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cysteine biosynthesis contributes to biofilm formation in Candida albicans
Fungal Genetics and Biology 51 (2013) 50–59
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ECM17-dependent methionine/cysteine biosynthesis contributes to biofilm formation in Candida albicans De-Dong Li, Yan Wang ⇑, Bao-Di Dai, Xing-Xing Li, Lan-Xue Zhao, Yong-Bing Cao, Lan Yan, Yuan-Ying Jiang ⇑ School of Pharmacy, Second Military Medical University, Shanghai 200433, China
a r t i c l e
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Article history: Received 1 August 2012 Accepted 28 November 2012 Available online 12 December 2012 Keywords: Candida albicans ECM17 Biofilm Cysteine Methionine
a b s t r a c t Candida albicans has become the fourth leading pathogen of nosocomial bloodstream infections largely due to biofilm formation on implanted medical devices. Previous microarray data indicated that almost all genes in methionine (Met)/cysteine (Cys) biosynthesis pathway were up-regulated during biofilm formation, especially during the adherence period. In this work, we studied the role of Met/Cys biosynthesis pathway by disrupting ECM17, a gene encoding sulfite reductase in C. albicans. It was found that the ecm17D/D mutant failed to catalyze the biochemical reaction from sulfite to H2S and hardly grew in media lacking Met and Cys. NaSH, the donor of H2S, dose-dependently improved the growth of ecm17D/D in media lacking a sulfur source. Sufficient Met/Cys supply inhibited the expression of ECM17 in a dose-dependent manner. These results validated the important role of ECM17 in Met/Cys biosynthesis. Interestingly, the ecm17D/D mutant showed diminished ability to form biofilm, attenuated adhesion on abiotic substrate and decreased filamentation on solid SLD medium, especially under conditions lacking Met/Cys. Further results indicated that ECM17 affected the expressions of ALS3, CSH1, HWP1 and ECE1, and that the cAMP–protein kinase A (PKA) pathway was associated with ECM17 and Met/Cys biosynthesis pathway. These results provide new insights into the role of Met/Cys biosynthesis pathway in regulating cAMP-PKA pathway and benefiting biofilm formation. Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction Candida albicans is the most common opportunistic fungal pathogen of humans, causing diseases varying from superficial mucosal infections to life-threatening systemic diseases (Karkowska-Kuleta et al., 2009). It has become the dominant pathogen in patients with candidemia, especially in Europe and USA (Falagas et al., 2010), largely due to the biofilm formation on implanted medical devices. There are three stages for biofilm formation: (i) attachment and adhesion to biomaterial surfaces, (ii) growth and proliferation of cells to form an anchoring layer, and (iii) growth of pseudohyphae, hyphae and extracellular matrix material to form a complex threedimensional structure (Nobile and Mitchell, 2006; Ramage et al., 2005; Seneviratne et al., 2008). Adhesion and filamentous growth are particularly important for biofilm formation (Ramage et al., 2009). More specifically, adhesion is mediated by both non-specific factors (including cell surface hydrophobicity) and specific
⇑ Corresponding authors. Address: School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433, China. Fax: +86 21 65490641. E-mail addresses: [email protected] (Y. Wang), [email protected] (Y.-Y. Jiang). 1087-1845/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.fgb.2012.11.010
adhesion genes (HWP1, HWP2, ALS1, ALS3, IFF4, EAP1 and CSH1) (Chaffin, 2008), and filamentous growth is mediated by some hypha-specific genes (HWP1, ECE1, SAP4, SAP5, SAP6, UME6, HGC1, EED1) (Sudbery, 2011). Among these adhesion-specific or hyphaspecific genes, ALS3, HWP1 and ECE1 are proven to be regulated by cAMP–PKA pathway (Hogan and Sundstrom, 2009). The mechanism of C. albicans biofilm formation is complex. Some groups of genes are co-regulated during the formation of biofilm (Seneviratne et al., 2008), but the underlying mechanisms remain unclear (Nobile and Mitchell, 2006). Genome-wide transcription profiles have provided plenty of valuable information for the better understanding about the mechanisms of biofilm formation (Garcia-Sanchez et al., 2004; Nobile and Mitchell, 2006). Notably, a group of methionine (Met)/cysteine (Cys) biosynthesis genes are up-regulated from the early adhesion stage and maintain for hours (Garcia-Sanchez et al., 2004; Murillo et al., 2005). Moreover, Met and Cys are sulfur-containing amino acids, and most sulfur assimilation genes are up-regulated simultaneously with the Met/Cys biosynthesis genes (Murillo et al., 2005). These data imply that Met/Cys biosynthesis may play an important role in biofilm formation. However, no study has focused on the relationship between Met/Cys biosynthesis pathway and biofilm formation.
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The predicted Met/Cys biosynthesis pathway of C. albicans is shown in Fig. 1. A search of this pathway in C. albicans genome database (http://www.candidagenome.org/ and http://genolist.pasteur.fr/CandidaDB/) reveals that ECM17 (also known as orf19.4099) encodes a putative enzyme that functions in sulfur amino acid biosynthesis. The ECM17 ortholog in Saccharomyces cerevisiae, known as MET5 (available at http://www.yeastgenome.org/), encodes the sulfite reductase beta subunit, which catalyzes the biochemical reaction from sulfite to H2S, a key step in Met and Cys biosynthesis (Thomas and Surdin-Kerjan, 1997). Our BLASTN result indicated that C. albicans ECM17 and S. cerevisiae MET5 have a 64% similarity of nucleotide sequence, and BLASTP result indicated that they have 52% similarity of amino acid sequence. These results imply that C. albicans ECM17 might play a role in Met and Cys biosynthesis. In this work, we developed an ecm17D/D mutant and our further investigations revealed that ECM17-dependent Met/Cys biosynthesis played a critical role in C. albicans biofilm formation. 2. Materials and methods 2.1. Strains and growth conditions C. albicans strains used in this study are listed in Table 1. All strains were routinely grown in YPD (1% yeast extract, 2% peptone and 2% dextrose) liquid medium at 30 °C in a shaking incubator. To observe the filamentous growth of the Candida cells, hyphainducing media (Spider and Lee) were prepared as described previously (Lee et al., 1975; Liu et al., 1994). For solid serum medium, 10% (vol/vol) fetal calf serum (FCS) was added to YPD after autoclaving. Synthetic low dextrose (SLD) medium is 0.17% YNB medium containing 0.5% (NH4)2SO4, 0.1% glucose, 1.5% agar and different concentrations of Met and Cys (Maidan et al., 2005). 2.2. Disruption and reintegration of ECM17 in C. albicans ECM17 was disrupted using a well-described strategy (Supplementary Fig. 1A and B) (Noble and Johnson, 2005). Briefly, the DNA fragments for homologous recombination were obtained through fusion PCR (Noble and Johnson, 2005) and the primers used to obtain the fragments are listed in Supplementary Table 1. The HIS1-containing fusion fragment upstream-HIS1-downstream was transformed into C. albicans strain SN152 using the lithium acetate method (Kawai et al., 2010). Transformants were plated
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on selective medium (yeast nitrogen base with amino acids except histidine), and positive clone KEC1 was verified to be correct with ECM17 one allele disrupted by PCR using genomic DNA as the template (Supplementary Fig. 2). To obtain a control strain, the HIS1 marker gene was transformed to the locus of RPS10 in SN152, resulting in strain SN10. Using the same strategy, the ARG4-containing fusion fragment upstream-ARG4-downstream was transformed into KEC1 to disrupt the other allele of ECM17. Positive clone KEC21 was verified to be correct with both alleles of ECM17 disrupted by PCR using genomic DNA as the template (Supplementary Fig. 2). To further obtain the control strain, ARG4 marker gene was transformed to the locus of ARG4 in SN10, resulting in strain SN101. Primers used to identify the strains are listed in Supplementary Table 2. Reintegration of ECM17 gene was performed using the SAT1 flipper tool (Reuss et al., 2004). The DNA fragment for homologous recombination was done as follows: A KpnI–XhoI fragment with the whole ORF of ECM17, 0.63 kb upstream and 0.25 kb downstream was amplified using primers ECM17 complement 1 and 2 (Supplementary Table 1), and the fragment was cloned to the upstream of the SAT1 flipper. A SacII–SacI fragment with the ECM17 ORF downstream was amplified using primers ECM17 complements 3 and 4 (Supplementary Table 1), and the fragment was cloned to the downstream of the SAT1 flipper, yielding plasmid pSFS2A–ECM17 (Supplementary Fig. 1C). Then the linearized pSFS2A–ECM17 plasmid was transformed into the strain KEC21. The transformants were spread on YPD plates containing 200 lg/ ml nourseothricin. Nourseothricin-resistant clones were streaked on arginine or histidine-lacking agar plates. In this study, the correct clones could grow on arginine-lacking agar plates but not on histidine-lacking agar plates, indicating that the HIS1 cassette had been replaced by our transformation fragment. Subsequently, transformants were grown in YPM medium (1% yeast extract, 2% peptone and 2% maltose) for 5 h to excise the SAT1 flipper cassette. One hundred to two hundred cells were then spread on YPD plates containing 25 lg/ml nourseothricin and grown for 2 days at 30 °C. Nourseothricin-sensitive clones, known as KEC3, were identified by their small clone size and confirmed by restreaking on YPD plates containing 200 lg/ml nourseothricin as described previously (Reuss et al., 2004). Finally, using the same SAT1 flipper strategy, HIS1 cassette was restored into KEC3, yielding strain KEC31. The reintegrated strain KEC31 was identified through PCR using primers as listed in Supplementary Table 3. 2.3. Detection of H2S generation H2S generation was detected using BiGGY agar (Oxoid, England) (Ugliano et al., 2011). BiGGY (Bismuth Sulfite Glucose Glycine Yeast) is a medium containing bismuth ammonium citrate. H2S can reduce the bismuth ammonium citrate and result in a brown color (Cordente et al., 2009; Ilkit et al., 2007). C. albicans cultures were deposited on the BiGGY agar plate separately. After 48-h incubation at 30 °C, the intensity of the brown color indicating the H2S generation was assessed. 2.4. Real-time RT-PCR
Fig. 1. Potential Met/Cys biosynthesis pathway of C. albicans. Sulfate/sulfite can be used as the sulfur source to synthesize Met and Cys, and ECM17 is speculated to catalyze the biochemical reaction from sulfite to H2S in this pathway.
