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Department of Pathobiology, University of Illinois, Urbana, IL 61802, USA
Correspondence
Lois L. Hoyer
lhoyer{at}uiuc.edu
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ABSTRACT |
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/als9 mutant phenotype in other assays demonstrated no significant difference from a control strain for adhesion to buccal epithelial cells or laminin-coated plastic plates. The als9/als9 mutant did not show significant differences from the control for adhesion to or destruction of cells in the reconstituted human epithelium (RHE) disease model, or for cell-wall defects, germ-tube formation or biofilm formation in a catheter model. Analysis of ALS9 allelic frequency in a collection of geographically diverse clinical isolates showed a distinct preference for ALS9-2 allelic sequences, within both the 5' and the 3' domain of the ALS9 coding region. These data suggest greater selective pressure to maintain the ALS9-2 allele in C. albicans isolates and imply its greater relative importance in host–pathogen interactions.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, 1998; Hoyer, 2001). Because of sequence similarities between Als1p and the Saccharomyces cerevisiae cell-surface adhesive glycoprotein 
-agglutinin, it was hypothesized that the Als proteins are adhesins (Hoyer et al., 1995). Isolation of ALS1 and ALS5 based on their ability to confer adhesive function on the relatively non-adherent S. cerevisiae supported this hypothesis (Gaur & Klotz, 1997; Fu et al., 1998). We have taken a systematic approach to investigating Als protein function that involves generating C. albicans mutant strains, each lacking the coding region for a single ALS gene. Consistent with our initial hypothesis, phenotypic testing of these C. albicans mutants has focused on adhesion assays and measured the contributions of Als1p, Als3p (Zhao et al., 2004), Als2p and Als4p (Zhao et al., 2005) to interactions between C. albicans and host surfaces.
Completion of the eight ALS gene sequences highlighted common features within the family (Hoyer, 2001). In the centre of each coding region is a domain composed entirely of tandemly repeated copies of a highly conserved 108 bp unit. The number of 108 bp copies present varies considerably between alleles. Flanking this central domain are a 5' domain of approximately 1.3 kb that is 55–90 % conserved across the ALS family and a 3' domain of variable length and sequence. The tandem repeat region and 3' domain encode serine/threonine-rich amino acid sequences that are heavily glycosylated in the mature Als protein (Kapteyn et al., 2000). These sequence features are consistent with a model of Als protein structure that includes a relatively non-glycosylated N-terminal domain that is displayed on the C. albicans cell surface by the remainder of the mature protein, which forms an extended stalk, due to its heavy glycosylation. For Als proteins that display adhesive activity in C. albicans and/or S. cerevisiae, data support the conclusion that most adhesive activity resides within the N-terminal domain (Loza et al., 2004; Zhao et al., 2006). However, recent work in S. cerevisiae suggests that the tandem repeat and C-terminal domain of Als5p may have more than a structural role (Rauceo et al., 2006).
The focus of this work is C. albicans ALS9, which is localized on chromosome 6, immediately downstream of the coding regions for ALS5 and ALS1 (Zhao et al., 2003). Analysis of ALS gene expression has shown that ALS9 is transcribed under a wide variety of in vitro growth conditions, and in both human clinical specimens and animal disease models (Cheng et al., 2005; Green et al., 2004, 2005a, b, 2006). Under the in vitro conditions assayed by real-time RT-PCR, however, maximal ALS9 transcript copy number is approximately 10- to 1000-fold less than the maximal copy number observed for other ALS genes such as ALS1, ALS2 or ALS3 (Green et al., 2005b). Perhaps the most notable feature of ALS9 is its allelic variability, which is defined based on the sequences of the two ALS9 alleles from C. albicans strain SC5314 (GenBank accession nos for ALS9-1 and ALS9-2 are AY269423 and AY269422, respectively). In general, the ALS family is known for its large degree of allelic variability, since each ALS gene exhibits this feature within the central tandem repeat domain (Hoyer, 2001). Certain ALS genes also display allelic sequence variability within the 5' domain (ALS5; Hoyer & Hecht, 2001) or 3' domain (ALS7; Zhang et al., 2003); however, ALS9 is the only gene with extensive sequence variability within each coding region domain (Zhao et al., 2003). The 5' domain sequences of ALS9-1 and ALS9-2 from strain SC5314 vary by 11 % at the nucleotide level (16 % at the amino acid level). Within the 3' domain, allele ALS9-2 encodes two sequence blocks, called variable block 1 (VB1) and variable block 2 (VB2), which are absent from the 3' domain of allele ALS9-1 (Zhao et al., 2003). In this work, we test the two ALS9 alleles in adhesion assays and demonstrate functional differences between them. Here, we associate adhesive function with Als9-2p and demonstrate that the ALS9-2 allele is more prevalent in C. albicans clinical isolates.
