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Department of Microbiology and Immunology, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA
Correspondence
W. LaJean Chaffin
LaJean.Chaffin{at}ttuhsc.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Present address: Department of Cell and Developmental Biology, Vanderbilt University Medical Center, U-3200 Medical Research Building III, Nashville, TN 37232, USA.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Kumamoto & Vinces, 2005; Mukherjee et al., 2005). Sixty-five percent of edentulous individuals suffer with denture stomatitis and 40 % of patients with intravenous catheters develop acute fungaemia due to the growth of C. albicans biofilms on the associated biomedical devices. Biofilms also show enhanced resistance to antifungal drugs.
In vitro, biofilms have been shown to form on catheter, polymethylmethacrylate (denture acrylic) and polystyrene surfaces (Douglas, 2003; Kumamoto & Vinces, 2005; Mukherjee et al., 2005). C. albicans forms a biofilm in three distinct developmental stages. The bottommost layer of adhered yeast cells act as founder cells, anchoring the developing biofilm to the substrate. The middle layer is composed of hyphae and pseudohyphae, and the topmost part of the biofilm consists mostly of a thicker and open hyphal layer and more extracellular matrix (ECM) (Baillie & Douglas, 1999; Chandra et al., 2001; Ramage et al., 2001). After 48 h, these biofilms range in thickness from 25 to >450 µm and are metabolically active communities of cells interspersed with ramifying water channels. The structural complexity of the biofilm may create a gradient of environmental conditions in which the C. albicans cells enter distinct physiological states. One such state may be equivalent to that of stationary-phase planktonic yeast cells, and, in particular, the founding yeast cells at the surface of the substratum may cease growing. The stationary phase and the genes involved in its progress and maintenance have not yet been well characterized in C. albicans, although several genes have been reported to show increased expression as active growth slows (Lamarre et al., 2001; Moreno et al., 2003). In this study, we investigated the expression patterns of the C. albicans homologues of the two most studied stationary-phase genes in Saccharomyces cerevisiae, SNO1 and SNZ1. We first determined whether the expression pattern of these genes, monitoring both RNA and protein, was associated with the stationary phase of planktonic yeast cells, hyphae and pseudohyphae. We then used these two genes as indicators of the stationary phase to study the physiological state of the cells in a biofilm.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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0.05). The viability of the recovered cells from three biofilms was determined at 20 days, as described above.
RNA extraction.
Total cellular RNA was isolated using the standard hot acid phenol method following grinding of the frozen cells using a mortar and pestle in liquid nitrogen (P. Uppuluri and others, unpublished results). We have found that grinding yields a better quality of intact RNA, particularly from late-stationary-phase cells. DNA contamination in the RNA was verified with the housekeeping gene EFB1 (Maneu et al., 2000). RNA quantity was estimated spectrophotometrically at 260 nm. RNA (10 µg) was electrophoresed under non-denaturing conditions in a 1.2 % agarose gel using Tris/acetate/EDTA buffer. The gel was stained with SYBR Green II (Sigma) and observed by UV light.
Real-time RT-PCR (RT-RTPCR).
