1 Department of Veterinary Pathobiology, University of Illinois at Urbana-Champaign, Urbana, IL 61802, USA
2 Department of Molecular and Cell Biology, University of Aberdeen, Institute of Medical Sciences, Foresterhill, Aberdeen, UK
Correspondence
Lois L. Hoyer
lhoyer{at}uiuc.edu
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ABSTRACT |
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The GenBank accession numbers for the sequences reported in this paper are AY223551 (ALS3 small allele) and AY223552 (ALS3 large allele).
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INTRODUCTION |
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One main goal of our research efforts is to understand the function of the various Als proteins and the role each plays in C. albicans biology and pathogenesis. The emergence of initial functional data for Als1p and Als5p is important because they have the most similar N-terminal domain sequences (87 % amino acid identity) within the ALS family (Hoyer et al., 1995; Gaur & Klotz, 1997
; Hoyer & Hecht, 2000
; Fu et al., 2002
). The sequence of Als3p is also closely related to Als1p (84 % amino acid identity) and Als5p (81 % amino acid identity) within the N-terminal domain (Hoyer et al., 1998
). Similarities in expression pattern have also been noted between ALS1 and ALS3, with expression of each gene being strongly upregulated under certain growth conditions such as inoculation into RPMI 1640 medium (Hoyer et al., 1998
). Comparisons between closely related Als proteins will begin to address functional similarities and differences within the Als family.
ALS3 was initially described as a hypha-specific gene in the ALS family (Hoyer et al., 1998). Previous unpublished work suggested the presence of a second ALS3-like locus in C. albicans, which was called ALS8. The ALS8 gene was isolated as a false positive in a screen for C. albicans transcription factors (P. Leng & A. J. P. Brown, unpublished data). ALS8 was believed to be identical to ALS3 and regulated in a similar fashion because four rounds of gene disruption were required to generate a null mutant, apparently generating a double als8/als8, als3/als3 mutant in the process (P. Leng & A. J. P. Brown, unpublished data). We recently revisited the construction of an als3/als3 mutant strain using a different approach. This process required only two rounds of disruption to generate the null mutant. These data suggest that ALS3 and ALS8 represent a single locus in the strain examined.
In this study, we used the als3/als3 mutant strains and a newly created als1/als1 mutant strain to compare the function of Als3p and Als1p in C. albicans. The direct comparison of two different als/als mutant strains reveals functional similarities between the Als proteins, but also highlights unique protein function and gene expression patterns. These observations begin to define the functional interrelationships among the Als proteins.
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METHODS |
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Growth rate measurements.
Cells were grown overnight in YPD (per litre: 10 g yeast extract, 20 g peptone, 20 g glucose) at 37 °C with 200 r.p.m. shaking. Cells were counted in duplicate and inoculated into 20 ml fresh YPD at a density of 1x106 cells ml1. Cultures were incubated at 37 °C with 200 r.p.m. shaking, and spectrophotometric readings were taken in triplicate every hour. Growth rates were measured on three different days from separate starting cultures. Rate of growth and doubling time were calculated from the linear portion of the growth curve using the exponential growth equation in nonlinear regression in GraphPad Prism (GraphPad Software). A mixed model analysis in two-way ANOVA was used to assess the statistical differences in the growth rates of the C. albicans strains. Statistical comparison of growth rates was completed using PROC MIXED in SAS (SAS Institute).
Evaluation of germ tube formation and cellular aggregation.
C. albicans strains were grown overnight in YPD at 37 °C with 200 r.p.m. shaking. Cells were washed in Dulbecco's phosphate-buffered saline without Ca2+ or Mg2+ (DPBS; Cambrex catalogue no. 17-513Q) and counted. An inoculum of 5x106 cells ml1 was added to 10 ml prewarmed RPMI 1640 without L-glutamine (RPMI; Invitrogen catalogue no. 11875-085) or to Lee medium (Lee et al., 1975) and incubated at 37 °C with 200 r.p.m. shaking. Cell growth was stopped by the addition of glutaraldehyde to a final concentration of 1 % and the culture kept on ice. A positive reading was assigned to cells that had a germ tube equal to or longer than one diameter of the mother yeast cell. A negative reading was assigned to cells that had a shorter germ tube or none at all. For each culture, 100 cells were evaluated and the number of positive cells was expressed as a percentage of the total. Cellular aggregation was also evaluated using the same cultures. The number of cells in the first 100 aggregates viewed was recorded. Cells that appeared individually were assigned a value of 1; other values represented the number of cells in the aggregate. Both assays were run in duplicate on 3 different days. The mean of these values was calculated and a mixed model analysis of variance (PROC MIXED in SAS) was used to assess differences in germ tube formation and cellular aggregation among the C. albicans strains.
