Saccharomyces Cerevisiae

Hindawi Publishing Corporation International Journal of Microbiology Volume 2010, Article ID 930465, 10 pages doi:10.1155/2010/930465 Research Article Boric Acid Disturbs Cell Wall Synthesis in Saccharomyces cerevisiae MartinSchmidt,JaronZ.Schaumberg,CourtneyM.Steen,andMichaelP.Boyer Biochemistry and Nutrition, Des Moines University, 3200 Grand Avenue, Des Moines, IA 50312, USA Correspondence should be addressed to Martin Schmidt, [email protected] Received 6 August 2010; Revised 20 October 2010; Accepted 16 November 2010 Academic Editor: Robert P. Gunsalus Copyright © 2010 Martin Schmidt et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Boric acid (BA) has broad antimicrobial activity that makes it a popular treatment for yeast vaginitis in complementary and alternative medicine. In the model yeast S. cerevisiae, BA disturbs the cytoskeleton at the bud neck and impairs the assembly of the septation apparatus. BA treatment causes cells to form irregular septa and leads to the synthesis of irregular cell wall protuberances that extend far into the cytoplasm. The thick, chitin-rich septa that are formed during BA exposure prevent separation of cells after abscission and cause the formation of cell chains and clumps. As a response to the BA insult, cells signal cell wall stress through the Slt2p pathway and increase chitin synthesis, presumably to repair cell wall damage. 1. Introduction ring (CAR) by the addition of myosin and actin, among other proteins [8, 9]. To complete abscission, the last phase In the right amounts, boron is an essential nutrient for of cytokinesis, the cells first separate mother and daughter animals, plants, and fungi [1–3]. However, at high concen- cells with a chitin primary septum. The deposition of ff trations boric acid (BA) becomes an e ective poison that glucan and mannoprotein-rich cell wall material on the is widely used for the killing of diverse organisms ranging mother and daughter side of the primary septum later from bacteria to rodents [4]. In medicine, BA is used as an completes the trilaminar septum that can be observed under alternative treatment for vaginal yeast infections [5]. While normal culture conditions. A disturbance in the assembly of the molecular details of BA action on yeast remain unclear, it the septation apparatus—the cohesive functional unit that was recently shown that BA interferes with morphogenesis, constructs the primary septum—leads to the formation of ff to the e ect that it inhibits the transition from the yeast to the highly aberrant septa [10, 11]. The septa formed under these hyphal form of the pathogenic yeast C. albicans [6]. Because conditions do not allow for the separation of cells after the ability to switch to hyphal growth is an important cytokinesis, leading to the formation of chains and clumps virulence factor in C. albicans [7], suppression of such of misshaped cells. Based on the observation that BA causes elongated growth by BA may in part explain its therapeutic such clumping and chain formation in S. cerevisiae, the ff ff e ect. The present study was undertaken to assess the e ect present study was initiated to assess the influence of BA on of BA on morphogenesis and cell wall synthesis in yeast, the function of the septation apparatus. using the well-established model organism Saccharomyces cerevisiae as a study subject. In S. cerevisiae, morphogenesis and cell wall synthesis 2. Methods depend on the correct assembly of cytoskeletal proteins. To guide cell wall synthesis during cytokinesis, a ring of 2.1. Strains and Culture Conditions. Growth media and septin filaments forms during the G1 phase of the cell cycle culture conditions were as described in [12]. Strains are listed and is subsequently completed into a contractile actomyosin in Table 1. 2 International Journal of Microbiology Table 1: Strains. with PBS. Cells were then pelleted and taken up in 50 μL ProLong Antifade reagent (Invitrogen) before mounting on Strain Genotype Source slides. BY4742 MATα his3Δ1leu2Δ 0lys2Δ0ura3Δ0 Invitrogen GFP-tagged Cdc3p and Myo1p were observed with the BY4742 MATα his3Δ1leu2Δ0lys2Δ 0ura3Δ 0 Invitrogen GFP-filter set (Zeiss) in cells transformed with pRS316CDC slt2::kanMX6 slt2::kanMX6 3GFP [12] and pMS55 [11]. MATa ura3-52 lys2-801 ade2-101 ECY46-1-8D [13] trp1-Δ63 his3-Δ200 leu2-Δ 1chs1::HIS3 2.4. Electron Microscopy. Electron microscopic examination MATa ura3-52 lys2-801 ade2-101 of yeast cell walls followed a previously published protocol YMS415 trp1-Δ63 his3-Δ200 leu2-Δ1 This study [18]. CHS3::3HA-HIS3 MATa ura3-52 lys2-801 ade2-101 YPH499 [14] 2.5. Western Blotting. Analysis of Slt2p phosphorylation and trp1-Δ63 his3-Δ200 leu2-Δ1 total Slt2p by western blotting followed established protocols [19, 20]. As a loading control, an antibody directed against Tub4p (goat χ-tubulin yK18, Santa Cruz Biotechnology) was