The amount of mRNA in the total RNA was quantified with the Poly(A) mRNA Detection System kit (Promega). cDNA was synthesized from known amounts of mRNA, and an equal amount of cDNA was used as starting template for RT-RTPCR. Analysis of transcripts was carried out using SYBR Green PCR Master Mix (Applied Biosystems) in an ABI Prism 7700 Sequence Detection System (Applied Biosystems). Each reaction was set up in triplicate in 25.0 µl volumes with 1.0 µl cDNA for 40 cycles (thermal cycling conditions: initial steps of 50 °C for 2 min and 95 °C for 10 min, and then 40 cycles of 95 °C for 15 s, followed by 60 °C for 1 min). The primers are given in Table 1. To quantify transcripts, a standard curve was constructed using DNA of each gene as standards. For this, genomic DNA was isolated from C. albicans SC5314 strain following a standard protocol (Adams et al., 1997) and an entire ORF was amplified by PCR with gene-specific primers (Table 1). PCR reactions were set up with 10 ng genomic DNA in a 50.0 µl reaction volume using 40 pmol of each primer, 200 µM each dNTP, 2.5 mM MgCl2, and 0.125 U Ampli-Taq DNA polymerase (Applied Biosystems) for 30 cycles (94 °C for 1 min, 52 °C for 1 min, and 72 °C for 1 min) and a final extension at 72 °C for 7 min. PCR products were separated in a 1.0 % agarose gel, and DNA was eluted from the gel and quantified spectrophotometrically. RT-RTPCR reactions for each gene were set up using different dilutions of the amplified ORFs as the DNA template. Due to the absence of reference genes for normalization of stationary-phase gene expression (P. Uppuluri and others, unpublished results), three independent biological and technical replicates were used for normalization. All the replicates yielded equivalent Ct values when analysed using ANOVA (P0.05).
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). For construction of the yellow fluorescent protein (YFP) fusion protein, the method of Gerami-Nejad et al. (2001) was used. Briefly, PCR was performed using primers with 5' ends corresponding to the SNZ1 or SNO1 target gene sequences and 3' ends that directed amplification of the YFP gene along with the selectable marker URA3. The primers used are listed in Table 1
. PCR was performed with 100 ng pYFP-URA3 (cassettes obtained from Cheryl Gale, University of Minnesota) as the template, 0.6 µM each primer, 3.5 mM MgCl2, 5 µl 10xPCR buffer, 0.4 mM each dNTP, and 2 U Expand High Fidelity Polymerase (Roche Applied Science). The 50 µl reactions were run for 94 °C for 4 min, then for 25 cycles at 94 °C for 1 min, 60 °C for 1 min and 72 °C for 3 min, followed by 72 °C for 10 min.
The products from 10 reactions were pooled, precipitated with ethanol, resuspended in 50 µl water, and used to transform C. albicans CAI4 and URA3 recombinants selected in YNB without uridine. Identification of transformants carrying the correctly integrated cassette was performed by PCR on total genomic DNA with a primer that annealed within the transformation module and a second primer annealing to the 3' region located outside the module (Table 1).
Fluorescence microscopy.
For fluorescence microscopy, cells were used without fixation. YFP-tagged proteins were visualized in live cells with an Olympus IX71 microscope with appropriate filters. Images were captured and documented using a Photometrics Cool Snap HQ digital camera and analysed with Meta Morph software. For localization, a bright-field image and a fluorescent image were first pseudo-coloured green and red, respectively. The resultant images were then merged.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Radonjic et al., 2005). Since most studies with C. albicans terminate experiments after 24–48 h of growth, we wanted to determine whether cells remain viable in culture for an extended period. We examined the ability of organisms cultured in YNB for 2 weeks to carry out cell division by determining the c.f.u. in the culture during the stationary phase (Fig. 1). Cells did not lose viability for 10 days. At 2 weeks, >60 % of the cells were viable, after which there was a progressive decline in the number of cells forming colonies. On day 9, there was appearance of pseudohyphae in an appreciable fraction of cells (
30 %) (data not shown).
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). A single SNZ and SNO gene pair was found in the annotated C. albicans genome (Arnaud et al., 2005) and a (Altschul et al., 1997) BLAST search did not reveal other unannotated pairs. SNZ1 (Orf19.2947) and SNO1 (Orf19.2948) were adjacent and divergently transcribed, with a sequence of 1.5 kb separating the translation initiation sites. Transcripts of the two genes were quantified at intervals during progression into the stationary phase (Fig. 2A). Although a small peak in expression was noted on day 2, the greatest expression for SNZ1 was reached on day 5, after which the transcription declined. There was a 67-fold increase in expression between day 1 and day 5. For SNO1, there was decreased expression up to day 4, followed by increased expression that peaked on day 6, after which expression declined. The increased expression at day 6 was 10.5-fold higher compared to that on day 1. In addition to a 1 day difference in peak expression, SNZ1 transcript (5.6x106–3.8x108) was more abundant than SNO1 transcript (4.2x105–4.2x106) at all intervals measured, and the increase between day 1 and the peak of expression was greater for SNZ1 (67-fold) compared to SNO1 (10.5-fold). The expression of these genes began to increase after the cell numbers stopped increasing and the pattern was consistent with that of genes with preferential expression in the stationary phase.