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METHODS |
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/als9 strain.. General methods for creating als/als strains have been described by Zhao et al. (2004). Details specific to the ALS9 studies are presented here. A diagram of the ALS9 alleles from strain SC5314 is shown in Fig. 1. An ALS9 disruption cassette was amplified from the plasmid pDDB57 template (a generous gift from Aaron Mitchell, Columbia University) with primers ALS9-5DR and ALS9-3DR (Table 1). The disruption cassette was transformed into C. albicans strain CAI4 (Table 2). Transformants were selected as described previously (Zhao et al., 2004) and screened by Southern blotting. The Southern blot probe, which hybridizes to nucleotides –473 to –55 upstream of ALS9, was PCR-amplified from SC5314 genomic DNA (Table 2) with primers ALS9upF and ALS9upR (Table 1). Correct transformants were screened by PCR using primer pair VB2F and VB2R (Table 1) to confirm Southern blot results and determine which ALS9 allele was deleted. Strain 1976, from which the ALS9-2 allele was deleted, was grown on medium containing 5-fluoroorotic acid (5-FOA; Boeke et al., 1984) to select a Uri– strain (Wilson et al., 2000). Strain 1978 (Table 2) was identified by Southern blotting and used for deletion of the ALS9-2 allele with the PCR-generated disruption cassette, as described above. Southern blotting indicated that both ALS9 alleles were deleted in strain 2028. The deletion region extended from 15 bp upstream of the ALS9 coding region to 29 bp downstream of the ALS9 stop codon. A Uri– version of strain 2028, named 2030 (Table 2), was used for subsequent transformation steps.
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). Primers QRT9-1F and QRT9-1R detected ALS9-1 transcript specifically, while primers QRT9-2F and QRT9-2R were specific for ALS9-2 (Table 1
). In strain CAI12, which is wild-type for ALS9, transcription of ALS9-1 was 1.1±0.2-fold of ALS9-2 transcript levels, suggesting that the alleles were transcribed equally. In strain 2028, transcription of ALS9-1 was below the detection limit of this assay, while transcription of ALS9-2 was 0.005±0.003-fold relative to strain CAI12. These results are consistent with the deletion of both ALS9 alleles in strain 2028.
Reintegration of wild-type ALS9 alleles into the als9/als9 strain.