Construction of green fluorescent protein (GFP) reporter constructs.
Reporter strains were constructed using the method of Gerami-Nejad et al. (2001). Plasmid pGFP-URA3 was used as template for a PCR reaction with primers specific for the ALS1 and ALS3 locus, respectively. The PALS1GFP cassette was amplified using primers ALS1-FPF and ALS1-FPR (Table 2
). Primers ALS3-FPF and ALS3-FPR were used to amplify an ALS3-specific cassette (Table 2
). PCR amplification reactions included 400 ng plasmid DNA as template. The PCR products were extracted with phenol/chloroform/isoamyl alcohol and precipitated with ethanol. DNA was resuspended in sterile water and used to transform spheroplasts of C. albicans strain CAI4 (Fonzi & Irwin, 1993
). Transformants were selected on SCUri agar plates with 1 M sorbitol. Correct transformants were verified by Southern blotting of BglII-digested genomic DNA for the ALS1 construct and EcoRV-digested DNA for the ALS3 construct. Probes upstream of ALS1 (produced by PCR using primers ALS1GFPF and ALS1GFPR) and ALS3 (produced using primers 3upABKpn and 3upABXho) were used. A GFP probe fragment amplified from pGFP-URA3 with primers GFPXhoI and GFPBglII was also used to verify constructs. Fluorescence of correct clones was monitored microscopically following growth in media conditions shown previously by Northern blotting to increase transcription from the ALS1 and ALS3 promoters (Hoyer et al., 1995
, 1998
). Expression patterns matching previous Northern blot results confirmed that the clones displayed GFP production under control of the correct promoter.
Flow cytometry analysis.
Strains CAI12 (control), 2185 (PALS3GFP) and 2225 (PALS1GFP) were grown to stationary phase in YPD. Cells were washed twice and then resuspended in DPBS. Cell stocks were counted and then inoculated at a density of 5x106 cells ml1 into prewarmed RPMI medium. Cultures were incubated at 37 °C with 200 r.p.m. shaking for 1 h, taking samples at 15 min intervals. Flow cytometry was performed, using a Beckman Coulter EPICS XL machine. This instrument is equipped with an argon laser with an excitation wavelength of 488 nm. For fluorescence analysis, a region was set on a histogram, which represented side-angle light scatter versus forward-angle light scatter for a population of CAI12 yeast cells. The fluorescence was then gated on this region. Ten thousand events were collected at a laser power of 15 mW at medium flow rate. Fluorescence was measured on the FL1 channel with a 525 nm bandpass filter. Geometric mean fluorescence values for each time point were calculated using WinList software (Verity) and graphs were generated using the Summit 3.1 analysis software (Cytomation).
RNA analysis.
RT-PCR analysis was conducted as described previously (Green et al., 2004). C. albicans strains were grown overnight in YPD at 37 °C with 200 r.p.m. shaking. Cells were washed twice in sterile water and counted. An inoculum of 1x107 cells ml1 was added to 100 ml RPMI medium that was prewarmed to 37 °C. The culture was incubated at 37 °C with 200 r.p.m. shaking for 90 min. Cells were collected by filtration, flash frozen in dry ice/ethanol and stored at 80 °C until RNA was extracted. RNA extraction used the method of Collart & Oliviero (1993)
; RNA was stored in ethanol at 80 °C. RT-PCR primers specific for ALS1 and ALS3 have been described previously (Green et al., 2004
). PCR products were run on 8 % acrylamide/TBE (Tris/borate/EDTA) gels and visualized by staining with ethidium bromide.
Endothelial cell and fibronectin adhesion assays.
