Glucanase Induces Filamentation of the Fungal Pathogen Candida albicans

Citation: Xu H, Nobile CJ, Dongari-Bagtzoglou A ( Glucanase Induces Filamentation of the Fungal Pathogen Candida albicans Hongbin Xu 0 Clarissa J. Nobile 0 Anna Dongari-Bagtzoglou 0 David R. Andes, University of Wisconsin Medical School, United States of America 0 1 Department of Oral Health and Diagnostic Sciences, University of Connecticut Health Center, Farmington, Connecticut, United States of America, 2 Department of Microbiology and Immunology, University of California San Francisco , San Francisco, California , United States of America Candida albicans is the most common human fungal pathogen. Many organisms, including C. albicans, secrete glucanases under different environmental conditions. Here, we report a novel role for beta-1, 3- glucanase in inducing Candida albicans to form filaments at 22uC and enhancing filamentation at 37uC in nutrient-rich medium. Quorum sensing, the efg1-signaling and cek1 MAP kinase pathways are involved in this process. Our data suggest that the natural antifungal agent betaglucanase may support morphologic transformation of Candida albicans at a wide range of ambient temperatures. - Funding: This research was supported by National Institutes for Health R01 DE13986. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Candida albicans is a fungus that can undergo multiple morphological transitions. C. albicans is known to infect a wide variety of hosts from warm-blooded animals to insects [1]. Hyphal formation is essential for virulence and invasion in a variety of hosts [2]. Many environmental stimuli can stimulate Candida albicans to form hyphae, including, but not limited to, serum, CO2, quorum sensing molecules, and pH [36]. However, most of these stimuli require incubation physiological temperature (37uC) for efficient hyphal growth. Although hyphal formation has been reported at lower temperatures, the conditions used were complex, requiring a matrix [7] or amino acid-containing liquid synthetic medium [8]. Many organisms have beta-1, 3-glucanase genes and secrete glucanases into the environment, including C. albicans [9,10]. C. albicans possesses three cell wall related exo-beta-1, 3-glucanases, Xog1, Exg2, and Spr1 [11]. CAMP65 was also identified as a putative beta-glucanase and is required for hyphal morphogenesis [12]. Although these hydrolytic enzymes are believed to play cell wall remodeling roles during growth and morphogenesis in filamentous fungi, they may play multiple physiological roles, which have not been fully elucidated in C. albicans. In this work, we show that beta-1, 3- glucanase, an enzyme produced by many bacteria, fungi and plants [1315], permits C. albicans to overcome the temperature requirement for hyphal transformation in yeast peptone dextrose (YPD) medium. Materials and Methods Candida albicans strains and growth conditions The wild type strain SC5314, and strains SN425 (reference stain), SGH284 (a biosensor reporter strain), HLC54 (egf1/cph1 double mutant), cph1 mutant, cek1 mutant, CJN2302 (efg1 mutant), cht2 mutant and CJN2318 (efg1 revertant) were grown in YPD medium and maintained on YPD agar plates (Table 1). These strains were described in detail elsewhere [2,1618]. To induce filamentation, the cells were grown to stationary phase overnight in YPD broth, and then inoculated in 5 ml glucanase-supplemented media (YPD, DMEM or RPMI) at 106 cells per