Because the large sequence differences between ALS9-1 and ALS9-2 might reflect functional differences in the encoded proteins, each ALS9 allele was reintegrated separately into the als9/als9 strain. To construct the ALS9-2 reintegration cassette, primers ALS9dnF2 and ALS9dnR (Table 1) were used to amplify 710 bp of sequence downstream of ALS9. This fragment was digested with SstII/NgoMIV and cloned into plasmid pUL (Zhao et al., 2004) to yield plasmid 1320. Full-length ALS9-2 with 602 bp of upstream sequence was amplified from SC5314 with primers ALS9FullF and ALS9R (Table 1) and cloned into pCRBlunt (Invitrogen) to yield plasmid 1499. Primers ALS9URCF and ALS9dnRCR2 (Table 1) were used to amplify the URA3 gene and ALS9 downstream sequence cassette from plasmid 1320. This product was digested with SpeI/HindIII and cloned into plasmid 1499 downstream of ALS9-2 to yield plasmid 1541. The ALS9-1 reintegration cassette was produced by amplifying full-length ALS9-1 with 475 bp of upstream sequence (primers ALS9F2 and ALS9R; Table 1) from strain 1976, which has an ALS9-2 deletion. The PCR product was digested with AvrII/XhoI and cloned into plasmid 1320 (Zhao et al., 2004) to yield plasmid 2729. Digestion with AvrII/NgoMIV released the ALS9-1 cassette from plasmid 2729; HindIII/XhoI released the ALS9-2 cassette from plasmid 1541. These fragments were transformed individually into strain 2030 to yield strain 2740 (ALS9-1 reintegrated) and strain 2064 (ALS9-2 reintegrated). Correct transformants were confirmed by Southern blotting. DNA sequencing of both the transformation cassette and PCR-amplified sequences from the correct transformants verified the integrity of the ALS9 alleles in these strains. PCR and DNA sequencing were used to confirm integration of ALS9-1 under control of its own promoter in strain 2740 and integration of ALS9-2 under control of its own promoter in strain 2064. Real-time RT-PCR (Green et al., 2005b) was used to demonstrate that transcription levels from the reintegrated genes were similar to those in the CAI12 control strain. Transcription of ALS9-1 in strain 2740 was 1.8±0.2-fold of CAI12 levels, while transcription of ALS9-2 in strain 2740 was 0.001±0.001-fold compared to that of CAI12. Transcription of ALS9-1 in strain 2064 was 0.005±0.004-fold of CAI12 levels, while transcription of ALS9-2 was 1.1±0.3-fold relative to CAI12.
Construction of a C. albicans strain that produces a cell-surface Als9-2p/Als9-1p fusion protein.
Approximately 500 bp of ALS9-2 upstream sequence and the 5'-most 1266 bp of the ALS9-2 coding region were amplified from plasmid 1499 using primers ALS9F2 and 1499-PinAIR (Table 1). Primer 1499-PinAIR includes a silent mutation (T to C at nt 1260 of the ALS9-2 coding region) that creates a PinAI site. The PCR product was digested with AvrII/XhoI and cloned into plasmid 2729 to replace the ALS9-1 sequence. The resulting construct was called plasmid 2760. The tandem repeat and 3' domains of ALS9-1 were amplified from plasmid 2096 using primers 2096-PinAIFm and ALS9-XhoIR (Table 1). The same silent mutation described above was included in the 2096-PinAIFm primer to create a PinAI site in the PCR product. The PCR product was digested with PinAI/XhoI and cloned into plasmid 2760 to yield the ALS9-2/ALS9-1 fusion plasmid 2765. This construct encodes a chimeric Als9 protein that includes amino acids 1–420 of Als9-2p followed by amino acids 421–1854 of Als9-1p. The fusion construct was verified by DNA sequencing. Digestion with AvrII/NgoMIV released the fragment that included the fused genes and the URA3 selectable marker, flanked by ALS9 upstream and downstream sequences to direct its integration into strain 2030 (Table 2). Southern blotting and DNA sequencing identified the correct transformant (strain 2767; Table 2). PCR demonstrated that the fusion construct was integrated under control of the ALS9-2 promoter. The real-time RT-PCR primers for ALS9-1 were used to measure transcription of the fused allele relative to ALS9-1 transcription in strain CAI12. Transcript levels for the fused allele were 1.8±0.5-fold of CAI12 levels, indicating similar transcriptional activity between the strains.
Germ-tube formation assay.
The germ-tube assay format was changed from the one used in our previous work in order to require fewer input cells and to include multiple time points. Strains for testing were streaked from –80 °C glycerol stocks to YPD plates (1 % yeast extract, 2 % peptone, 2 % glucose with 2 % agar for plates), incubated for 24 h at 37 °C and stored at 4 °C for no more than 1 week. A single C. albicans colony was inoculated into 10 ml YPD and incubated for 16 h at 37 °C with 200 r.p.m. shaking. Cells were washed in Dulbecco's PBS without calcium or magnesium (DPBS; Cambrex catalogue no. 17-512Q) and counted, and 5x105 cells were added to 3 ml of either RPMI 1640 with L-glutamine (Invitrogen catalogue no. 11875-085) or Lee medium (Lee et al., 1975) pre-warmed to 37 °C in a 25 ml Erlenmeyer flask. The culture flasks were incubated at 37 °C with 200 r.p.m. shaking for 20, 40 or 60 min. The entire volume of the flask was collected by vacuum filtration over a 1 µm pore-size polycarbonate filter (Fisher Scientific). Filters were washed with 5 ml DPBS to remove residual medium. Filters were removed from the vacuum filtration unit, inverted onto glass slides and dried. Following removal of the filter from the slide, slides were heat-fixed, stained with crystal violet for 2 min, washed with tap water and dried. C. albicans yeast cells with a germ tube longer than one diameter of the mother cell were considered positive. The percentage of cells forming germ tubes was calculated by visually counting all cells present on five randomly selected fields on the slide. Approximately 200 cells were counted from each slide. Duplicate assays for each condition were conducted on two different days. Statistical significance was assessed using PROC MIXED in SAS (SAS Institute) with P
0.05 considered significant.