These assays were conducted in a six-well plate format using modifications of the method described by Ibrahim et al. (1995). Endothelial cells were purchased from Cambrex and grown according to the distributor's instructions in EGM-2 medium without addition of antimicrobials. Cells formed confluent monolayers in a six-well tissue culture-treated polystyrene plate (Fisher; catalogue no. 07-200-83). The growth medium of the endothelial monolayer was changed with a fresh medium 1 day prior to the adhesion assay. C. albicans strains were inoculated from the stock plate into 10 ml liquid YPD and grown for 16 h at 37 °C with 200 r.p.m. shaking. Cells were washed twice with DPBS and counted. In order to induce germ tube formation, 104 C. albicans cells were inoculated into 10 ml prewarmed RPMI in a 37 °C incubator. After 1 h, each endothelial monolayer was rinsed twice with 37 °C RPMI and 1 ml RPMI containing 103 C. albicans germ tubes was added to at least six replicate wells. The inoculated plate was placed in the 37 °C/5 % CO2 incubator for 30 min to allow yeast cells to adhere to endothelial cells. To remove unattached C. albicans cells, the plate was tilted at a 20° angle and 5 ml DPBS was gently allowed to run across the cell monolayer with simultaneous aspiration from the bottom of the well. The plate was turned 180° and another 5 ml DPBS was applied to each well as described above and the well was covered with 4 ml YPD top agar. Viability of C. albicans was verified by plating 100 µl C. albicans inoculum on YPD plates in triplicate. The six-well plate and the YPD plates were incubated overnight at 37 °C and c.f.u. were counted. The percentage adherence of each investigated C. albicans strain was calculated as (mean adherent c.f.u./mean total c.f.u.)x100.
Adhesion to fibronectin was tested in a similar manner using fibronectin-coated six-well plates (BD Biosciences; catalogue no. 354402). C. albicans cells were grown and prepared in the same way as for the endothelial adhesion assay. After counting, 5x103 cells were inoculated in 20 ml prewarmed RPMI medium and incubated for 1 h at 37 °C for germ tube formation. The fibronectin-coated plates were prewarmed at 37 °C and 2 ml RPMI medium containing 103 yeast cells was added into six replicate coated wells. After 30 min incubation at 37 °C/5 % CO2, the initial RPMI solution was aspirated and 2 ml DPBS was applied to each well. To remove the nonadherent C. albicans, the plate was gently shaken manually and DPBS was aspirated. The same washing was repeated one more time and then 4 ml YPD top agar was added to each well. The viability of C. albicans was evaluated by plating 200 µl inoculum on a YPD plate in triplicate. The percentage adherence of each C. albicans strain was calculated as described previously. All adhesion assays were performed in at least six wells per day on three different days. Results were evaluated statistically using a mixed model analysis of variance (PROC MIXED in SAS). Separation of means was performed using the LSMEANS option.
Buccal epithelial cell (BEC) adhesion assays.
BEC were collected from five human donors and pooled. Each donor provided written consent for participation in the study and collection procedures followed the guidelines of the University of Illinois Institutional Review Board. Cells were washed twice with DPBS and counted. Cells were resuspended at a concentration of 8x104 cells ml1 and kept on ice. C. albicans strains were inoculated from a stock YPD plate into 10 ml liquid YPD and grown for 16 h at 37 °C with 200 r.p.m. shaking. Cells were counted and 2x106 fungal cells were inoculated into 4 ml RPMI in a 25 ml sterile Erlenmeyer flask. Cultures were incubated at 37 °C with 200 r.p.m. shaking for 1 h to allow germ tube formation. At that time, 250 µl DPBS containing 2x104 BEC was added to each flask. Adhesion progressed for 30 min at 37 °C with 200 r.p.m. shaking. Cell mixtures were vacuum filtered across 12 µm pore size Nuclepore polycarbonate filters (Corning; catalogue no. 111116). Filters were washed dropwise with 25 ml DPBS to remove nonadherent C. albicans cells. Filters were removed from the vacuum filtration device, inverted onto glass microscope slides and dried. Following removal of the filter from the slide, slides were heat fixed, stained with crystal violet, washed with tap water, dried and examined microscopically. The number of germ tubes adhering to the first 50 BEC observed on the centre of each slide was recorded. Replicates for each strain were run on three separate days using a different pool of BEC on each day. Results are expressed as the mean number of C. albicans germ tubes that adhere to each BEC. A mixed model analysis of variance was used to study the differences in adherence to BEC. The mean number of adherent germ tubes for each replicate within a strain per day was analysed using PROC MIXED in SAS. Separation of means was performed with the LSMEANS option.
