-GFP fusion construct was amplified from pGP51 with the primer pair UTR2-5′-GFP-SalI/UTR2-3′-GFP-NheI and cloned into CIpACT-C-ZZ under the control of the ACT1 promoter to yield pGP52. This plasmid was digested with StuI and transformed into the utr2Δ strain (GPY04) to yield GPY101. The UTR2-GFP construct could yield a functional protein as it complemented the phenotypes (hypersusceptibility to cell wall damaging agents) of the utr2Δ strain (data not shown). Fluorescence Microscopy—Fluorescence and contrast interference microscopy were performed using a Zeiss Axioplan microscope (Zeiss) equipped for epifluorescence microscopy with a 100-watt mercury high pressure bulb and Zeiss filter set 15. A Kappa DX30 digital camera with high resolution was used to record images, which were processed further using the computer program Adobe Photoshop 6.0. For chitin staining, cells were incubated for 5 min at room temperature in the presence of 0.5 μg/ml CFW, and cells were examined directly for fluorescence after washing off excess CFW. Mouse Infection Assays—Animal experiments were performed as described previously (13Karababa M. Valentino E. Pardini G. Coste A.T. Bille J. Sanglard D. Mol. Microbiol. 2006; 9: 1429-1451Crossref Scopus (180) Google Scholar). Briefly, C. albicans isolates grown overnight at 30 °C in YEPD were diluted to 106 cells/ml into 50 ml of YEPD and incubated for another 24 h at 30 °C. Cells were washed twice with PBS, and cell concentrations were adjusted to 2 × 106 CFU/ml in PBS. To confirm the accuracy of the infection dose, samples of inoculum suspensions were plated onto YEPD agar. Mice were infected intravenously with each strain at a dose of 5 × 105 CFU in 250 μl of PBS via the lateral tail vein. For reintroduction of UTR2, CRH11, and CRH12 into the utr2Δ/crh11Δ/crh12Δ mutant strain GPY103, pMK83 containing the CaSAT1 marker was used (32Reuss O. Vik A. Kolter R. Morschhauser J. Gene (Amst.). 2004; 341: 119-127Crossref PubMed Scopus (566) Google Scholar). UTR2, CRH11, and CRH12 were amplified by PCR using the primer pairs listed in supplemental Table I and cloned into compatible restriction sites of pMK83 to yield pGP55 (containing UTR2), pGP56 (containing CRH11), and pGP57 (containing CRH12). These plasmids were linearized by HpaI (pGP55 and pGP56) and MluI (pGP57) in order to facilitate integration in