Testing for cell-wall defects.
C. albicans strains were tested for defects in the C. albicans cell wall by growing them on YPD agar plates containing one of the following compounds: NaCl (1, 1.5 M), SDS (0.02, 0.1 %), Calcofluor White (Sigma; 20, 50 µg ml–1), Congo red (Sigma; 100, 250 µg ml–1) or Hygromycin B (Invitrogen; 75, 200 µg ml–1). C. albicans strains were grown for 16 h at 37 °C and 200 r.p.m. shaking, as described above. Cells were harvested, washed in DPBS, counted and resuspended at 1x108 cells ml–1. Tenfold serial dilutions were prepared and 5 µl of each spotted onto the YPD agar plates with the appropriate test substance included. Plates were incubated for 24 h and growth of the test strains was compared to growth of a CAI12 positive control.
Other phenotypic assays.
Key strains used in this analysis were evaluated to ensure that their growth rate was not statistically different from that of the CAI12 control strain (Table 2), and that the degree of cellular aggregation observed did not increase compared to that of CAI12. Zhao et al. (2004) have described methods for these analyses. Formation and analysis of biofilm on a silicone elastomer catheter substrate followed the method of Kuhn et al. (2002). Methods for measuring adhesion to vascular endothelial cells, adhesion to buccal epithelial cells and relative pathogenicity in the reconstituted human epithelium (RHE) tissue model have been published (Zhao et al., 2004). Adhesion and cellular aggregation assays used C. albicans yeast forms inoculated from a YPD stock plate prepared as described above. A single colony was resuspended in 1 ml YPD; 10 µl of the suspension was inoculated into a 10 ml culture, which was incubated for 16 h at 37 °C and 200 r.p.m. shaking. Cells were washed in DPBS prior to the assay. Methods for real-time RT-PCR analysis of ALS gene expression have been published (Green et al., 2005b).
Adhesion to laminin was evaluated in a six-well plate format using modifications of the fibronectin adhesion assay described by Zhao et al. (2004). Briefly, laminin (Sigma catalogue no. L-2020) was diluted to 10 µg ml–1 in DPBS and 1 ml added to each tissue-culture well. Plates were incubated overnight at 37 °C. C. albicans yeast forms were cultured in YPD as described above, washed in DPBS and resuspended at a density of 500 cells ml–1. Laminin solution was removed immediately before the assay and each well rinsed twice with 2 ml DPBS. One millilitre of the yeast suspension was added to each coated well and plates were incubated at 37 °C for 30 min. Washing and counting of adherent colonies, calculation of percentage adherence, experimental replication and statistical analysis were the same as described for the fibronectin adhesion assay (Zhao et al., 2004).
Collection of clinical C. albicans isolates and PCR genotyping reactions.