Strain YMS415 (CHS3::HA-HIS3) was constructed by the used at 1/1,000 dilution. For the determination of Chs3p- short flanking homology method [15]. The CHS3::HA-HIS3 HA, cell membranes were isolated according to Orlean [21]. fragment was amplified by PCR from plasmid pFA6a-3HA- One volume of mercaptoethanol-free 2x sample buffer with His3MX6 with primers 5-TATTCTCAATCGGAAGGA- 2% SDS (Bio-Rad) was added to the protein extracts and GGAAAGTGACTCCTTCGTTGCAGGTGGAGGTGG- samples were loaded on 6% SDS polyacrylamide gels with- AGGTGGAGGTGGACGGATCCCCGGGTTAATTAA- out boiling. Incubations with antibodies were performed 3 and 5-TCAACTTGTAAGTATCACAGTAAAAAT- as described above [18]. Antibodies used were a 1/5,000 ATTTTCATACTGTGAATTCGAGCTCGTTTAAAC-3. dilution of anti-HA/1/5,000 dilution of goat antimouse HRP Integration of the fragment was verified by PCR and western (Santa Cruz Biotechnology). Four independent experiments blotting. Transformants showed a wild-type like distribution were performed and representative results are shown. Mem- of chitin in calcofluor white stained preparations, demon- branes were stained in 0.02% Coomassie blue R250 in 40% strating full functionality of the Chs3p-HA fusion protein. Methanol, 5% acetic acid to serve as loading controls. 2.2. Determination of BA Sensitivity. A visual representation 2.6. Enzyme Measurements. For the determination of of BA sensitivity was obtained by serially diluting an enzyme activities, cell membranes were isolated as previously overnight culture of yeast grown in YPD and spotting 5 μL described [21]. of cell dilutions on YPD plates with the indicated concentra- Determination of glucan synthase activity followed a tions of BA. Growth was analyzed after 3-day incubation at protocol by Mol and coworkers [22]. A 40 μLglucan ◦ 30 C. Minimal inhibitory concentrations were determined synthase assay contained 20 μL of membrane suspension at according to CLSI standards with the broth microdilution a protein concentration of 1 mg mL−1,5mM14C-UDP- assay [16]. glucose (activity 1 × 109 cpm mmol−1), 75 mM Tris-Cl pH 7.5, 25 mM KF, and—when indicated—with 20 μMGTP-γ- 2.3. Staining Procedures and Fluorescence Microscopy. Viabil- SforthedeterminationofmaximalGSactivity.Thereaction ◦ ity staining of yeast cultures was performed by incubating was incubated for 1 hour at 30 C and then stopped by adding cells in 0.2 mg/mL methylene blue in 50 mM KH2PO4 for 5 1 mL 10% trichloroacetic acid (TCA). Reaction mixtures minutes at RT. were filtered through a type A/E glass fiber filter (Pall). In order to visualize the distribution of chitin and glucan Filters were washed twice with 1 mL 10% TCA and four in the cell wall, cells were washed and incubated for 5 minutes times with 70% ethanol. Filters were dried and taken up in in 0.01% calcofluor white or 0.5% aniline blue, respectively 10 mL cytoscint ES scintillation fluid (ICN), and activity was [17]. Fluorescence was observed with a standard diamidino- recorded in a scintillation counter. Activities were calculated − − 2-phenylindol (DAPI) filter set (Zeiss). as c.p.m. incorporated h 1μgprotein 1. Filamentous actin was visualized by Alexa 568-phalloidin Chitin synthesis was determined as described by Choi staining of yeast cells. Fifty mL yeast cultures at a titer and Cabib [23]. To measure chitin synthase 2 and 3 activities, of 1–5∗106 cells/mL in YPD were fixed by the addition experiments were performed in the chs1 deletion strain of formaldehyde to a final concentration of 4% and ECY46-1-8D. The chs1 deletion was found to have no incubated for 10 minutes at room temperature. Cells were influence on BA sensitivity (Figure 1(a)). The chitin synthase pelleted and incubated for 1 hour in phosphate buffered assay mixture contained 20 μL of membrane suspension −1 saline (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, at 1 mg protein mL ,5μLof0.5MTris/ClpH7.8,5μL 14 1.76 mM KH2PO4, pH 7.4) with 4% formaldehyde. After of 20 mM cobalt acetate, 5 μLof10mM C-UDP-GlcNAc 2 washes in PBS, cells were suspended in 100 μL PBS (5000 c.p.m. μL−1), and 2 μL of trypsin solutions (Sigma) at with 10 μL of an Alexa 568-phalloidin solution (6.6 μMin concentrations from 0.25 to 2.0 mg mL−1 in a total volume methanol; Invitrogen) and 1 μLof1mg/mLcalcofluorwhite. of 46 μL. For determination of chitin synthase 3 activity, Cells were incubated for 1 hour in the dark and washed twice 5 μLofwaterweresubstitutedwith5μLof50mM International Journal of Microbiology 3 nickel acetate. Proteolysis was stopped after incubating for 100 15 minutes at 30◦C by adding 2 μL of a soybean trypsin inhibitor solution at 1.5x the concentration of the trypsin solution. Chitin synthesis was initiated by adding 2 μLof 80 0.8 M GlcNAc. After incubating for 60 minutes at 30◦C, chitin synthesis was stopped by adding 1 mL 10% TCA.

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