RNA isolation was performed as described previously (Wang et al., 2006) and treated with DNase I (TaKaRa, Biotechnology, Dalian, PR China) to remove genomic DNA contamination. cDNA was obtained through reverse transcription reaction using a reverse transcription kit (TaKaRa). Real-time PCR was performed with SYBR Green I (TaKaRa), using LightCycler Real-Time PCR system (Roche diagnostics, GmbH Mannheim, Germany). Gene-specific primers are shown in Supplementary Table 4. The thermal cycling conditions comprised an initial step at 95 °C for 2 min, followed by
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Table 1 C. albicans strains used in this study.
a
Strain
Parent
Relevant genotypea
Reference
SN152 SN10 SN101 KEC1 KEC11 KEC21 KEC3 KEC31
SN152 SN10 SN152 KEC1 KEC1 KEC21 KEC3
leu2/leu2 his1/his1 arg4/arg4 URA3/ura3::imm434 IRO1/iro1::imm434 leu2/leu2 his1/his1 arg4//arg4 URA3/ura3::imm434 IRO1/iro1::imm434 rps10:: C.d.HIS1/RPS10 leu2/leu2 his1/his1 arg4/arg4::ARG4 URA3/ura3::imm434 IRO1/iro1::imm434 rps10:: C.d.HIS1/RPS10 leu2/leu2 his1/his1 arg4/arg4 URA3/ura3::imm434 IRO1/iro1 ::imm434 ECM17/ecm17::C.d HIS1 leu2/leu2 his1/his1 arg4/arg4::ARG4 URA3/ura3::imm434 IRO1/iro1 ::imm434 ECM17/ecm17::C.d HIS1 leu2/leu2 his1/his1 arg4/arg4 URA3/ura3::imm434 IRO1/iro1::imm434 ecm17::C.d.HIS1/ecm17::C.d.ARG4 leu2/leu2 his1/his1 arg4/arg4 URA3/ura3::imm434 IRO1/iro1::imm434 ecm17::C.d.ARG4/ecm17:: ECM17 leu2/leu2 his1/his1 arg4/arg4 URA3/ura3::imm434 IRO1/iro1::imm434 rps10:: C.d.HIS1/RPS10 ecm17::C.d.ARG4/ecm17:: ECM17
Noble and Johnson (2005) This study This study This study This study This study This study This study
C. d., C. dubliniensis; C. m., C. maltosa.
40 cycles at 95 °C for 10 s, 60 °C for 20 s, and 72 °C for 30 s. Change in fluorescence of SYBR Green I in every cycle was monitored, and the threshold cycle (CT) was measured. 18S rRNA was used as an internal control. Relative fold change of the gene expression level for the mutants, compared to SN101, was calculated using the formula 2 DDCT (Xu et al., 2009). 2.5. In vitro biofilm formation assay Standardized C. albicans cells (1.0 106 cells/ml in SC medium) were introduced into the wells of 96-well tissue culture plates (Corning Inc., Corning, NY) and incubated at 37 °C. After the initial 2-h adhesion, the medium was aspirated and non-adherent cells were removed. Fresh SC medium was then added to adherent cells. The plates were incubated for a further 48 h at 37 °C (Cao et al., 2008). A semiquantitative measure of biofilm formation was calculated by using an XTT [2,3-bis (2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide]-reduction assay (Klotz et al., 1985). Briefly, adherent cells were washed twice with PBS and then incubated with 0.5 mg/ml XTT and 10 lM menadione in PBS at 37 °C for 90 min. Optical density at 490 nm (OD490) was determined using a microtiter plate reader. 2.6. In vitro adhesion assay The adhesion of C. albicans was measured by cell-silicon sheet adhesion assay as described previously (Nobile et al., 2006) with minor modifications. Briefly, strains were grown overnight in SC medium with different concentrations of Met and/or Cys at 30 °C, diluted to an OD600 of 0.2, and added to a sterile 12-well plate with a prepared silicone square (1.5 1.5 cm cut from Cardiovascular Instrument silicone sheets [PR72034-06N, Bentec Medical Inc., United States]). The silicon square was pre-treated with bovine serum (Sigma–Aldrich) overnight and washed with PBS. The inoculated plate was incubated with gentle agitation (150 rpm) for 90 min at 37 °C for adhesion to occur. The squares were washed with 2 ml PBS to remove un-adhered cells, and then moved to a fresh 12-well plate containing 2 ml fresh SC medium. The plate was incubated at 37 °C for 48 h at 75 rpm agitation to allow biofilm formation. After incubation, the plate was agitated at 200 rpm for additional 5 min for detection of adhesion strength of the biofilms. Silicon squares with biofilms were photographed.
of 1.2 ml suspension from each group was pipetted into a clean glass tube and overlaid with 0.3 ml octane. The mixture was vortexed for 3 min for phase separation. Soon after the two phases were separated, OD600 of the aqueous phase was determined. OD600 for the group without the octane overlay in YPD medium was used as the negative control. Three repeats were performed for each group. The relative hydrophobicity was obtained as [(OD600 of the control minus OD600 after octane overlay)/OD600 of the control] 100%. 2.8. Determination of cAMP level Determination of intracellular cAMP level was performed as described previously (Miwa et al., 2004). Briefly, fungal cells were routinely grown overnight, washed with PBS and then transferred to SC medium (with different concentrations of Met and Cys). With an initial OD600 of 0.1, the cells were incubated at 37 °C for 6 h and then collected. After one wash with water and one wash with MES buffer (10 mM MES [morpholineethanesulfonic acid] containing 0.1 mM EDTA; pH 6), cells were re-suspended with MES buffer to an OD600 of 8 and 500 ll aliquots were taken from the suspension. Samples were transferred to 1.5-ml microcentrifuge tubes containing 0.5 g glass beads and 500 ll 10% trichloroacetic acid, briefly vortexed, and frozen immediately in liquid nitrogen. The samples were thawed on ice and sonicated under chilled conditions (twice at 130 W for 2.5 min). After centrifugation, trichloroacetic acid was extracted four times with water-saturated ether. The cAMP content was measured using the cAMP Enzyme Immunoassay Kit (Sigma– Aldrich) according to the manufacturer’s instructions. 3. Results 3.1. ecm17D/D is defective to produce H2S The role of ECM17 in H2S biosynthesis was first investigated using a BIGGY-agar (Cordente et al., 2009), with brown color of the colony indicating the generation of H2S. SN101 (ECM17/ ECM17) presented a dark brown color on BiGGY agar plate, indicating abundant H2S production, while the ecm17D/D mutant was white, indicating deficient H2S production (Fig. 2). The reintegration of ECM17 restored the wild-type phenotype on BiGGY agar plate.
2.7. Cellular surface hydrophobicity assay
3.2. ecm17D/D is defective to synthesize Met and Cys
Cellular surface hydrophobicity of C. albicans was measured by water–hydrocarbon two-phase assay as described previously (Klotz et al., 1985). Briefly, C. albicans strains were cultured as described above in biofilm formation assay, and the cells of the biofilms were removed from the flask surfaces with a sterile scraper to prepare a cell suspension (OD600 = 1.0 in YPD medium). A total
To investigate the role of ECM17 in Met and Cys biosynthesis, we observed the growth of the ecm17D/D mutant on solid SC medium lacking Met/Cys. Spot assay results indicated that the ecm17D/ D mutant could grow on solid SC medium lacking Met or Cys, but it was defective to grow when both Met and Cys were lacking (SC– Met–Cys medium). In contrast, the wild-type strain SN101 and
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Fig. 2. BiGGY plate assay to test the generation of H2S from fungal cells. H2S can reduce the bismuth ammonium citrate in BiGGY medium and result in brown color of fungal cells. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 3. Growth comparison of C. albicans strains. Overnight-cultured strains were suspended in PBS to an OD600 of 0.5, and 5 ll of each suspension was deposited on various auxotrophic SC plates with sufficient or insufficient Met/Cys supply. Growth difference was detected after 48-h incubation at 30 °C.
the reintegrated strain KEC31 could grow normally on solid SC– Met–Cys medium (Fig. 3). These results indicated that ECM17 was a key gene in Met and Cys biosynthesis. As the basic function of ECM17 is to catalyze the biochemical reaction for H2S generation, it is assumed that defective growth of ecm17D/D on solid SC–Cys–Met medium is caused by insufficient H2S generation. To verify this assumption, we tested the influence of H2S donor NaSH on the growth of ecm17D/D. The growth curves indicated that NaSH improved the growth of ecm17D/D in SC–Cys–Met liquid medium in a dose-dependent manner (Fig. 4). In accordance with the spot assay results shown above, ecm17D/D could not grow in SC–Cys–Met liquid medium (Fig. 4). Addition of 8 lg/ml of NaSH improved its growth remarkably, with the cell density of ecm17D/D reached 3 107 cells/ml after 48-h culture (Fig. 4). Addition of 16 lg/ml NaSH further improved its growth, and after 48-h culture, the cell density of ecm17D/D reached 4.5 107 cells/ml, which was the same as the wild-type strain (Fig. 4). We further examined the impact of Met and Cys supply on ECM17 expression. Real-time RT-PCR indicated that the expression of ECM17 with 20 lg/ml Cys was less than 50% of that without Met/ Cys, the relative fold change being 0.41. The expression of ECM17 with 80 lg/ml Cys was about 1/6 of that without Met/Cys, the relative fold change being 0.16. Consistently, the expression of ECM17 with 40 lg/ml Met was about 2/3 of that without Met/Cys, the relative fold change being 0.62. The expression of ECM17 with 160 lg/ ml Cys was about 1/9 of that without Met/Cys, the relative fold change being 0.11 (Fig. 5). These results indicated that the Met
Fig. 4. Influence of NaSH supply on the growth of ecm17D/D mutant. Strains were cultured in SC–Met–Cys auxotrophic medium with various concentrations of NaSH. No NaSH was added in the control group. With an initial OD600 of 0.001, fungi were cultured at 30 °C under constant shaking (200 rpm) and OD600 was determined at the designated time points after culture. Three independent experiments were performed.
Fig. 5. Real-time RT-PCR analysis of ECM17 in C. albicans strain SN101. Gene expression is indicated as a fold change relative to that of the control group, i.e., SN101 grown in medium without Met and Cys. The data are shown as mean ± SD from three independent experiments.
and Cys supply could repress the expression of ECM17 in a dosedependent manner. 3.3. ecm17D/D is defective to form biofilms As mentioned in Section 1, ECM17 was assumed to play a role in biofilm formation, so we further investigated the impact of ECM17 disruption on biofilm formation. Biofilm formation was evaluated by XTT reduction assay using OD490 value to indicate biofilm formation. Our data indicated that although 10 lg/ml Met or 20 lg/ ml Cys was sufficient for planktonic growth of ecm17D/D (Fig. 6),
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Fig. 6. Planktonic growth of C. albicans strains in SC–Met–Cys auxotrophic medium with different concentrations of Met/Cys. With 1.5 104 cells/ml as the initial density, fungi were cultured at 30 °C under constant shaking (200 rpm) and C. albicans cells were counted using a hemocytometer at the designated time points. Three independent experiments were performed.
ecm17D/D showed significant diminishment of biofilm formation even with 20 lg/ml Met and 40 lg/ml Cys (p < 0.05, Fig. 7). With decreased Met/Cys supply, the impact of ECM17 on biofilm formation was enlarged. With only 10 lg/ml Met supply and without Cys, biofilm formed by ecm17D/D was about 50% of that by SN101, and with only 20 lg/ml Cys supply and without Met, biofilm formed by ecm17D/D was about 1/5 of that by SN101. The reintegration of ECM17 restored the ability KEC31 to form biofilm (Fig. 7). These results indicated that ECM17 played an important role in biofilm formation, especially under conditions lacking Met/Cys supply. We further investigated the association between Met/Cys biosynthesis pathway and biofilm formation. The expression of MET4 was examined to indicate the status of Met/Cys biosynthesis pathway because MET4 encodes an important activator of this pathway (Lafaye et al., 2005). Interestingly during biofilm formation, MET4 was notably up-regulated in the ecm17D/D mutant compared with the wild-type SN101 with Met/Cys supply insufficiency (Fig. 8). More specifically, MET4 was up-regulated 13-fold with only 10 lg/ml Met supply and without Cys, and up-regulated 54-fold with only 20 lg/ml Cys supply and without Met (Fig. 8). These results confirmed the association between ECM17 and Met/
Fig. 8. Real-time RT-PCR analysis of MET4 in C. albicans. Strains were cultured in SC medium with different concentrations of Met/Cys. Gene expression is indicated as a fold change relative to SN101. (A) Planktonic cells obtained after overnight culture with constant shaking were collected and RNA was isolated. (B) Biofilm cells obtained after overnight incubation without shaking were collected and RNA was isolated. The data are shown as mean ± SD from three independent experiments.