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, 2000; Chandra et al., 2001). To determine if some organisms in a biofilm reach a physiologic state in which the SNZ1 and SNO1 markers for stationary phase are expressed, we examined biofilms formed under two conditions. Biofilms were formed on pieces of acrylic in YNB from a yeast-cell inoculum under static conditions, i.e. maintained without shaking for the duration of the experiment. There was an 1.5- and fourfold increase in SNZ1 and SNO1 expression, respectively, between day 1 and day 6 (Fig. 2B). However, overall expression of both genes in biofilm conditions at day 6 was at least 25 times less than the maximum that was recorded under planktonic conditions.
The reduced expression in the biofilm compared to planktonic cells could be attributable to the heterogeneous nature of the biofilm, which contains hyphal, pseudohyphal and yeast organisms, or to the presence of growing organisms responsible for the release of primarily yeast cells from the biofilm. To address these possibilities, we used a different system for biofilm formation. Biofilms were formed under flow conditions that replenished medium and permitted the biofilm to be maintained for 15 days. To answer the question of whether the bottommost layer of the biofilm formed from founder yeast cells reaches the stationary phase earlier than the rest of the biofilm, we separated two layers of the biofilm. We collected the bottommost adhered layer and the upper layers of the biofilm separately to monitor gene expression of the two stationary-phase genes, again, at various time points (Fig. 2C, D). We found that, in flow conditions, the level of expression of both the genes in the upper layers of the biofilm decreased over 15 days. When gene expression changes were monitored in the bottommost adhered cells of the biofilm, a different pattern of expression was revealed. Expression of both the genes was observed on day 1 and increased over the 15 days.
Protein localization of Snz1p–YFP and Sno1p–YFP in planktonic cells
We used YFP cassettes to tag SNZ1 and SNO1 in C. albicans, and observed the localization and expression of the two encoded proteins under different growth conditions and morphologies. Fluorescence was observed for both proteins in yeast cells (Fig. 3A, B). The proteins were then localized within the cells. When bright-field and fluorescent images were compared visually, fluorescence could be easily localized within the cytoplasm (Fig. 4A, B). The two images were pseudocoloured green and red using the Meta Morph software and then merged (Fig. 4C). This method confirmed that the two proteins localized to the cytoplasm.
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). About 25 % of cells expressed Snz1p–YFP on day 4 and >90 % expressed the protein on day 5. On day 6, there was a greater than threefold decrease in the fluorescent cells. Expression of Sno1p–YFP began a day later than Snz1p–YFP. About 40 % of cells expressed Sno1p–YFP on day 5, while >90 % expressed it on day 6. The expression of Sno1p–YFP did not decrease in the subsequent 7 days (data not shown). We next examined the fate of fluorescence when yeast cells resumed growth. Fluorescent cells, expressing either gene when inoculated into fresh medium, lost fluorescence within 24 h (Fig. 5B).