The collection of 196 isolates used in this study was obtained from three previously analysed populations (Pujol et al., 1997, 2002; Blignaut et al., 2002). The collection included 100 bloodstream isolates, 81 oral isolates, 10 vaginal isolates and five from other body sites. PCR genotyping was used to determine which ALS9 domains were present in each strain. Primers ALS9-1F and ALS9-1R were used to detect the ALS9-1 5' domain, while primers ALS9-2F and ALS9-2R were used to detect the ALS9-2 5' domain (Table 1, Fig. 1). The size of VB1 was determined using primers VB1F and VB1R (Table 1, Fig. 1). A smaller product is characteristic of the ALS9-1 prototype allele as defined in strain SC5314 (GenBank accession no. AY269423), while a larger product is associated with the ALS9-2 allele (GenBank accession no. AY269422; Zhao et al., 2003). The presence of both PCR product sizes suggests that both allelic sequences are present, but does not associate a specific VB1 sequence with a particular 5' domain. Similarly, the size of VB2 was assessed using primers VB2F and VB2R with a smaller product amplified from the ALS9-1 allele and a larger product from the ALS9-2 allele (Fig. 1). Amplification of both product sizes was observed for heterozygous strains. PCR products were separated on agarose gels using a SC5314 standard to judge relative fragment sizes and assign allelic designations.
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RESULTS |
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/als9 mutant (strain 2028) was to assess its adhesion to cell types relevant to C. albicans host–pathogen interactions. These assays used C. albicans yeast forms that were grown at 37 °C in YPD medium for 16 h, as described in Methods. ALS9 is transcribed in C. albicans cells grown in this manner (Green et al., 2005b), suggesting that the growth condition is appropriate for assessing Als9p function. The als9/als9 strain was significantly less adherent to human vascular endothelial cell monolayers than the control strain CAI12 (P=0.05; Fig. 2a). Although reintegration of a wild-type ALS9-1 allele did not complement the mutant phenotype (P=0.02), transformation of ALS9-2 into the mutant strain restored wild-type adhesion levels (Fig. 2a). These results suggest functional differences between the proteins encoded by the ALS9 alleles. One possible explanation for this result is that the reintegrated ALS9-1 allele had defects and did not produce a viable protein. Since antibodies specific for Als9p are not available, other verification steps were pursued. DNA sequencing of ALS9-1 that was PCR-amplified from the reintegrant strain verified that its sequence was identical to that derived from the parental strain. Further, the adhesion phenotype of strain 1976 was tested. Strain 1976 is hemizygous (ALS9-1/als9-2) and was derived during construction of the als9/als9 mutant (Table 1). Compared to the percentage adherence for the CAI12 control (15.8±1.6), the percentage adherence for the hemizygous strain 1976 (9.7±1.6) was similar to those of the mutant strain 2028 (9.1±1.6) and the ALS9-1 reintegrant strain 2740 (9.9±1.6). The percentage adherence for strains 1976, 2028 and 2740 was significantly lower than that of the CAI12 control (P0.006). These results are consistent with the conclusion that there is an unequal contribution of these ALS9 alleles to adhesion between C. albicans and human vascular endothelial cells.
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/als9 strain 2028 (Fig. 1). The resulting strain, called 2767, had wild-type adhesion levels, suggesting that the fusion protein restored the missing Als9p function. This result suggests that adhesive function for Als9-2p largely resides within the N-terminal domain.
The als9/als9 mutant was also tested for adhesion to buccal epithelial cells (Fig. 2b) and laminin-coated plastic plates (Fig. 2c). Testing for adhesion to laminin was prompted by a report that S. cerevisiae becomes adherent to laminin when overexpressing ALS9 (Sheppard et al., 2004). In each assay, deletion of ALS9 did not have a significant effect on the phenotype of the C. albicans strain, suggesting that although Als9-2p may play a measurable role in adhesion to endothelial cells, the Als9 proteins are not important for buccal cell or laminin adhesion.
Other phenotypic analyses of the C. albicans als9/als9 strain
The C. albicans als9/als9 strain 2028 was tested in other assays to assess its phenotype. Inoculation of the buccal RHE model with the als9/als9 mutant and a control strain showed no difference between the strains with respect to epithelial destruction, adherence or hypha formation (Fig. 3). Comparison between the als9/als9 strain and the CAI12 control strain showed no differences in growth on YPD agar that contained cell-wall-stressing agents, including NaCl, SDS, Calcofluor White, Congo red or Hygromycin B. The lack of an effect of these agents on growth of the als9/als9 mutant suggests that cell-wall structure and glycosylation are not affected by ALS9 deletion (Elorza et al., 1983; Kopecka & Gabriel, 1992; Dean, 1995; Machi et al., 2004). The als9/als9 mutant strain also did not exhibit any significant differences in germ-tube formation over the course of 1 h when grown in RPMI 1640 or Lee medium (Table 3). Finally, although growth of the als9/als9 strain in a model of biofilm formation on a silicone elastomer catheter surface (Kuhn et al., 2002) resulted in decreased biomass compared to the CAI12 control strain (1.21±0.16 versus 1.4±0.16 mg, respectively), this difference was not significant statistically (P=0.06). Compensatory changes in expression of other ALS genes in the als9/als9 strain were ruled out using real-time RT-PCR analysis of ALS gene expression (Green et al., 2005b).