Cys biosynthesis pathway and implied that more Met/Cys were needed in the ecm17D/D mutant compared with the wild-type cells. Of note, the significant up-regulation of MET4 in the ecm17D/D mutant was only observed during biofilm formation, but not in planktonic cells (Fig. 8), which indicated the association between Met/Cys biosynthesis pathway and biofilm formation. 3.4. ecm17D/D is defective to adhere and grow filamentously
Fig. 7. In vitro biofilm formation assay of C. albicans strains. Strains were cultured in media with different concentrations of Met/Cys. After 48-h incubation, the biofilm biomass was quantitated using XTT reduction assay. The data are shown as mean ± SD from three independent experiments. indicates P < 0.05 and indicates P < 0.01, compared with SN101.
Knowing that adhesion and filamentous growth are particularly important for biofilm formation, we investigated the role of ECM17 in adhesion and filamentous growth. The results indicated that the ecm17D/D mutant failed to adhere to silicon squares in the absence of sufficient Met/Cys supply in the medium (Fig. 9). After 48-h incubation for biofilm formation, vigorous agitation of 200 rpm ruined the ecm17D/D biofilms formed in the medium with only 20 lg/ml Cys or 10 lg/ml Met (Fig. 9), while the vigorous agitation did not affect the biofilms of wild-type strain SN101 or reintegrated strain KEC31 (Fig. 9), indicating a less adhesive strength of the ecm17D/D biofilms. Knowing that there is a positive correlation between cellular surface hydrophobicity (CSH) and adhesion (Luo and Samaranayake, 2002; Pompilio et al., 2008; Samaranayake et al., 1995), we further investigated the impact of ECM17 disruption on CSH. In SC medium with 40 lg/ml Cys and 20 lg/ml Met, the CSH of ecm17D/D decreased significantly compared with that of the wild-type strain SN101 (p < 0.05; Fig. 10), and the reintegration of ECM17 restored CSH of KEC31. Insufficient Cys/Met supply en-
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Fig. 9. In vitro adhesion assay of C. albicans strains. Strains were cultured in media with different concentrations of Met/Cys. After 48-h incubation for biofilm formation, plates with silicon squares were agitated at 200 rpm for 5 min to detect the adhesion strength of the biofilms. The silicon squares with biofilms were photographed.
larged the difference between ecm17D/D and SN101, which is consistent with the result of adhesion assay (Fig. 10). In SC medium with only 10 lg/ml Met without Cys, the CSH of ecm17D/D was only 1/4 of that of SN101, while in SC medium with only 20 lg/ ml Cys without Met, the ratio was 1/3 (Fig. 10). The reintegration of ECM17 improved the CSH of C. albicans, and there was no significant difference between KEC31 and SN101 (p > 0.05, Fig. 10). We further investigated the impact of ECM17 disruption on filamentous growth, and the results showed that the ecm17D/D mutant was defective in hyphal formation on SLD agar plates with insufficient Met/Cys supply (Fig. 11). Wild-type SN101 formed filaments on SLD plates lacking Met/Cys (10 lg/ml Met without Cys or 20 lg/ml Cys without Met) as well as with sufficient Met/ Cys supply (40 lg/ml Cys and 20 lg/ml Met) (Fig. 11), while the ecm17D/D mutant strain was defective to form filaments with Met/Cys supply insufficiency. More specifically, SN101 formed colonies with radial hyphae on the Met-lacking SLD plate, while ecm17D/D formed smooth colonies (Fig. 11C and F). The integra-
tion of ECM17 in KEC31 improved the filamentous growth of C. albicans (Fig. 11H and I). We also investigated the filamentous growth of the C. albicans strain in some other hypha-inducing media, including Spider, lee and serum media (solid and liquid), but no significant difference was observed between the ecm17D/D strain and SN101 (data not shown), indicating that the filamentation defect of ecm17D/D was condition-dependent. 3.5. ecm17D/D is defective to express some adhesion/filamentation genes To understand the mechanism of Ecm17-promoted adherence and hyphal formation, we further compared the expression of the known adhesion-specific or hypha-specific genes in strains SN101 and ecm17D/D (Fig. 12). The results showed no significant difference in the expression of most adhesion-specific or hyphaspecific genes (ALS1, ALS3, HWP1, HWP2, CSH1, IFF4, EAP1, ECE1, SAP4, SAP5, SAP6, UME6, HGC1 and EED1) between ecm17D/D and SN101 (>0.5 and <2 of relative fold) (Fig. 12). Interestingly, the expression of ALS3, CSH1, HWP1 and ECE1 was significantly down-regulated in the ecm17D/D mutant compared with the wild-type strain SN101 under conditions lacking Met or Cys. The relative fold was 0.33, 0.21, 0.05 and 0.16 respectively in medium with 20 lg/ml Cys and without Met (Figs. 12 and 13), and 0.32, 0.15, 0.12 and 0.47 respectively in medium with 10 lg/ml Met and without Cys (Figs. 12 and 13). Even with sufficient Met and Cys supply (20 lg/ml Met and 40 lg/ml Cys) in the medium, the relative fold was about 0.5 (Fig. 13). The reintegration of ECM17 restored the expression of ALS3, CSH1, HWP1 and ECE1. These results indicated that the expression of ALS3, CSH1, HWP1 and ECE1 was affected by ECM17 disruption significantly. 3.6. cAMP–PKA pathway is associated with ECM17 and Met/Cys biosynthesis pathway
Fig. 10. Cell surface hydrophobicity (CSH) assay of C. albicans strains. Strains were cultured in media with different concentrations of Met/Cys for 48-h. Cellular surface hydrophobicity of C. albicans was measured by water–hydrocarbon twophase assay The data are shown as mean ± SD from three independent experiments. indicates P < 0.05 and indicates P < 0.01, compared with SN101.
Since ALS3, HWP1 and ECE1 are known genes regulated by cAMP–PKA pathway, we further investigated the impact of ECM17 disruption on intracellular cAMP level. As was expected,
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Fig. 11. Filamentous growth of C. albicans strains on SLD agar plates. (A–I): Strains were grown on plates with different concentrations of Met/Cys. (a–i): Strains were grown on various plates with addition of exogenous cAMP. The growth was monitored after 8-day incubation at 30 °C.
the intracellular cAMP level, and no significant difference was observed between KEC31 and SN101. These results suggested that ECM17 and Met/Cys may regulate the expression of adhesion-specific and hypha-specific genes including HWP1, ALS3 and ECE1 by affecting the intracellular cAMP level. Subsequently, we tested the influence of exogenous cAMP supply on the filamentous growth of ecm17D/D. As was expected, the filamentous growth ability of ecm17D/D was recovered with the addition of 5 mM exogenous cAMP on SLD plates lacking Met or Cys, and there was no obvious difference between the wild-type strain and the ecm17D/D strain (Fig. 11e and f). We also tested the influence of exogenous cAMP supply on biofilm formation and adhesion of ecm17D/D, but no obvious effect was observed (Figs. 15 and 16). These results indicated that cAMP–PKA pathway was associated with ECM17 and Met/Cys biosynthesis pathway, especially for filamentous growth.
4. Discussion
Fig. 12. Real-time RT-PCR analysis of 14 genes involved in adhesion and/or filamentous growth. Gene expression is indicated as a fold change relative to SN101. The data are shown as mean ± SD from three independent experiments. (A) SC medium with 20 lg/ml Cys without Met; (B) SC medium with 10 lg/ml Met without Cys.
intracellular cAMP level was decreased significantly in the ecm17D/D mutant under all conditions tested (p < 0.05, Fig. 14). Interestingly, intracellular cAMP level of ecm17D/D decreased significantly even with 40 lg/ml Cys and 20 lg/ml Met as nutrition supply, and it was about 3/5 of that by SN101 (p < 0.05, Fig. 14). With Met and Cys supply decreasing, the impact of ECM17 on intracellular cAMP level was enlarged. With only 20 lg/ml Cys without Met, or with only 10 lg/ml of Met without Cys in the medium, the intracellular cAMP level of ecm17D/D was about 1/2 of that by SN101 (p < 0.01, Fig. 14), indicating a positive correlation between the ECM17-dependent Met/Cys biosynthesis pathway and the intracellular cAMP level. The reintegration of ECM17 increased
In this study, we investigated the role of ECM17 in Met/Cys biosynthesis and the contribution of Met/Cys to biofilm formation. It is the first report showing that the C. albicans ecm17D/D mutant was unable to catalyze the biochemical reaction from sulfite to H2S, and hardly grew in medium lacking Met and Cys. With the supply of H2S donor, the growth of ecm17D/D was improved in the Met/Cys-lacking medium. Meanwhile, the expression of ECM17 was inhibited with the addition of Met/Cys. These results confirmed the important role of ECM17 in Met and Cys biosynthesis. Further results also validated the important role of ECM17 in biofilm formation. Biofilm formation was attenuated by ecm17D/ D mutant compared with the wild-type or reintegrated strains under the same incubation conditions. In addition, adhesion and filamentous growth of ecm17D/D declined, especially under conditions lacking Met or Cys. Further results indicated that the defective biofilm formation of ecm17D/D was possibly related to the down-regulation of ALS3, CSH1, HWP1 and ECE1, four important genes in adherence and filamentous growth. It is known that HWP1, ALS3 and ECE1 are regulated by cAMP–PKA pathway, and our further results indicated that disruption of ECM17 decreased cAMP level under all conditions in this study. In addition, the filamentous growth of ecm17D/D on SLD lacking Met or Cys was
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Fig. 14. Determination of intracellular cAMP level. Strains were cultured with the supply of different concentrations of Met/Cys. The data are shown as mean ± SD from three independent experiments. indicates P < 0.05 and indicates P < 0.01, compared with SN101.
Fig. 15. Influence of exogenous cAMP on biofilm formation of ecm17D/D. Strains were cultured in SC medium with different concentrations of Met/Cys and different concentrations of exogenous cAMP added. After 48-h incubation, the biofilm biomass was quantitated using XTT reduction assay. The data are shown as mean ± SD from three independent experiments. indicates P < 0.01, compared with SN101.