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Protein expression in biofilm organisms
We first examined expression of both Snz1p–YFP and Sno1p–YFP in a static model of biofilm formed on acrylic placed in the well of a polystyrene plate. On day 6, there were more fluorescent cells (P0.01) in the bottommost layer of adhered cells (25 %) than in the upper biofilm layer (11 %). Biofilms were formed in the second model system under flow conditions in which medium was continuously replenished (Fig. 6A–C). No fluorescence was observed in the uppermost layer which mainly contained hyphae, even though hyphae had been present in the biofilm from the first day. A few fluorescent organisms were observed in the middle layer, which had mixed morphologies. As in the static biofilm formation, 25 % of the bottommost adhered cells were fluorescent at day 6; the additional days of growth in the flow system showed that the number of fluorescent cells increased to 40 % on day 15. The bottommost layer of adhered yeast cells recovered from the biofilm retained 88 % viability (1x107 out of 1.2x107 cells), even up to 20 days.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Chandra et al., 2001; Ramage et al., 2001). This 450 nm-thick mature biofilm is also interspersed with water channels and is sheltered by an ECM. Thus, such a varied, closely packed community of cells may lead to a gradient of environmental conditions within the biofilm, in which the C. albicans cells may enter distinct physiological states. The goal of this study was to determine if C. albicans cells in a biofilm reach a physiologically similar state to that of planktonic stationary-phase C. albicans cells. However, the stationary phase in C. albicans has not been characterized, and our first steps were to confirm that cells remained viable in planktonic culture after the increase in cell number ceased (Fig. 1), and to identify a marker for the planktonic C. albicans stationary phase. Drawing on some paradigms represented by S. cerevisiae, we initiated a study to verify the expression patterns of the C. albicans homologues of the two most studied stationary-phase genes in S. cerevisiae, SNO1 and SNZ1. In S. cerevisiae, there are three pairs of SNO and SNZ genes. The genes of each pair are adjacent and divergently transcribed (Balakrishnan et al., 2002). The pairs are coordinately regulated with both the SNO2–SNZ2 and SNO3–SNZ3 pairs which are expressed prior to diauxic shift and the stationary phase (Arnaud et al., 2005). Only a single pair of SNO–SNZ genes was found in C. albicans, and they were adjacent and divergently transcribed.
We found that, in planktonic-grown C. albicans, the expression of SNZ1 and SNO1 appeared during entry into the stationary phase, peaking several days later (Fig. 2A). Expression of SNZ1 peaked on day 5, 1 day before SNO1 peak expression, and the level of SNZ1 expression and the magnitude of increase were greater than those of SNO1. This paralleled the observations in S. cerevisiae for SNZ1 and SNO1 (Braun et al., 1996). However, in a mutant strain of S. cerevisiae, in which SNO1–SNZ1 is the only pair of genes present, the genes are expressed prior to diauxic shift (Braun et al., 1996). Based on this analogy, we might have expected the C. albicans SNO1 and SNZ1 expression to parallel that of the S. cerevisiae mutant strain. This was not observed. Thus, it would seem that the function of SNZ and SNO genes prior to the stationary phase is dispensable in C. albicans. Snz1p–YFP expression was detected at 3 days (Fig. 5), perhaps reflecting the transient increase in transcript level seen on day 2 (Fig. 2A). The peak protein expression was observed on day 5, coincident with peak transcript level (Fig. 3A, B). This suggests that the increase in transcript level is derived from an increase in most cells of the population rather than in only a few cells. Sno1p–YFP expression began increasing on day 2 (Fig. 5A), at the same time that transcription level showed a small decrease. Maximum expression was reached on day 6, coincident with the peak transcription level. Unlike Snz1–YFP, Sno1p–YFP continued to be observed in cells, even though the transcription level began to decrease on day 7. The greater stability of Sno1p–YFP compared to that of Snz1p–YFP may explain the increase in the number of fluorescent cells at low transcript levels, as the protein accumulates and the fluorescent cells persist even when the peak transcript level declines. However, when stationary-phase planktonic yeast cells resumed growth, the number of fluorescent cells began decreasing immediately, such that only a few fluorescent cells were detected in the growing culture (Fig. 5B). The fluorescent cells decreased at a similar rate for both proteins, suggesting that, when the cell resumed growth, the proteins expressed for the stationary phase were lost. When protein expression was examined in hyphae, no fluorescence was observed (data not shown). Subapical compartments are arrested in G1 phase, are extensively vacuolated, and have very little cytoplasm (Barelle et al., 2003). Two possibilities for the lack of fluorescence in hyphae are that the G1-arrested, non-growing state of subapical hyphal cells is different from that of the G1 stationary-phase yeast cells, or that the expression in the small amount of cytoplasm of these subapical cells is below the level of detection. When pseudohyphae were observed in planktonic yeast cultures, they were fluorescent, but daughter buds were not. Since the buds were growing, this is consistent with the loss of fluorescence when cells resume growth, and also suggests that the partition of cytoplasm between parent and daughter cells did include the same level of stationary-phase protein found in the mother cell.