ml and incubated at 22uC, 30uC or 37uC, for 18 h without shaking. For glucanase dose response experiments beta-1, 3- glucanase/lyticase, purified from two microbial sources (from Arthrobacter luteus or Trichoderma harzianum, Sigma), was added in YPD at concentrations ranging between 0.1100 mg/ml and organisms were inoculated and incubated as described above. Glucanase inactivated by heating at 95uC for 10 minutes was used as control. To induce true hyphae, 10% FBS was used as positive control and organisms were grown at 37uC in an aerobic incubator with 5% CO2. Hyphal units were enumerated under phase contrast microscopy. Fluorescence Microscopy For staining the cell wall or nuclear material, the cells were collected by centrifugation, washed with PBS and stained with Calcofluor white (Sigma) or Hoechst 33258 (BIO-RAD), respectively, for 10 minutes. After washing in PBS the cells were mounted on slides and observed under a fluorescence microscope. To examine the effect of glucanase treatment on the cell wall glucan, cells were stained with a monoclonal antibody highly specific for (1R6) branched, (1R3)-b-D-glucans (BFDiv, Biothera), as we previously described [19]. To examine cell viability, a fluorescence LIVE-DEAD viability stain kit (Molecular Probes) was used to stain the cells, according to manufacturers instructions. In some experiments farnesol or tyrosol were added to the media at 10200 mM (Sigma) [20,21]. Quantitative Real- time PCR Assay The assay was described in detail previously [22]. Briefly, 5 ml cell cultures were grown as described above and total RNA was extracted using the RiboPure yeast kit (Ambion, Inc.). RNA concentrations and quality were determined by measuring the absorbance at 260 nm and 280 nm (ND-1000 spectrophotometer, NanoDrop Technologies). Equal amounts of RNA (3 mg in 20 ml reactions) were reverse transcribed with oligo(dT) primers using Superscript reverse transcriptase II (Invitrogen). Primers used were as follows: EFB1, Forward: 59- CAT TGA TGG TAC TAC TGC CAC -39; Reverse: 59- TTT ACC GGC TGG CAA GTC TT -39. HWP1, Forward: 59- TGG TCC AGG TGC TTC TTC TT -39; Reverse: 59- GGT TGC ATG AGT GGA ACT GA-39. ALS3, Forward: 59- CCA CTT CAC AAT CCC CAT C -39; Reverse: 59- CAG CAG TAG TAA CAG TAG TTT CAT C -39. HYR1, Forward: 59- CGT CAA CCT GAC TGT TAC ATC -39; Reverse: 59- TCT ACG GTG GTA TGT GGA AC -39.UME6, Forward: 59- CAG TGG TAA TGG CAC TAA CAC C -39; Reverse: 59- GCA CAA CCT CCA CAA ATT GGT G -39. CHT2, Forward: 59- CAA ACC ACT TCC TAC CCT GTTG39; Reverse: 59-GAT GTT GGG TAT GTA ACT GGGG -39. CHT3, Forward: 59- CAA CTT CGT CGA CAA GTT TATC ura3D::limm434 ARG4 his1::hisG::pHIS1-pTDH3-GFP-tADH1 HWP1::pHWP1-RFP-tADH1-URA3, ura3D:: limm434 arg4::hisG his1::hisG HWP1 [26] ura3D:: limm434::URA3-IRO1 arg4::hisG his1::hisG leu2::hisG::CdARG4 efg1D::CmLEU2, ura3D:: limm434 arg4::hisG his1::hisG leu2::hisG efg1D::CdHIS1 ura3D:: limm434::URA3-IRO1 arg4::hisG his1::hisG leu2::hisG::EFG1-CdARG4, efg1D::CmLEU2, ura3D:: limm434 arg4::hisG his1::hisG leu2::hisG efg1D::CdHIS1 ura3D:: limm434::URA3-IRO1 arg4::hisG::CdARG4 his1::hisG leu2::hisG::CdHIS1, ura3D:: limm434 arg4::hisG his1::hisG leu2::hisG::CmLEU2 ura3::1 imm434/ura3::1 imm434 cph1::hisG/cph1::hisG efg1::hisG/efg1::hisG-URA3 hisG his1D/his1D, leu2D/leu2D, arg4D/arg4D, URA3/ura3D::imm434,IRO1/iro1D::imm434, orf19.4433D::C.dubliniensisHIS1/ orf19.4433D::C.maltosaLEU2 his1D/his1D, leu2D/leu2D, arg4D/arg4 (...truncated)