Fig. 13. Real-time RT-PCR analysis of ALS3, CSH1, HWP1 and ECE1 in C. albicans strains. Gene expression is indicated as a fold change relative to SN101. The data are shown as mean ± SD from three independent experiments.
improved markedly with the addition of exogenous cAMP, indicating that cAMP–PKA pathway is associated with ECM17 and Met/ Cys biosynthesis pathway, especially for filamentous growth. To the best of our knowledge, this is the first report focusing the role of ECM17 in C. albicans, and our findings indicate that ECM17 plays a critical role in Met/Cys biosynthesis by controlling the synthesis of H2S. Based on the results of bioinformatics analysis (Fig. 1), it could be inferred that the disruption of ECM17 would unfavorably affect the ability of C. albicans to make use of sulfate/sulfite as the sulfur source to synthesize H2S and further inhi-
bit the biosynthesis of Met and Cys. This is in accordance with the following findings in the present study: (i) The ecm17D/D mutant was unable to produce H2S normally as the wild-type strain (Fig. 2). (ii) The ecm17D/D mutant rarely grew in medium lacking Met and Cys (Fig. 3). (iii) A H2S donor NaSH could improve the growth of ecm17D/D dose-dependently (Fig. 4). Furthermore, addition of Met/Cys inhibited the expression of ECM17 dose-dependently (Fig. 5). This seems to be a Met/Cys feedback regulating mechanism on ECM17 expression. This work demonstrated the important role of ECM17-dependent Met/Cys biosynthesis in biofilm formation. As was expected, the ability of the ecm17D/D mutant to form biofilms declined (Fig. 7). More interestingly, this decline was enlarged under conditions with insufficient Met/Cys supply, suggesting that ECM17 is involved in biofilm formation through regulating the biosynthesis of Met and Cys (Fig. 7). We also noticed that the Met and Cys concentrations (10 lg/ml Met; 20 lg/ml Cys) used in this study were sufficient for planktonic growth of all the strains, including ecm17D/D mutant (Fig. 6). Thus the growth defect of ecm17D/D mutant in developing biofilm suggests that more Met and Cys
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Fig. 16. Influence of exogenous cAMP on adhesion of C. albicans. Strains were cultured in SC medium with different concentrations of Met/Cys and different concentrations of exogenous cAMP added. After 48-h incubation for biofilm formation, plates with silicon squares were agitated at 200 rpm for 5 min to detect the adhesion strength of the biofilms. The silicon squares with biofilms were photographed.
are needed in the biofilm growth process as compared with planktonic growth. In accordance, the significant up-regulation of MET4 in ecm17D/D was only observed during biofilm formation, but not in planktonic cells. These results are consistent with the microarray finding that the Met/Cys biosynthesis genes were up-regulated from the early adhesion stage and this up-regulation state could be maintained for hours during biofilm formation (Murillo et al., 2005). Filamentous growth defect of ecm17D/D was observed on solid SLD medium but not other hypha-inducing media tested, which may be due to the rich organic sulfur in Spider, serum and Lee media. More specifically, there are 100 lg/ml methionine in Lee medium (Lee et al., 1975). Although the exact concentrations of methionine and cysteine in Spider and serum media are unknown, it is known that there are 1% nutrient broth in Spider medium and 1% yeast extract in serum medium (Maidan et al., 2005), and both nutrient broth and yeast extract are rich of organic sulfur (Blaszczyk, 1993). Besides, filamentous growth defect of ecm17D/D was only observed on solid SLD but not liquid medium, which indicates that the mechanisms of filamentation are different in liquid and solid media. Some previous findings (Lorenz et al., 2000) have provided a clue: A putative G-protein-coupled receptor Gpr1 regulates morphogenesis and hyphal formation in C. albicans, and the Cagpr1D/D mutant is defective in yeast-to-hypha transition only on solid medium but not in liquid medium, which is similar to our findings in this study. Interestingly, Gpr1 was reported to be involved in the nutrient sensing, and therefore we hypothesize that Cys and Met may regulate hypha formation through CaGPR1. To explore the underlying mechanism contributing to adhesion decline and filamentous growth defects of ecm17D/D, we investigated the expression of some most important adhesion and hypha-specific genes including the ALS family genes (ALS1 and ALS3), HWP1, HWP2, CSH1, IFF4, EAP1, ECE1, SAP4, SAP5, SAP6, UME6, HGC1 and EED1 (Chaffin, 2008; Sudbery, 2011) (Fig. 12). Interestingly, we found that ALS3, CSH1, HWP1 and ECE1 were associated with ECM17 (Fig. 13). ALS3, an ALS family gene, plays an essential role in the adherence stage of C. albicans to human umbilical vein endothelial cells and buccal epithelial cells (Zhao et al., 2006, 2004). Csh1p, encoded by CSH1, is a hydrophobic surface protein mediating binding to host target proteins. Disruption of
CSH1 decreased cell surface hydrophobicity and adhesion between C. albicans and fibronectin (Singleton and Hazen, 2004; Singleton et al., 2001). HWP1 is a unique adhesion gene expressing on the hyphal surface. Biofilms lacking HWP1 gene were prone to detach from the abiotic substrate (Chaffin, 2008; Nobile et al., 2006). ECE1 is highly expressed in hyphae and its expression correlates with the extent of hyphal cell elongation (Birse et al., 1993). The down-regulation of these four genes may decrease adhesion and hyphal formation of C. albicans. Our study further provides new insights into the association between ECM17-dependent Met/Cys biosynthesis pathway and cAMP–PKA signaling pathway. As was expected, the intracellular cAMP level declined in ecm17D/D mutant compared with the wild-type strain (Fig. 14). More interestingly, the phenomenon of cAMP decline in ecm17D/D mutant was obviously enlarged under conditions with insufficient Met/Cys supply. This result is consistent with the previous finding that Met could activate the cAMP– PKA signaling pathway in C. albicans cells (Maidan et al., 2005). Noticeably, our results indicated that as was the case with Met, the lack of Cys also decreased the intracellular cAMP level. This may result from the direct regulatory role of Cys on cAMP–PKA signaling pathway, or possibly result from the biochemical conversion between Met and Cys. As shown in Fig. 1, Met can be converted to Cys, and Cys can be converted to Met. Under conditions without Cys, some Met might be converted to Cys to meet the requirement of cells, thus decreasing the concentration of Met and then the intracellular cAMP level. Collectively, our findings indicate that Met/Cys may act as signal molecules to regulate intracellular cAMP level, and Met/Cys biosynthesis pathway is associated with cAMP– PKA pathway in C. albicans. In addition to the cAMP–PKA pathway, some other mechanisms may also be involved in the defect of ecm17D/D to form biofilms. We notice that although the addition of exogenous cAMP can improve the filamentous growth of ecm17D/D on solid SLD medium, cAMP cannot significantly improve the biofilm defect and adhesion decline of ecm17D/D (Fig. 15 and Fig. 16). The other possible mechanisms include: (i) Down-regulation of CSH1 may play a critical role in ecm17D/D. No documentary evidence has shown the association between CSH1 and cAMP–PKA pathway. Our unpublished data indicate that cAMP could not regulate the expression of
D.-D. Li et al. / Fungal Genetics and Biology 51 (2013) 50–59
CSH1, and exogenous addition of cAMP could not up-regulate its expression. CSH1 may be involved in biofilm formation independently with cAMP–PKA pathway. (ii) As Met and Cys are involved in the formation of disulfide bonds, and the cysteine residues are important to maintain the functions of many proteins (Frade-Perez et al., 2010; Perez et al., 2006; Popolo et al., 2008; Tirelli et al., 2010), we speculate that plenty of proteins are affected by the lacking of Met and Cys, and nutrition shortage, besides the adjustment of signal pathways, may also contribute to the biofilm defect of ecm17D/D. Collectively, we demonstrated that ECM17 regulated the biosynthesis of Met and Cys by controlling the generation of H2S, and ECM17-dependent Met/Cys biosynthesis played an important role in biofilm formation through affecting adhesion and filamentous growth. This work provides new insights into the role of Met/Cys as signal molecules and is helpful to better understand the associations between Met/Cys biosynthesis pathway, cAMP– PKA pathway and biofilm formation. Acknowledgments This work was supported by the National Natural Science Foundation of China (81072678, 30825041 and 90913008); the National Science and Technology Major Project of the Ministry of Science and Technology of China (2011ZX09102-002-01); Shanghai Educational Development Foundation (2007CG51); and Shanghai Science and Technology Major Project (10431902200). We would like to thank Dr. Alexander D. Johnson (Department of Microbiology and Immunology, University of California–San Francisco, California) for providing C. albicans strain SN152, plasmid pSN40 and plasmid pSN69. We also thank Dr. Joachim Morschhauser (Institute fur Molekulare Infektionsbiologie, University of Wurzburg, Germany) for kindly providing plasmid pSFS2A. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.fgb.2012.11.010. References Birse, C.E. et al., 1993. Cloning and characterization of ECE1, a gene expressed in association with cell elongation of the dimorphic pathogen Candida albicans. Infection and Immunity 61, 3648–3655. Blaszczyk, M., 1993. Effect of medium composition on the denitrification of nitrate by Paracoccus denitrificans. Applied and Environment Microbiology 59, 3951– 3953. Cao, Y. et al., 2008. In vitro activity of baicalein against Candida albicans biofilms. International Journal of Antimicrobial Agents 32, 73–77. Chaffin, W.L., 2008. Candida albicans cell wall proteins. Microbiology and Molecular Biology Reviews 72, 495–544. Cordente, A.G. et al., 2009. Isolation of sulfite reductase variants of a commercial wine yeast with significantly reduced hydrogen sulfide production. FEMS Yeast Research 9, 446–459. Falagas, M.E. et al., 2010. Relative frequency of albicans and the various nonalbicans Candida spp. among candidemia isolates from inpatients in various parts of the world: a systematic review. International Journal of Infectious Diseases 14, e954–e966. Frade-Perez, M.D. et al., 2010. Biochemical characterization of Candida albicans alpha-glucosidase I heterologously expressed in Escherichia coli. Antonie van Leeuwenhoek 98, 291–298. Garcia-Sanchez, S. et al., 2004. Candida albicans biofilms: a developmental state associated with specific and stable gene expression patterns. Eukaryotic Cell 3, 536–545. Hogan, D.A., Sundstrom, P., 2009. The Ras/cAMP/PKA signaling pathway and virulence in Candida albicans. Future Microbiology 4, 1263–1270. Ilkit, M. et al., 2007. Evaluation of albicans ID2 and biggy agar for the isolation and direct identification of vaginal yeast isolates. Journal of Medical Microbiology 56, 762–765.