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). In a previous study, PCR genotyping of the ALS9 alleles in 30 C. albicans clinical isolates showed that, in each strain, at least one allele had ALS9-2 sequences in the 5' domain (Zhao et al., 2003). This analysis was expanded to 196 C. albicans clinical isolates from various body sites (Table 4). PCR primers were used that could distinguish between the ALS9-1 and ALS9-2 sequences within the 5' domain and also within the VB1 and VB2 regions of the 3' domain (Table 1, Fig. 1). Within the 5' domain, approximately half of the isolates were positive for ALS9-1, while all isolates had at least one ALS9-2 allele (Table 4). Therefore, among the C. albicans isolates tested, approximately half were heterozygous (ALS9-1/ALS9-2), while the others appeared to be ALS9-2 homozygotes, although it is formally possible for these strains to encode other ALS9 alleles. Within VB1 of the 3' domain, ALS9-2 sequences were common, while ALS9-1 sequences were rather rare (Table 4). Nearly half of the C. albicans isolates examined had both ALS9-1 and ALS9-2 sequences within VB1. Within the VB2 region, ALS9-2 sequences were far more common than ALS9-1 sequences, and nearly one-quarter of the strains examined had both ALS9-1 and ALS9-2 sequence types (Table 4). Within both VB1 and VB2, a low number of strains had sequences that did not conform to either the ALS9-1 or ALS9-2 types that were defined from strain SC5314. This result suggests even greater sequence variability within the ALS9 3' domain.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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/als9 mutant strain showed that the most notable effect of deleting ALS9 was a significant decrease in adhesion to human vascular endothelial cell monolayers. Reintegration of the ALS9-2 allele complemented the mutation, while the ALS9-1 allele did not. Fusion of the Als9-2p N-terminal domain (amino acids 1–420) to the tandem repeats and C-terminal domain of Als9-1p (amino acids 421–1854) produced a protein that was also capable of complementing the mutant phenotype. This observation is consistent with the conclusion that adhesive function resides within the Als9p N-terminal domain. In previous work, fusion of the Als3p N-terminal domain (amino acids 1–430) to the C-terminal 323 amino acids of S. cerevisiae -agglutinin was sufficient to increase adhesion of a C. albicans als3/als3 strain to buccal epithelial cells (Zhao et al., 2006). Although most adhesive activity is believed to be associated with the N-terminal domain, experiments that involve overexpression of ALS genes in S. cerevisiae suggest that some adhesive activity may be associated with other domains in Als proteins (Loza et al., 2004; Rauceo et al., 2006).
Assay of C. albicans als/als mutant strains and overexpression of ALS genes in S. cerevisiae are the two main methods that have been utilized to evaluate Als protein function (Loza et al., 2004; Rauceo et al., 2006; Zhao et al., 2004, 2005). In some cases, results from the two methods are in agreement; however, more often, the two methods provide different views of Als protein function. Advantages of the S. cerevisiae system include working in a tractable model system using relatively simple genetic manipulations, and the ability to address hypotheses regarding adhesive function in a low-adhesion background. These advantages are gained at the expense of differences in genetic code (Santos & Tuite, 1995) and glycosylation patterns (Herrero et al., 2002), the tendency to draw functional conclusions from testing a single ALS allele, the need to utilize artificial gene-expression levels and patterns, the potential for protein overproduction to create cell-wall artefacts including mislocalized products, the inability to address C. albicans-specific Als protein function and/or interaction of Als proteins with others, and the limited ability to address non-adhesive phenotypes. While these disadvantages of S. cerevisiae-based studies are not present when working with the native C. albicans, C. albicans is inherently sticky, which complicates adhesion assays. Working in C. albicans also requires an understanding of ALS gene-expression patterns to select assay conditions, and phenotypic changes may be masked by compensatory gene-expression changes (Zhao et al., 2005). Differences in results between the C. albicans- (Fig. 2) and S. cerevisiae-based (Sheppard et al., 2004) approaches to studying Als9p function are obvious. When overexpressed in S. cerevisiae, ALS9 did not confer adhesion to vascular endothelial cell monolayers (Sheppard et al., 2004). Because the allele tested was not specified, it is possible that ALS9-1 was used, which would account for the difference in results compared to our C. albicans analysis (Fig. 2). Overexpression of ALS9 in S. cerevisiae, which presumably coats the cell surface in heterologous protein, resulted in adhesion to laminin (Sheppard et al., 2004). Although statistically significant, this percentage adhesion was very low (
3 %) suggesting, at best, a minimal contribution to laminin adhesion from the Als9 protein.