Reconstituted human epithelium model.
Reconstituted human epithelium (RHE) is a product of SkinEthic Laboratories (Nice, France). The product consists of human epithelial cell lines cultured on polycarbonate filters in vitro at the airliquid interface in a serum-free chemically defined medium. The experiments here used oral RHE (derived from the TR146 cell line) in maintenance medium without antimicrobials. The maintenance medium is based on the MCDB-153 of Clonetics and contains 5 µg insulin ml1. Preparation of the inoculum strains followed the protocol described by Green et al. (2004). An inoculum of 50 µl C. albicans yeasts/PBS suspension (2x106 cells total) was added onto the surface of each RHE sample, which was incubated at 37 °C, 5 % CO2 and saturated humidity for either 1, 4 or 8 h. Replicate samples were collected at the two later time points. Upon harvesting the specimens, each plastic well containing RHE was placed into 1 ml Karnovsky's fixative in a 12-well plate and the RHE layer was covered by another 1 ml of fixative. Samples were fixed for 1 h at room temperature and stored at 4 °C until processed. Prior to processing for microscopy, the Karnovsky's fixative was removed from the surface of each RHE specimen and the specimen was washed twice with 1 ml DPBS per wash. Washing was intended to remove nonadherent organisms. Processing for microscopy followed the methods of Green et al. (2004)
. Tissue blocks were cut into four longitudinal fragments and sections taken from each of these points in the specimen. Five sections from each of these locations in the sample were evaluated for RHE damage, the number of C. albicans cells adherent to the RHE layer and percentage germination of C. albicans cells present. Percentage germination of the C. albicans strains in RHE maintenance medium was observed following the methods for evaluation of germ tube formation described above. Specimens were examined using a Nikon Eclipse E600 microscope fitted with a Spot camera (Diagnostic Instruments). Images of representative sections were collected using Metamorph software (Universal Imaging Corporation) and processed with Adobe Photoshop.
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RESULTS |
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Construction of an als1/als1 mutant strain
We constructed an als1/als1 strain in which the entire coding region of the gene was deleted. The resulting strain, 1467, was validated using Southern blots (Fig. 2) and PCR. PCR primers ALS1upF and ALS1dn were used to amplify genomic DNA from strain 1467. DNA sequencing of the resulting PCR product verified that the ALS1 coding region was deleted from 56 bp upstream of the ALS1 start codon to 3 bp downstream of the stop codon (data not shown). A replacement strain, 2151, was constructed in the 1467 background using the large ALS1 allele from strain SC5314, which contains 20 copies of the 108 bp tandem repeat sequence in the central domain (Fig. 2
). These strains were used in functional analyses described below for comparisons to the als3/als3 mutants and provided the first direct comparison of Als protein function in C. albicans.
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DISCUSSION |
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Previous work showed an increase in the transcriptional activity of ALS3 and ALS1 as yeasts form germ tubes, particularly in RPMI medium (Hoyer et al., 1995, 1998
; Fu et al., 2002
). These results are confirmed here using GFP reporter constructs. With these constructs, we were able to separate transcription of the two genes temporally, showing a large increase in transcription from the ALS1 promoter within minutes after the cells are transferred to RPMI medium. In contrast, ALS3 transcription does not increase until germ tubes are visible. These observations suggest a role for Als1p in very early events during the change in cellular morphology while the function of Als3p is likely to be associated with a more mature germ tube. Understanding the relationship between transcriptional activity of these two genes and their connection with changes in cellular morphology will allow more appropriate design of microarray analyses to better understand the effects of als/als mutation on C. albicans. These experiments are likely to identify cellular pathways in which the ALS genes function.
Because previous publications have suggested that Als proteins might serve in aggregation of C. albicans cells (Fu et al., 2002; Gaur & Klotz, 1997
) and cellular aggregation can potentially have a large effect on adhesion assays, we included experiments to ascertain whether removal of Als proteins creates a phenotype different from wild-type controls. Data presented here showed that cellular aggregation was statistically the same as wild-type for als3/als3 or als1/als1 strains forming germ tubes in RPMI or Lee medium. Mutation of ALS3 also did not affect the ability of the strain to form germ tubes in either medium. In contrast, the als1/als1 strain showed a slowed germ tube formation in Lee medium, consistent with previous reports (Fu et al., 2002
). This defect was not apparent in RPMI medium, indicating that the defect is growth-medium-specific. The als1/als1 filamentation defect was also noted in vivo at early time points in a murine model of disseminated candidiasis (Fu et al., 2002
), but not in a murine model of oropharyngeal candidiasis (Kamai et al., 2002
). Collectively, these results suggest that local environment plays a large role in the function of Als1p.