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The G protein-coupled receptor gpr1 is a nutrient sensor that regulates pseudohyphal differentiation in Saccharomyces cerevisiae. Genetics 154, 609–622. Luo, G., Samaranayake, L.P., 2002. Candida glabrata, an emerging fungal pathogen, exhibits superior relative cell surface hydrophobicity and adhesion to denture acrylic surfaces compared with Candida albicans. APMIS 110, 601–610. Maidan, M.M. et al., 2005. The G protein-coupled receptor Gpr1 and the Galpha protein Gpa2 act through the cAMP–protein kinase A pathway to induce morphogenesis in Candida albicans. Molecular Biology of the Cell 16, 1971– 1986. Miwa, T. et al., 2004. Gpr1, a putative G-protein-coupled receptor, regulates morphogenesis and hypha formation in the pathogenic fungus Candida albicans. Eukaryotic Cell 3, 919–931. Murillo, L.A. et al., 2005. Genome-wide transcription profiling of the early phase of biofilm formation by Candida albicans. Eukaryotic Cell 4, 1562–1573. Nobile, C.J. et al., 2006. Critical role of Bcr1-dependent adhesins in C. albicans biofilm formation in vitro and in vivo. PLoS Pathogens 2, e63. Nobile, C.J., Mitchell, A.P., 2006. Genetics and genomics of Candida albicans biofilm formation. Cellular Microbiology 8, 1382–1391. Noble, S.M., Johnson, A.D., 2005. Strains and strategies for large-scale gene deletion studies of the diploid human fungal pathogen Candida albicans. Eukaryotic Cell 4, 298–309. Perez, A. et al., 2006. Biofilm formation by Candida albicans mutants for genes coding fungal proteins exhibiting the eight-cysteine-containing CFEM domain. FEMS Yeast Research 6, 1074–1084. Pompilio, A. et al., 2008. Factors associated with adherence to and biofilm formation on polystyrene by Stenotrophomonas maltophilia: the role of cell surface hydrophobicity and motility. FEMS Microbiology Letters 287, 41–47. Popolo, L. et al., 2008. Disulfide bond structure and domain organization of yeast beta(1, 3)-glucanosyltransferases involved in cell wall biogenesis. 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ECM17-dependent methionine/cysteine biosynthesis contributes to biofilm formation in Candida albicans De-Dong Li, Yan Wang ⇑, Bao-Di Dai, Xing-Xing Li, Lan-Xue Zhao, Yong-Bing Cao, Lan Yan, Yuan-Ying Jiang ⇑ School of Pharmacy, Second Military Medical University, Shanghai 200433, China
a r t i c l e
i n f o
Article history: Received 1 August 2012 Accepted 28 November 2012 Available online 12 December 2012 Keywords: Candida albicans ECM17 Biofilm Cysteine Methionine
a b s t r a c t Candida albicans has become the fourth leading pathogen of nosocomial bloodstream infections largely due to biofilm formation on implanted medical devices. Previous microarray data indicated that almost all genes in methionine (Met)/cysteine (Cys) biosynthesis pathway were up-regulated during biofilm formation, especially during the adherence period. In this work, we studied the role of Met/Cys biosynthesis pathway by disrupting ECM17, a gene encoding sulfite reductase in C. albicans. It was found that the ecm17D/D mutant failed to catalyze the biochemical reaction from sulfite to H2S and hardly grew in media lacking Met and Cys. NaSH, the donor of H2S, dose-dependently improved the growth of ecm17D/D in media lacking a sulfur source. Sufficient Met/Cys supply inhibited the expression of ECM17 in a dose-dependent manner. These results validated the important role of ECM17 in Met/Cys biosynthesis. Interestingly, the ecm17D/D mutant showed diminished ability to form biofilm, attenuated adhesion on abiotic substrate and decreased filamentation on solid SLD medium, especially under conditions lacking Met/Cys. Further results indicated that ECM17 affected the expressions of ALS3, CSH1, HWP1 and ECE1, and that the cAMP–protein kinase A (PKA) pathway was associated with ECM17 and Met/Cys biosynthesis pathway. These results provide new insights into the role of Met/Cys biosynthesis pathway in regulating cAMP-PKA pathway and benefiting biofilm formation. Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction Candida albicans is the most common opportunistic fungal pathogen of humans, causing diseases varying from superficial mucosal infections to life-threatening systemic diseases (Karkowska-Kuleta et al., 2009). It has become the dominant pathogen in patients with candidemia, especially in Europe and USA (Falagas et al., 2010), largely due to the biofilm formation on implanted medical devices. There are three stages for biofilm formation: (i) attachment and adhesion to biomaterial surfaces, (ii) growth and proliferation of cells to form an anchoring layer, and (iii) growth of pseudohyphae, hyphae and extracellular matrix material to form a complex threedimensional structure (Nobile and Mitchell, 2006; Ramage et al., 2005; Seneviratne et al., 2008). Adhesion and filamentous growth are particularly important for biofilm formation (Ramage et al., 2009). More specifically, adhesion is mediated by both non-specific factors (including cell surface hydrophobicity) and specific
⇑ Corresponding authors. Address: School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433, China. Fax: +86 21 65490641. E-mail addresses: [email protected] (Y. Wang), [email protected] (Y.-Y. Jiang). 1087-1845/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.fgb.2012.11.010
adhesion genes (HWP1, HWP2, ALS1, ALS3, IFF4, EAP1 and CSH1) (Chaffin, 2008), and filamentous growth is mediated by some hypha-specific genes (HWP1, ECE1, SAP4, SAP5, SAP6, UME6, HGC1, EED1) (Sudbery, 2011). Among these adhesion-specific or hyphaspecific genes, ALS3, HWP1 and ECE1 are proven to be regulated by cAMP–PKA pathway (Hogan and Sundstrom, 2009). The mechanism of C. albicans biofilm formation is complex. Some groups of genes are co-regulated during the formation of biofilm (Seneviratne et al., 2008), but the underlying mechanisms remain unclear (Nobile and Mitchell, 2006). Genome-wide transcription profiles have provided plenty of valuable information for the better understanding about the mechanisms of biofilm formation (Garcia-Sanchez et al., 2004; Nobile and Mitchell, 2006). Notably, a group of methionine (Met)/cysteine (Cys) biosynthesis genes are up-regulated from the early adhesion stage and maintain for hours (Garcia-Sanchez et al., 2004; Murillo et al., 2005). Moreover, Met and Cys are sulfur-containing amino acids, and most sulfur assimilation genes are up-regulated simultaneously with the Met/Cys biosynthesis genes (Murillo et al., 2005). These data imply that Met/Cys biosynthesis may play an important role in biofilm formation. However, no study has focused on the relationship between Met/Cys biosynthesis pathway and biofilm formation.
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The predicted Met/Cys biosynthesis pathway of C. albicans is shown in Fig. 1. A search of this pathway in C. albicans genome database (http://www.candidagenome.org/ and http://genolist.pasteur.fr/CandidaDB/) reveals that ECM17 (also known as orf19.4099) encodes a putative enzyme that functions in sulfur amino acid biosynthesis. The ECM17 ortholog in Saccharomyces cerevisiae, known as MET5 (available at http://www.yeastgenome.org/), encodes the sulfite reductase beta subunit, which catalyzes the biochemical reaction from sulfite to H2S, a key step in Met and Cys biosynthesis (Thomas and Surdin-Kerjan, 1997). Our BLASTN result indicated that C. albicans ECM17 and S. cerevisiae MET5 have a 64% similarity of nucleotide sequence, and BLASTP result indicated that they have 52% similarity of amino acid sequence. These results imply that C. albicans ECM17 might play a role in Met and Cys biosynthesis. In this work, we developed an ecm17D/D mutant and our further investigations revealed that ECM17-dependent Met/Cys biosynthesis played a critical role in C. albicans biofilm formation. 2. Materials and methods 2.1. Strains and growth conditions C. albicans strains used in this study are listed in Table 1. All strains were routinely grown in YPD (1% yeast extract, 2% peptone and 2% dextrose) liquid medium at 30 °C in a shaking incubator. To observe the filamentous growth of the Candida cells, hyphainducing media (Spider and Lee) were prepared as described previously (Lee et al., 1975; Liu et al., 1994). For solid serum medium, 10% (vol/vol) fetal calf serum (FCS) was added to YPD after autoclaving. Synthetic low dextrose (SLD) medium is 0.17% YNB medium containing 0.5% (NH4)2SO4, 0.1% glucose, 1.5% agar and different concentrations of Met and Cys (Maidan et al., 2005). 2.2. Disruption and reintegration of ECM17 in C. albicans ECM17 was disrupted using a well-described strategy (Supplementary Fig. 1A and B) (Noble and Johnson, 2005). Briefly, the DNA fragments for homologous recombination were obtained through fusion PCR (Noble and Johnson, 2005) and the primers used to obtain the fragments are listed in Supplementary Table 1. The HIS1-containing fusion fragment upstream-HIS1-downstream was transformed into C. albicans strain SN152 using the lithium acetate method (Kawai et al., 2010). Transformants were plated
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on selective medium (yeast nitrogen base with amino acids except histidine), and positive clone KEC1 was verified to be correct with ECM17 one allele disrupted by PCR using genomic DNA as the template (Supplementary Fig. 2). To obtain a control strain, the HIS1 marker gene was transformed to the locus of RPS10 in SN152, resulting in strain SN10. Using the same strategy, the ARG4-containing fusion fragment upstream-ARG4-downstream was transformed into KEC1 to disrupt the other allele of ECM17. Positive clone KEC21 was verified to be correct with both alleles of ECM17 disrupted by PCR using genomic DNA as the template (Supplementary Fig. 2). To further obtain the control strain, ARG4 marker gene was transformed to the locus of ARG4 in SN10, resulting in strain SN101. Primers used to identify the strains are listed in Supplementary Table 2. Reintegration of ECM17 gene was performed using the SAT1 flipper tool (Reuss et al., 2004). The DNA fragment for homologous recombination was done as follows: A KpnI–XhoI fragment with the whole ORF of ECM17, 0.63 kb upstream and 0.25 kb downstream was amplified using primers ECM17 complement 1 and 2 (Supplementary Table 1), and the fragment was cloned to the upstream of the SAT1 flipper. A SacII–SacI fragment with the ECM17 ORF downstream was amplified using primers ECM17 complements 3 and 4 (Supplementary Table 1), and the fragment was cloned to the downstream of the SAT1 flipper, yielding plasmid pSFS2A–ECM17 (Supplementary Fig. 1C). Then the linearized pSFS2A–ECM17 plasmid was transformed into the strain KEC21. The transformants were spread on YPD plates containing 200 lg/ ml nourseothricin. Nourseothricin-resistant clones were streaked on arginine or histidine-lacking agar plates. In this study, the correct clones could grow on arginine-lacking agar plates but not on histidine-lacking agar plates, indicating that the HIS1 cassette had been replaced by our transformation fragment. Subsequently, transformants were grown in YPM medium (1% yeast extract, 2% peptone and 2% maltose) for 5 h to excise the SAT1 flipper cassette. One hundred to two hundred cells were then spread on YPD plates containing 25 lg/ml nourseothricin and grown for 2 days at 30 °C. Nourseothricin-sensitive clones, known as KEC3, were identified by their small clone size and confirmed by restreaking on YPD plates containing 200 lg/ml nourseothricin as described previously (Reuss et al., 2004). Finally, using the same SAT1 flipper strategy, HIS1 cassette was restored into KEC3, yielding strain KEC31. The reintegrated strain KEC31 was identified through PCR using primers as listed in Supplementary Table 3. 2.3. Detection of H2S generation H2S generation was detected using BiGGY agar (Oxoid, England) (Ugliano et al., 2011). BiGGY (Bismuth Sulfite Glucose Glycine Yeast) is a medium containing bismuth ammonium citrate. H2S can reduce the bismuth ammonium citrate and result in a brown color (Cordente et al., 2009; Ilkit et al., 2007). C. albicans cultures were deposited on the BiGGY agar plate separately. After 48-h incubation at 30 °C, the intensity of the brown color indicating the H2S generation was assessed. 2.4. Real-time RT-PCR
Fig. 1. Potential Met/Cys biosynthesis pathway of C. albicans. Sulfate/sulfite can be used as the sulfur source to synthesize Met and Cys, and ECM17 is speculated to catalyze the biochemical reaction from sulfite to H2S in this pathway.