A key issue in extrapolating S. cerevisiae-based Als protein functional data to the C. albicans system is how well the S. cerevisiae model mimics native C. albicans. High-level overexpression of ALS genes in S. cerevisiae would best mimic C. albicans growth stages at which a specific ALS gene is highly expressed. While some ALS genes are known to undergo large changes in expression level from nearly undetectable to very high (ALS1 and ALS3, for example; Zhao et al., 2004), others appear to be transcribed only at very low levels (ALS6 and ALS7; Green et al., 2004, 2005a, b, 2006). ALS9 ranks in the middle of this ALS gene-expression hierarchy: clearly transcribed under all of the conditions tested, but to date, observed at expression levels that are well below those of the most highly expressed ALS genes (Green et al., 2004, 2005a, b, 2006). For each of the Als proteins, the protein abundance and localization on the cell surface are almost unknown. Many variations in Als protein localization can be envisioned, from large areas or small focal patches of high protein concentration, to diffuse arrangements of a sparse number of proteins across the cell surface. Knowing the Als protein localization on the C. albicans cell surface would provide a better understanding of how well the S. cerevisiae model mimics the native system.
In many of the phenotypic assays conducted, the als9/als9 strain failed to show a difference from the wild-type control strain. One potential explanation for these results is compensatory expression of other C. albicans ALS genes to confer a wild-type phenotype. This possibility is suggested by work that shows a relationship between expression of ALS2 and ALS4 in which a reduction in the transcriptional level of one gene results in increased transcription of the other (Zhao et al., 2005). Compensatory ALS gene expression in response to ALS9 deletion has been ruled out by real-time RT-PCR analysis (Green et al., 2005b). Compensatory expression of non-ALS genes is possible, but if applicable to the als9/als9 mutant, would have to involve genes that function in all processes tested except endothelial cell adhesion. These limitations decrease the likelihood that compensatory expression is a factor in functional analyses of the als9/als9 strain.
Within the ALS family, ALS9 is the most extreme example of allelic variability that has been described. While all eight ALS genes have variability in the number of tandem-repeat copies within the central coding domain, sequence variability in the non-tandem-repeat domains is less documented. Examples include ALS5, which has sequence variability within the 5' domain that surpasses the mean sequence variability present in a representative list of C. albicans genes (Hoyer & Hecht, 2001), and ALS7, which has a complex repeated structure within the 3' domain that lends considerable allelic variability to this locus (Zhang et al., 2003). For ALS3, the presence of 12 instead of nine tandem-repeat copies in the central domain results in a higher percentage adherence of C. albicans to both endothelial and epithelial cell monolayers (Oh et al., 2005). For ALS9, allele-specific function is associated with the large sequence differences (11 % at the nucleotide level; 16 % at the amino acid level) within the ALS9 5' domain. These differences may affect key residues within the Als9p N-terminal domain that are involved in binding to endothelial cells. Structural analysis of the Als N-terminal domain will answer such questions and could exploit the sequence and functional differences described here to better understand Als9p interaction with host cells.
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ACKNOWLEDGEMENTS |
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Edited by: M. Molina
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Received 7 December 2006;
revised 28 February 2007;
accepted 2 March 2007.
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