As expression of these two genes is a marker for stationary-phase planktonic yeast cells, we used the two genes to determine if cells within a biofilm reach a physiological state in which these genes are expressed. Biofilms were formed on denture acrylic under static conditions and under conditions of continuous medium flow. When formed under static conditions, expression increased over the 6 days of observation. As in planktonic yeast cells, SNO1 was expressed at a higher level than SNZ1 (Fig. 2B). However, both genes were expressed at 4 % of the maximum expression of planktonic yeast cells. Under conditions of medium replenishment, in which the biofilm could be observed for >2 weeks, expression was determined in the upper layers of the biofilm and in the cells adhered to the substrate (Fig. 2C, D). Expression from the upper-level biofilm organisms decreased by day 6 and remained at lower levels. In contrast, expression in the adhered cells increased and was about 100-fold higher than that in the upper layers and 5–11-fold higher than that in the static biofilm. At 15 days, levels of SNZ1 and SNO1 in the adhered cells were only five- and 2.4-fold less than their peak expression levels in 5- and 6-day-old planktonic cells, respectively. These adhered cells are likely to be founder cells of the biofilm and therefore older, and may show less proliferation than cells at the periphery of the biofilm.
When protein expression was examined in cells from different portions of the biofilm, the organisms at the top were almost exclusively hyphal and non-fluorescent, as seen in planktonic cultures (Fig. 6). Most of the fluorescent cells were found adhered to the substrate. The number of fluorescent cells increased between days 3 and 6, as did gene expression (Fig. 2). The 15-day continuously fed biofilm, with 40 % of the adhered yeast cells showing fluorescence and a 2.5–5-fold reduction in gene expression level for the cell population, suggests that the level of expression in the fluorescent cells may be similar to that of planktonic, 5–6-day stationary-phase yeast cells. Although hyphae were present in the biofilm from day 1, no fluorescence was observed and, as with planktonic hyphae, this may have arisen from a difference in the G1 state of subapical compartments, or an inability to detect fluorescence in the reduced cytoplasm of these cells.
The proteins were localized in the cytoplasm (Fig. 4). In a genome-wide S. cerevisiae study, Snz1p could not be localized by green fluorescent protein (GFP) fusion, due to low GFP expression signals or to other technical difficulties, while a low-level cytoplasmic fluorescence was noted for Sno1p (Huh et al., 2003). The greater level of fluorescence in this study may reflect a higher level of expression of these proteins in C. albicans, or the successful tagging of the protein.
In summary, C. albicans has a single pair of SNZ and SNO genes that was expressed in the stationary phase of planktonic yeast cells but not in hyphae. Proteins were localized in the cytoplasm and >90 % of 5- and 6-day stationary-phase yeast cells expressed the proteins. Expression of these genes was less in biofilms, whether formed under static or medium-flow conditions. Expression of the genes increased during biofilm formation and was primarily associated with founder yeast cells adhered to the substrate. This finding suggests that some cells at the base of a biofilm either were in the stationary phase or had reached a physiological state in which genes associated with the stationary-phase planktonic yeast cells were expressed.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 5 December 2005;
revised 3 March 2006;
accepted 9 March 2006.
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