RNA isolation was performed as described previously (Wang et al., 2006) and treated with DNase I (TaKaRa, Biotechnology, Dalian, PR China) to remove genomic DNA contamination. cDNA was obtained through reverse transcription reaction using a reverse transcription kit (TaKaRa). Real-time PCR was performed with SYBR Green I (TaKaRa), using LightCycler Real-Time PCR system (Roche diagnostics, GmbH Mannheim, Germany). Gene-specific primers are shown in Supplementary Table 4. The thermal cycling conditions comprised an initial step at 95 °C for 2 min, followed by
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Table 1 C. albicans strains used in this study.
a
Strain
Parent
Relevant genotypea
Reference
SN152 SN10 SN101 KEC1 KEC11 KEC21 KEC3 KEC31
SN152 SN10 SN152 KEC1 KEC1 KEC21 KEC3
leu2/leu2 his1/his1 arg4/arg4 URA3/ura3::imm434 IRO1/iro1::imm434 leu2/leu2 his1/his1 arg4//arg4 URA3/ura3::imm434 IRO1/iro1::imm434 rps10:: C.d.HIS1/RPS10 leu2/leu2 his1/his1 arg4/arg4::ARG4 URA3/ura3::imm434 IRO1/iro1::imm434 rps10:: C.d.HIS1/RPS10 leu2/leu2 his1/his1 arg4/arg4 URA3/ura3::imm434 IRO1/iro1 ::imm434 ECM17/ecm17::C.d HIS1 leu2/leu2 his1/his1 arg4/arg4::ARG4 URA3/ura3::imm434 IRO1/iro1 ::imm434 ECM17/ecm17::C.d HIS1 leu2/leu2 his1/his1 arg4/arg4 URA3/ura3::imm434 IRO1/iro1::imm434 ecm17::C.d.HIS1/ecm17::C.d.ARG4 leu2/leu2 his1/his1 arg4/arg4 URA3/ura3::imm434 IRO1/iro1::imm434 ecm17::C.d.ARG4/ecm17:: ECM17 leu2/leu2 his1/his1 arg4/arg4 URA3/ura3::imm434 IRO1/iro1::imm434 rps10:: C.d.HIS1/RPS10 ecm17::C.d.ARG4/ecm17:: ECM17
Noble and Johnson (2005) This study This study This study This study This study This study This study
C. d., C. dubliniensis; C. m., C. maltosa.
40 cycles at 95 °C for 10 s, 60 °C for 20 s, and 72 °C for 30 s. Change in fluorescence of SYBR Green I in every cycle was monitored, and the threshold cycle (CT) was measured. 18S rRNA was used as an internal control. Relative fold change of the gene expression level for the mutants, compared to SN101, was calculated using the formula 2 DDCT (Xu et al., 2009). 2.5. In vitro biofilm formation assay Standardized C. albicans cells (1.0 106 cells/ml in SC medium) were introduced into the wells of 96-well tissue culture plates (Corning Inc., Corning, NY) and incubated at 37 °C. After the initial 2-h adhesion, the medium was aspirated and non-adherent cells were removed. Fresh SC medium was then added to adherent cells. The plates were incubated for a further 48 h at 37 °C (Cao et al., 2008). A semiquantitative measure of biofilm formation was calculated by using an XTT [2,3-bis (2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide]-reduction assay (Klotz et al., 1985). Briefly, adherent cells were washed twice with PBS and then incubated with 0.5 mg/ml XTT and 10 lM menadione in PBS at 37 °C for 90 min. Optical density at 490 nm (OD490) was determined using a microtiter plate reader. 2.6. In vitro adhesion assay The adhesion of C. albicans was measured by cell-silicon sheet adhesion assay as described previously (Nobile et al., 2006) with minor modifications. Briefly, strains were grown overnight in SC medium with different concentrations of Met and/or Cys at 30 °C, diluted to an OD600 of 0.2, and added to a sterile 12-well plate with a prepared silicone square (1.5 1.5 cm cut from Cardiovascular Instrument silicone sheets [PR72034-06N, Bentec Medical Inc., United States]). The silicon square was pre-treated with bovine serum (Sigma–Aldrich) overnight and washed with PBS. The inoculated plate was incubated with gentle agitation (150 rpm) for 90 min at 37 °C for adhesion to occur. The squares were washed with 2 ml PBS to remove un-adhered cells, and then moved to a fresh 12-well plate containing 2 ml fresh SC medium. The plate was incubated at 37 °C for 48 h at 75 rpm agitation to allow biofilm formation. After incubation, the plate was agitated at 200 rpm for additional 5 min for detection of adhesion strength of the biofilms. Silicon squares with biofilms were photographed.
of 1.2 ml suspension from each group was pipetted into a clean glass tube and overlaid with 0.3 ml octane. The mixture was vortexed for 3 min for phase separation. Soon after the two phases were separated, OD600 of the aqueous phase was determined. OD600 for the group without the octane overlay in YPD medium was used as the negative control. Three repeats were performed for each group. The relative hydrophobicity was obtained as [(OD600 of the control minus OD600 after octane overlay)/OD600 of the control] 100%. 2.8. Determination of cAMP level Determination of intracellular cAMP level was performed as described previously (Miwa et al., 2004). Briefly, fungal cells were routinely grown overnight, washed with PBS and then transferred to SC medium (with different concentrations of Met and Cys). With an initial OD600 of 0.1, the cells were incubated at 37 °C for 6 h and then collected. After one wash with water and one wash with MES buffer (10 mM MES [morpholineethanesulfonic acid] containing 0.1 mM EDTA; pH 6), cells were re-suspended with MES buffer to an OD600 of 8 and 500 ll aliquots were taken from the suspension. Samples were transferred to 1.5-ml microcentrifuge tubes containing 0.5 g glass beads and 500 ll 10% trichloroacetic acid, briefly vortexed, and frozen immediately in liquid nitrogen. The samples were thawed on ice and sonicated under chilled conditions (twice at 130 W for 2.5 min). After centrifugation, trichloroacetic acid was extracted four times with water-saturated ether. The cAMP content was measured using the cAMP Enzyme Immunoassay Kit (Sigma– Aldrich) according to the manufacturer’s instructions. 3. Results 3.1. ecm17D/D is defective to produce H2S The role of ECM17 in H2S biosynthesis was first investigated using a BIGGY-agar (Cordente et al., 2009), with brown color of the colony indicating the generation of H2S. SN101 (ECM17/ ECM17) presented a dark brown color on BiGGY agar plate, indicating abundant H2S production, while the ecm17D/D mutant was white, indicating deficient H2S production (Fig. 2). The reintegration of ECM17 restored the wild-type phenotype on BiGGY agar plate.
2.7. Cellular surface hydrophobicity assay
3.2. ecm17D/D is defective to synthesize Met and Cys
Cellular surface hydrophobicity of C. albicans was measured by water–hydrocarbon two-phase assay as described previously (Klotz et al., 1985). Briefly, C. albicans strains were cultured as described above in biofilm formation assay, and the cells of the biofilms were removed from the flask surfaces with a sterile scraper to prepare a cell suspension (OD600 = 1.0 in YPD medium). A total
To investigate the role of ECM17 in Met and Cys biosynthesis, we observed the growth of the ecm17D/D mutant on solid SC medium lacking Met/Cys. Spot assay results indicated that the ecm17D/ D mutant could grow on solid SC medium lacking Met or Cys, but it was defective to grow when both Met and Cys were lacking (SC– Met–Cys medium). In contrast, the wild-type strain SN101 and
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Fig. 2. BiGGY plate assay to test the generation of H2S from fungal cells. H2S can reduce the bismuth ammonium citrate in BiGGY medium and result in brown color of fungal cells. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 3. Growth comparison of C. albicans strains. Overnight-cultured strains were suspended in PBS to an OD600 of 0.5, and 5 ll of each suspension was deposited on various auxotrophic SC plates with sufficient or insufficient Met/Cys supply. Growth difference was detected after 48-h incubation at 30 °C.
the reintegrated strain KEC31 could grow normally on solid SC– Met–Cys medium (Fig. 3). These results indicated that ECM17 was a key gene in Met and Cys biosynthesis. As the basic function of ECM17 is to catalyze the biochemical reaction for H2S generation, it is assumed that defective growth of ecm17D/D on solid SC–Cys–Met medium is caused by insufficient H2S generation. To verify this assumption, we tested the influence of H2S donor NaSH on the growth of ecm17D/D. The growth curves indicated that NaSH improved the growth of ecm17D/D in SC–Cys–Met liquid medium in a dose-dependent manner (Fig. 4). In accordance with the spot assay results shown above, ecm17D/D could not grow in SC–Cys–Met liquid medium (Fig. 4). Addition of 8 lg/ml of NaSH improved its growth remarkably, with the cell density of ecm17D/D reached 3 107 cells/ml after 48-h culture (Fig. 4). Addition of 16 lg/ml NaSH further improved its growth, and after 48-h culture, the cell density of ecm17D/D reached 4.5 107 cells/ml, which was the same as the wild-type strain (Fig. 4). We further examined the impact of Met and Cys supply on ECM17 expression. Real-time RT-PCR indicated that the expression of ECM17 with 20 lg/ml Cys was less than 50% of that without Met/ Cys, the relative fold change being 0.41. The expression of ECM17 with 80 lg/ml Cys was about 1/6 of that without Met/Cys, the relative fold change being 0.16. Consistently, the expression of ECM17 with 40 lg/ml Met was about 2/3 of that without Met/Cys, the relative fold change being 0.62. The expression of ECM17 with 160 lg/ ml Cys was about 1/9 of that without Met/Cys, the relative fold change being 0.11 (Fig. 5). These results indicated that the Met
Fig. 4. Influence of NaSH supply on the growth of ecm17D/D mutant. Strains were cultured in SC–Met–Cys auxotrophic medium with various concentrations of NaSH. No NaSH was added in the control group. With an initial OD600 of 0.001, fungi were cultured at 30 °C under constant shaking (200 rpm) and OD600 was determined at the designated time points after culture. Three independent experiments were performed.