In the work presented here, the RHE model of oral candidiasis was used to assess the effects of the als1/als1 and als3/als3 strains on epithelial adhesion and destruction. This model has been used previously to assess adhesion and early events in epithelial damage using strains lacking the two-component histidine kinase, CHK1, or a response regulator, CSSK1 (Li et al., 2002). Inoculation of RHE with the als1/als1 mutant strain resulted in a slight decrease in epithelial damage compared to the wild-type and replacement strain controls. In contrast, inoculation with the als3/als3 strain resulted in a marked decrease in epithelial damage. These conclusions paralleled results from in vitro BEC adhesion assays, which showed a slight change in adhesion for the als1/als1 mutant and a large decrease in adhesion for the als3/als3 strain (Fig. 5
). Two previous reports address the ability of Als1p to adhere to epithelial cells. In the first report, ALS1 was overexpressed in S. cerevisiae, converting the normally nonadherent organism to one that adhered to epithelial cells (Fu et al., 1998
). Although use of the S. cerevisiae model system may be attractive for isolating and characterizing the function of C. albicans adhesins, results need to be interpreted carefully because of differences in codon usage (Santos & Tuite, 1995
) and glycosylation (Herrero et al., 2002
) that exist between the organisms. An adhesive phenotype might be created by the stickiness of denatured or improperly glycosylated protein, or by disruption of normal cell-wall structure by overproduction of a large, heavily glycosylated protein. The second publication describing Als1p adhesion to epithelial surfaces used a murine model of oropharyngeal candidiasis and demonstrated that the als1/als1 strain was less adherent to oral tissues and to the tongue ex vivo (Kamai et al., 2002
). In this model, the complex environment of the oral cavity may affect interaction of Als1p with host surfaces and adhesion may not be directly to epithelial ligands. The epithelial cell adhesion comparisons for als1/als1 and als3/als3 strains presented here predict that als3/als3 strains should show an even greater reduction in oral pathology than als1/als1 strains. This relationship remains to be tested.
Construction of the als1/als1 mutant described here and the one tested in previous studies (Fu et al., 2002; Kamai et al., 2002
) differs in a way that may be significant to conclusions about adhesion and pathogenesis. In the previously published strain, disruption of the ALS1 coding region left the 5' end of one allele intact. Transcription from the ALS1 promoter in this strain can result in export of a protein that includes the N-terminal 285 amino acids of Als1p. This portion of the N-terminal domain of Als proteins has been proposed to be involved in adhesive interactions by comparisons to the structure of the S. cerevisiae cell-surface adhesion glycoprotein
-agglutinin (Chen et al., 1995
; Hoyer, 2001
). Under conditions where ALS1 is transcribed, the exported N-terminal fragment would be delivered to the local site occupied by C. albicans, with the potential to interfere with adhesive interactions between C. albicans and other cells or surfaces. Therefore, pathogenesis effects observed with this strain may reflect more factors than simple loss of cell-surface-bound Als1p. To avoid the potential problems of creating soluble N-terminal Als fragments, the ALS coding regions in the mutant strains constructed in this work were completely removed. The relative pathogenesis of C. albicans strains that produce N-terminal Als fragments is being investigated (C. B. Green & L. L. Hoyer, unpublished results).
The studies described here outline similarities and differences between Als3p and Als1p, two of the most closely related proteins in the Als family. The direct comparison of the als1/als1 and als3/als3 mutant strains in the adhesion assays presented here could lead to the conclusion that Als3p is the stronger adhesin. However, in the context of the Als family, it is possible that some of the proteins have redundant function. Another protein may partially compensate for the loss of Als1p, resulting in a less severe phenotype for the als1/als1 strain. Phenotypic analysis of our collection of strains mutant in the other ALS genes and construction of selected double mutant strains will reveal the interrelationships between the Als proteins and their roles in C. albicans biology and pathogenesis.
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ACKNOWLEDGEMENTS |
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Received 26 November 2003;
revised 8 April 2004;
accepted 16 April 2004.