Fig. 5. Real-time RT-PCR analysis of ECM17 in C. albicans strain SN101. Gene expression is indicated as a fold change relative to that of the control group, i.e., SN101 grown in medium without Met and Cys. The data are shown as mean ± SD from three independent experiments.
and Cys supply could repress the expression of ECM17 in a dosedependent manner. 3.3. ecm17D/D is defective to form biofilms As mentioned in Section 1, ECM17 was assumed to play a role in biofilm formation, so we further investigated the impact of ECM17 disruption on biofilm formation. Biofilm formation was evaluated by XTT reduction assay using OD490 value to indicate biofilm formation. Our data indicated that although 10 lg/ml Met or 20 lg/ ml Cys was sufficient for planktonic growth of ecm17D/D (Fig. 6),
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Fig. 6. Planktonic growth of C. albicans strains in SC–Met–Cys auxotrophic medium with different concentrations of Met/Cys. With 1.5 104 cells/ml as the initial density, fungi were cultured at 30 °C under constant shaking (200 rpm) and C. albicans cells were counted using a hemocytometer at the designated time points. Three independent experiments were performed.
ecm17D/D showed significant diminishment of biofilm formation even with 20 lg/ml Met and 40 lg/ml Cys (p < 0.05, Fig. 7). With decreased Met/Cys supply, the impact of ECM17 on biofilm formation was enlarged. With only 10 lg/ml Met supply and without Cys, biofilm formed by ecm17D/D was about 50% of that by SN101, and with only 20 lg/ml Cys supply and without Met, biofilm formed by ecm17D/D was about 1/5 of that by SN101. The reintegration of ECM17 restored the ability KEC31 to form biofilm (Fig. 7). These results indicated that ECM17 played an important role in biofilm formation, especially under conditions lacking Met/Cys supply. We further investigated the association between Met/Cys biosynthesis pathway and biofilm formation. The expression of MET4 was examined to indicate the status of Met/Cys biosynthesis pathway because MET4 encodes an important activator of this pathway (Lafaye et al., 2005). Interestingly during biofilm formation, MET4 was notably up-regulated in the ecm17D/D mutant compared with the wild-type SN101 with Met/Cys supply insufficiency (Fig. 8). More specifically, MET4 was up-regulated 13-fold with only 10 lg/ml Met supply and without Cys, and up-regulated 54-fold with only 20 lg/ml Cys supply and without Met (Fig. 8). These results confirmed the association between ECM17 and Met/
Fig. 8. Real-time RT-PCR analysis of MET4 in C. albicans. Strains were cultured in SC medium with different concentrations of Met/Cys. Gene expression is indicated as a fold change relative to SN101. (A) Planktonic cells obtained after overnight culture with constant shaking were collected and RNA was isolated. (B) Biofilm cells obtained after overnight incubation without shaking were collected and RNA was isolated. The data are shown as mean ± SD from three independent experiments.
Cys biosynthesis pathway and implied that more Met/Cys were needed in the ecm17D/D mutant compared with the wild-type cells. Of note, the significant up-regulation of MET4 in the ecm17D/D mutant was only observed during biofilm formation, but not in planktonic cells (Fig. 8), which indicated the association between Met/Cys biosynthesis pathway and biofilm formation. 3.4. ecm17D/D is defective to adhere and grow filamentously
Fig. 7. In vitro biofilm formation assay of C. albicans strains. Strains were cultured in media with different concentrations of Met/Cys. After 48-h incubation, the biofilm biomass was quantitated using XTT reduction assay. The data are shown as mean ± SD from three independent experiments. indicates P < 0.05 and indicates P < 0.01, compared with SN101.
Knowing that adhesion and filamentous growth are particularly important for biofilm formation, we investigated the role of ECM17 in adhesion and filamentous growth. The results indicated that the ecm17D/D mutant failed to adhere to silicon squares in the absence of sufficient Met/Cys supply in the medium (Fig. 9). After 48-h incubation for biofilm formation, vigorous agitation of 200 rpm ruined the ecm17D/D biofilms formed in the medium with only 20 lg/ml Cys or 10 lg/ml Met (Fig. 9), while the vigorous agitation did not affect the biofilms of wild-type strain SN101 or reintegrated strain KEC31 (Fig. 9), indicating a less adhesive strength of the ecm17D/D biofilms. Knowing that there is a positive correlation between cellular surface hydrophobicity (CSH) and adhesion (Luo and Samaranayake, 2002; Pompilio et al., 2008; Samaranayake et al., 1995), we further investigated the impact of ECM17 disruption on CSH. In SC medium with 40 lg/ml Cys and 20 lg/ml Met, the CSH of ecm17D/D decreased significantly compared with that of the wild-type strain SN101 (p < 0.05; Fig. 10), and the reintegration of ECM17 restored CSH of KEC31. Insufficient Cys/Met supply en-
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Fig. 9. In vitro adhesion assay of C. albicans strains. Strains were cultured in media with different concentrations of Met/Cys. After 48-h incubation for biofilm formation, plates with silicon squares were agitated at 200 rpm for 5 min to detect the adhesion strength of the biofilms. The silicon squares with biofilms were photographed.
larged the difference between ecm17D/D and SN101, which is consistent with the result of adhesion assay (Fig. 10). In SC medium with only 10 lg/ml Met without Cys, the CSH of ecm17D/D was only 1/4 of that of SN101, while in SC medium with only 20 lg/ ml Cys without Met, the ratio was 1/3 (Fig. 10). The reintegration of ECM17 improved the CSH of C. albicans, and there was no significant difference between KEC31 and SN101 (p > 0.05, Fig. 10). We further investigated the impact of ECM17 disruption on filamentous growth, and the results showed that the ecm17D/D mutant was defective in hyphal formation on SLD agar plates with insufficient Met/Cys supply (Fig. 11). Wild-type SN101 formed filaments on SLD plates lacking Met/Cys (10 lg/ml Met without Cys or 20 lg/ml Cys without Met) as well as with sufficient Met/ Cys supply (40 lg/ml Cys and 20 lg/ml Met) (Fig. 11), while the ecm17D/D mutant strain was defective to form filaments with Met/Cys supply insufficiency. More specifically, SN101 formed colonies with radial hyphae on the Met-lacking SLD plate, while ecm17D/D formed smooth colonies (Fig. 11C and F). The integra-
tion of ECM17 in KEC31 improved the filamentous growth of C. albicans (Fig. 11H and I). We also investigated the filamentous growth of the C. albicans strain in some other hypha-inducing media, including Spider, lee and serum media (solid and liquid), but no significant difference was observed between the ecm17D/D strain and SN101 (data not shown), indicating that the filamentation defect of ecm17D/D was condition-dependent. 3.5. ecm17D/D is defective to express some adhesion/filamentation genes To understand the mechanism of Ecm17-promoted adherence and hyphal formation, we further compared the expression of the known adhesion-specific or hypha-specific genes in strains SN101 and ecm17D/D (Fig. 12). The results showed no significant difference in the expression of most adhesion-specific or hyphaspecific genes (ALS1, ALS3, HWP1, HWP2, CSH1, IFF4, EAP1, ECE1, SAP4, SAP5, SAP6, UME6, HGC1 and EED1) between ecm17D/D and SN101 (>0.5 and <2 of relative fold) (Fig. 12). Interestingly, the expression of ALS3, CSH1, HWP1 and ECE1 was significantly down-regulated in the ecm17D/D mutant compared with the wild-type strain SN101 under conditions lacking Met or Cys. The relative fold was 0.33, 0.21, 0.05 and 0.16 respectively in medium with 20 lg/ml Cys and without Met (Figs. 12 and 13), and 0.32, 0.15, 0.12 and 0.47 respectively in medium with 10 lg/ml Met and without Cys (Figs. 12 and 13). Even with sufficient Met and Cys supply (20 lg/ml Met and 40 lg/ml Cys) in the medium, the relative fold was about 0.5 (Fig. 13). The reintegration of ECM17 restored the expression of ALS3, CSH1, HWP1 and ECE1. These results indicated that the expression of ALS3, CSH1, HWP1 and ECE1 was affected by ECM17 disruption significantly. 3.6. cAMP–PKA pathway is associated with ECM17 and Met/Cys biosynthesis pathway
Fig. 10. Cell surface hydrophobicity (CSH) assay of C. albicans strains. Strains were cultured in media with different concentrations of Met/Cys for 48-h. Cellular surface hydrophobicity of C. albicans was measured by water–hydrocarbon twophase assay The data are shown as mean ± SD from three independent experiments. indicates P < 0.05 and indicates P < 0.01, compared with SN101.
Since ALS3, HWP1 and ECE1 are known genes regulated by cAMP–PKA pathway, we further investigated the impact of ECM17 disruption on intracellular cAMP level. As was expected,
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Fig. 11. Filamentous growth of C. albicans strains on SLD agar plates. (A–I): Strains were grown on plates with different concentrations of Met/Cys. (a–i): Strains were grown on various plates with addition of exogenous cAMP. The growth was monitored after 8-day incubation at 30 °C.
the intracellular cAMP level, and no significant difference was observed between KEC31 and SN101. These results suggested that ECM17 and Met/Cys may regulate the expression of adhesion-specific and hypha-specific genes including HWP1, ALS3 and ECE1 by affecting the intracellular cAMP level. Subsequently, we tested the influence of exogenous cAMP supply on the filamentous growth of ecm17D/D. As was expected, the filamentous growth ability of ecm17D/D was recovered with the addition of 5 mM exogenous cAMP on SLD plates lacking Met or Cys, and there was no obvious difference between the wild-type strain and the ecm17D/D strain (Fig. 11e and f). We also tested the influence of exogenous cAMP supply on biofilm formation and adhesion of ecm17D/D, but no obvious effect was observed (Figs. 15 and 16). These results indicated that cAMP–PKA pathway was associated with ECM17 and Met/Cys biosynthesis pathway, especially for filamentous growth.
4. Discussion
Fig. 12. Real-time RT-PCR analysis of 14 genes involved in adhesion and/or filamentous growth. Gene expression is indicated as a fold change relative to SN101. The data are shown as mean ± SD from three independent experiments. (A) SC medium with 20 lg/ml Cys without Met; (B) SC medium with 10 lg/ml Met without Cys.
intracellular cAMP level was decreased significantly in the ecm17D/D mutant under all conditions tested (p < 0.05, Fig. 14). Interestingly, intracellular cAMP level of ecm17D/D decreased significantly even with 40 lg/ml Cys and 20 lg/ml Met as nutrition supply, and it was about 3/5 of that by SN101 (p < 0.05, Fig. 14). With Met and Cys supply decreasing, the impact of ECM17 on intracellular cAMP level was enlarged. With only 20 lg/ml Cys without Met, or with only 10 lg/ml of Met without Cys in the medium, the intracellular cAMP level of ecm17D/D was about 1/2 of that by SN101 (p < 0.01, Fig. 14), indicating a positive correlation between the ECM17-dependent Met/Cys biosynthesis pathway and the intracellular cAMP level. The reintegration of ECM17 increased
In this study, we investigated the role of ECM17 in Met/Cys biosynthesis and the contribution of Met/Cys to biofilm formation. It is the first report showing that the C. albicans ecm17D/D mutant was unable to catalyze the biochemical reaction from sulfite to H2S, and hardly grew in medium lacking Met and Cys. With the supply of H2S donor, the growth of ecm17D/D was improved in the Met/Cys-lacking medium. Meanwhile, the expression of ECM17 was inhibited with the addition of Met/Cys. These results confirmed the important role of ECM17 in Met and Cys biosynthesis. Further results also validated the important role of ECM17 in biofilm formation. Biofilm formation was attenuated by ecm17D/ D mutant compared with the wild-type or reintegrated strains under the same incubation conditions. In addition, adhesion and filamentous growth of ecm17D/D declined, especially under conditions lacking Met or Cys. Further results indicated that the defective biofilm formation of ecm17D/D was possibly related to the down-regulation of ALS3, CSH1, HWP1 and ECE1, four important genes in adherence and filamentous growth. It is known that HWP1, ALS3 and ECE1 are regulated by cAMP–PKA pathway, and our further results indicated that disruption of ECM17 decreased cAMP level under all conditions in this study. In addition, the filamentous growth of ecm17D/D on SLD lacking Met or Cys was
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Fig. 14. Determination of intracellular cAMP level. Strains were cultured with the supply of different concentrations of Met/Cys. The data are shown as mean ± SD from three independent experiments. indicates P < 0.05 and indicates P < 0.01, compared with SN101.
Fig. 15. Influence of exogenous cAMP on biofilm formation of ecm17D/D. Strains were cultured in SC medium with different concentrations of Met/Cys and different concentrations of exogenous cAMP added. After 48-h incubation, the biofilm biomass was quantitated using XTT reduction assay. The data are shown as mean ± SD from three independent experiments. indicates P < 0.01, compared with SN101.
Fig. 13. Real-time RT-PCR analysis of ALS3, CSH1, HWP1 and ECE1 in C. albicans strains. Gene expression is indicated as a fold change relative to SN101. The data are shown as mean ± SD from three independent experiments.
improved markedly with the addition of exogenous cAMP, indicating that cAMP–PKA pathway is associated with ECM17 and Met/ Cys biosynthesis pathway, especially for filamentous growth. To the best of our knowledge, this is the first report focusing the role of ECM17 in C. albicans, and our findings indicate that ECM17 plays a critical role in Met/Cys biosynthesis by controlling the synthesis of H2S. Based on the results of bioinformatics analysis (Fig. 1), it could be inferred that the disruption of ECM17 would unfavorably affect the ability of C. albicans to make use of sulfate/sulfite as the sulfur source to synthesize H2S and further inhi-
bit the biosynthesis of Met and Cys. This is in accordance with the following findings in the present study: (i) The ecm17D/D mutant was unable to produce H2S normally as the wild-type strain (Fig. 2). (ii) The ecm17D/D mutant rarely grew in medium lacking Met and Cys (Fig. 3). (iii) A H2S donor NaSH could improve the growth of ecm17D/D dose-dependently (Fig. 4). Furthermore, addition of Met/Cys inhibited the expression of ECM17 dose-dependently (Fig. 5). This seems to be a Met/Cys feedback regulating mechanism on ECM17 expression. This work demonstrated the important role of ECM17-dependent Met/Cys biosynthesis in biofilm formation. As was expected, the ability of the ecm17D/D mutant to form biofilms declined (Fig. 7). More interestingly, this decline was enlarged under conditions with insufficient Met/Cys supply, suggesting that ECM17 is involved in biofilm formation through regulating the biosynthesis of Met and Cys (Fig. 7). We also noticed that the Met and Cys concentrations (10 lg/ml Met; 20 lg/ml Cys) used in this study were sufficient for planktonic growth of all the strains, including ecm17D/D mutant (Fig. 6). Thus the growth defect of ecm17D/D mutant in developing biofilm suggests that more Met and Cys
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Fig. 16. Influence of exogenous cAMP on adhesion of C. albicans. Strains were cultured in SC medium with different concentrations of Met/Cys and different concentrations of exogenous cAMP added. After 48-h incubation for biofilm formation, plates with silicon squares were agitated at 200 rpm for 5 min to detect the adhesion strength of the biofilms. The silicon squares with biofilms were photographed.
are needed in the biofilm growth process as compared with planktonic growth. In accordance, the significant up-regulation of MET4 in ecm17D/D was only observed during biofilm formation, but not in planktonic cells. These results are consistent with the microarray finding that the Met/Cys biosynthesis genes were up-regulated from the early adhesion stage and this up-regulation state could be maintained for hours during biofilm formation (Murillo et al., 2005). Filamentous growth defect of ecm17D/D was observed on solid SLD medium but not other hypha-inducing media tested, which may be due to the rich organic sulfur in Spider, serum and Lee media. More specifically, there are 100 lg/ml methionine in Lee medium (Lee et al., 1975). Although the exact concentrations of methionine and cysteine in Spider and serum media are unknown, it is known that there are 1% nutrient broth in Spider medium and 1% yeast extract in serum medium (Maidan et al., 2005), and both nutrient broth and yeast extract are rich of organic sulfur (Blaszczyk, 1993). Besides, filamentous growth defect of ecm17D/D was only observed on solid SLD but not liquid medium, which indicates that the mechanisms of filamentation are different in liquid and solid media. Some previous findings (Lorenz et al., 2000) have provided a clue: A putative G-protein-coupled receptor Gpr1 regulates morphogenesis and hyphal formation in C. albicans, and the Cagpr1D/D mutant is defective in yeast-to-hypha transition only on solid medium but not in liquid medium, which is similar to our findings in this study. Interestingly, Gpr1 was reported to be involved in the nutrient sensing, and therefore we hypothesize that Cys and Met may regulate hypha formation through CaGPR1. To explore the underlying mechanism contributing to adhesion decline and filamentous growth defects of ecm17D/D, we investigated the expression of some most important adhesion and hypha-specific genes including the ALS family genes (ALS1 and ALS3), HWP1, HWP2, CSH1, IFF4, EAP1, ECE1, SAP4, SAP5, SAP6, UME6, HGC1 and EED1 (Chaffin, 2008; Sudbery, 2011) (Fig. 12). Interestingly, we found that ALS3, CSH1, HWP1 and ECE1 were associated with ECM17 (Fig. 13). ALS3, an ALS family gene, plays an essential role in the adherence stage of C. albicans to human umbilical vein endothelial cells and buccal epithelial cells (Zhao et al., 2006, 2004). Csh1p, encoded by CSH1, is a hydrophobic surface protein mediating binding to host target proteins. Disruption of
CSH1 decreased cell surface hydrophobicity and adhesion between C. albicans and fibronectin (Singleton and Hazen, 2004; Singleton et al., 2001). HWP1 is a unique adhesion gene expressing on the hyphal surface. Biofilms lacking HWP1 gene were prone to detach from the abiotic substrate (Chaffin, 2008; Nobile et al., 2006). ECE1 is highly expressed in hyphae and its expression correlates with the extent of hyphal cell elongation (Birse et al., 1993). The down-regulation of these four genes may decrease adhesion and hyphal formation of C. albicans. Our study further provides new insights into the association between ECM17-dependent Met/Cys biosynthesis pathway and cAMP–PKA signaling pathway. As was expected, the intracellular cAMP level declined in ecm17D/D mutant compared with the wild-type strain (Fig. 14). More interestingly, the phenomenon of cAMP decline in ecm17D/D mutant was obviously enlarged under conditions with insufficient Met/Cys supply. This result is consistent with the previous finding that Met could activate the cAMP– PKA signaling pathway in C. albicans cells (Maidan et al., 2005). Noticeably, our results indicated that as was the case with Met, the lack of Cys also decreased the intracellular cAMP level. This may result from the direct regulatory role of Cys on cAMP–PKA signaling pathway, or possibly result from the biochemical conversion between Met and Cys. As shown in Fig. 1, Met can be converted to Cys, and Cys can be converted to Met. Under conditions without Cys, some Met might be converted to Cys to meet the requirement of cells, thus decreasing the concentration of Met and then the intracellular cAMP level. Collectively, our findings indicate that Met/Cys may act as signal molecules to regulate intracellular cAMP level, and Met/Cys biosynthesis pathway is associated with cAMP– PKA pathway in C. albicans. In addition to the cAMP–PKA pathway, some other mechanisms may also be involved in the defect of ecm17D/D to form biofilms. We notice that although the addition of exogenous cAMP can improve the filamentous growth of ecm17D/D on solid SLD medium, cAMP cannot significantly improve the biofilm defect and adhesion decline of ecm17D/D (Fig. 15 and Fig. 16). The other possible mechanisms include: (i) Down-regulation of CSH1 may play a critical role in ecm17D/D. No documentary evidence has shown the association between CSH1 and cAMP–PKA pathway. Our unpublished data indicate that cAMP could not regulate the expression of
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CSH1, and exogenous addition of cAMP could not up-regulate its expression. CSH1 may be involved in biofilm formation independently with cAMP–PKA pathway. (ii) As Met and Cys are involved in the formation of disulfide bonds, and the cysteine residues are important to maintain the functions of many proteins (Frade-Perez et al., 2010; Perez et al., 2006; Popolo et al., 2008; Tirelli et al., 2010), we speculate that plenty of proteins are affected by the lacking of Met and Cys, and nutrition shortage, besides the adjustment of signal pathways, may also contribute to the biofilm defect of ecm17D/D. Collectively, we demonstrated that ECM17 regulated the biosynthesis of Met and Cys by controlling the generation of H2S, and ECM17-dependent Met/Cys biosynthesis played an important role in biofilm formation through affecting adhesion and filamentous growth. This work provides new insights into the role of Met/Cys as signal molecules and is helpful to better understand the associations between Met/Cys biosynthesis pathway, cAMP– PKA pathway and biofilm formation. Acknowledgments This work was supported by the National Natural Science Foundation of China (81072678, 30825041 and 90913008); the National Science and Technology Major Project of the Ministry of Science and Technology of China (2011ZX09102-002-01); Shanghai Educational Development Foundation (2007CG51); and Shanghai Science and Technology Major Project (10431902200). We would like to thank Dr. Alexander D. Johnson (Department of Microbiology and Immunology, University of California–San Francisco, California) for providing C. albicans strain SN152, plasmid pSN40 and plasmid pSN69. We also thank Dr. Joachim Morschhauser (Institute fur Molekulare Infektionsbiologie, University of Wurzburg, Germany) for kindly providing plasmid pSFS2A. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.fgb.2012.11.010. References Birse, C.E. et al., 1993. Cloning and characterization of ECE1, a gene expressed in association with cell elongation of the dimorphic pathogen Candida albicans. Infection and Immunity 61, 3648–3655. Blaszczyk, M., 1993. Effect of medium composition on the denitrification of nitrate by Paracoccus denitrificans. Applied and Environment Microbiology 59, 3951– 3953. Cao, Y. et al., 2008. In vitro activity of baicalein against Candida albicans biofilms. International Journal of Antimicrobial Agents 32, 73–77. Chaffin, W.L., 2008. Candida albicans cell wall proteins. Microbiology and Molecular Biology Reviews 72, 495–544. Cordente, A.G. et al., 2009. Isolation of sulfite reductase variants of a commercial wine yeast with significantly reduced hydrogen sulfide production. FEMS Yeast Research 9, 446–459. Falagas, M.E. et al., 2010. Relative frequency of albicans and the various nonalbicans Candida spp. among candidemia isolates from inpatients in various parts of the world: a systematic review. International Journal of Infectious Diseases 14, e954–e966. Frade-Perez, M.D. et al., 2010. Biochemical characterization of Candida albicans alpha-glucosidase I heterologously expressed in Escherichia coli. Antonie van Leeuwenhoek 98, 291–298. Garcia-Sanchez, S. et al., 2004. Candida albicans biofilms: a developmental state associated with specific and stable gene expression patterns. Eukaryotic Cell 3, 536–545. Hogan, D.A., Sundstrom, P., 2009. The Ras/cAMP/PKA signaling pathway and virulence in Candida albicans. Future Microbiology 4, 1263–1270. Ilkit, M. et al., 2007. Evaluation of albicans ID2 and biggy agar for the isolation and direct identification of vaginal yeast isolates. Journal of Medical Microbiology 56, 762–765.
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