1 Department of Veterinary Pathobiology, University of Illinois at Urbana-Champaign, Urbana, IL 61802, USA
2 Department of Biological Sciences, The University of Iowa, Iowa City, IA 52242, USA
Correspondence
Lois L. Hoyer
lhoyer{at}uiuc.edu
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ABSTRACT |
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INTRODUCTION |
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Recently, adhesive function was demonstrated for Als3p, a protein that is produced on C. albicans germ tubes and hyphae (Hoyer et al., 1998; Zhao et al., 2004
). Disruption of ALS3 results in cells with reduced adherence to vascular endothelial and buccal epithelial cells, as well as a marked reduction in destruction of epithelial cells in the reconstituted human epithelium in vitro model of candidiasis (Zhao et al., 2004
). Allele-specific lengths of the tandem repeat domain in ALS3 were reported previously (Hoyer et al., 1998
; Zhao et al., 2004
). In strain SC5314, which is used most often for constructing C. albicans mutants (Fonzi & Irwin, 1993
), DNA sequencing verified that one ALS3 allele encodes 9 copies of the tandem repeat sequence, while the other encodes 12 copies (Zhao et al., 2004
). Similar variability within the tandem repeat domain has been reported for ALS1 (Lott et al., 1999
), where the number of repeat sequences per allele ranged from 4 to 37, with 16 copies the most frequent. Analysis of the tandem repeat domain of ALS7 detected alleles that varied from 1 to 33 tandem copies, with 17 copies the most frequent (Zhang et al., 2003
). Both of these studies demonstrated that some ALS alleles are more common than others and that, among alleles of a specific ALS gene, variability in the number of tandem repeat copies can be large.
During efforts to functionally characterize Als3p, we created C. albicans strains heterozygous for each ALS3 allele. In the studies presented here, we demonstrate variability in adhesion of these strains, with greater adherence associated with the presence of the larger ALS3 allele. These results suggested functional differences between Als3p proteins in strain SC5314 depending on the number of tandem repeat copies in the central domain. This observation prompted examination of the distribution of ALS3 alleles, discriminated by the number of tandem repeat copies, among a larger population of C. albicans strains. For this work, we focused on genetically distinct groups of C. albicans that were assigned to five major clades (I, II, III, SA and E) by DNA fingerprinting with the species-specific complex probe Ca3 (Pujol et al., 1997, 2002
; Soll, 2000
; Blignaut et al., 2002
; Soll & Pujol, 2003
). One of these clades, SA, is relatively specific to South Africa, while a second clade, E, is relatively specific to Europe. A third clade, II, appears to be absent in the Southwest USA. Recently, it was demonstrated that all tested strains of C. albicans naturally resistant to 5-fluorocytosine (MIC
32 µg ml1) and the great majority of strains less susceptible to 5-fluorocytosine (MIC
5 µg ml1) are members of clade I (Pujol et al., 2004
) and that clade-specific 5-fluorocytosine resistance is due to a single nucleotide change in the FUR1 gene (Dodgson et al., 2004
). The maintenance of deep-rooted clades side by side in the same geographical area, as well as the relative uniformity of the less susceptible 5-fluorocytosine phenotype in clade I, has led to the suggestion that recombination may occur frequently within a clade, but infrequently between clades (Soll & Pujol, 2003
; Pujol et al., 2004
). This hypothesis suggested that other clade-specific differences, perhaps for ALS3 alleles, are likely to exist across the general population of C. albicans strains.
Using PCR-based methods to discriminate the number of tandem repeat copies in the central domain of the ALS3 alleles of representative strains in each of the five major C. albicans clades, we demonstrate dramatic clade-specific differences in the distribution of ALS3 alleles. Our results also reveal that, on average, the two ALS3 alleles of a strain encode one long and one short Als3p, suggesting the possibility of a functional role for the long and short combination. These data, combined with observed differences in adhesion contributed by shorter and longer ALS3 alleles in strain SC5314, suggest that the variability in ALS3 alleles among the five clades identified in this study may also reflect functional differences.
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METHODS |
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Methods for constructing strains heterozygous for each ALS3 allele and als3/als3
null strains were described by Zhao et al. (2004)
. The set of strains was constructed twice in its entirety, starting with strain CAI4 (Fonzi & Irwin, 1993
). Strain CAI12, used as a control for the studies described here, was created by reintegration of URA3 into its native locus in the CAI4 background (Porta et al., 1999
). Both CAI4 and CAI12 were generous gifts from Dr William Fonzi (Georgetown University, Washington, DC, USA). C. albicans SC5314 encodes a larger ALS3 allele (ALS3(12); 12 copies of the 108 bp tandem repeat sequence in the central domain; GenBank accession no. AY223552) and a smaller ALS3 allele (ALS3(9); 9 tandem repeat copies; GenBank accession no. AY223551). Strains resulting from two independent construction efforts were 1704 and 1893 (ALS3(12)/als3
(9) URA3), 1702 and 1897 (als3
(12)/ALS3(9) URA3), and 1843 and 1954 (als3
(12)/als3
(9) URA3). There was no statistically significant difference between any of the strains for growth rate, the ability to form germ tubes in RPMI 1640 medium, or cellular aggregation. Methods described by Zhao et al. (2004)
were used to evaluate these characteristics.
C. albicans adhesion to human cell monolayers.
The method to measure adherence of C. albicans strains to human umbilical vein endothelial cells was described by Zhao et al. (2004). These assays were conducted in a six-well tissue culture plate format using modifications of the method described by Ibrahim et al. (1995)
. Six-well plate assays were also conducted to measure adhesion of C. albicans to human pharyngeal epithelial cells. The epithelial cell line was purchased from the American Type Culture Collection (HTB-43, also called FaDu) and grown in Minimal Essential Medium (MEM; Gibco) containing 10 % fetal bovine serum (Gibco). Growth medium was supplemented with 2 mM L-glutamine and 1 mM sodium pyruvate; no antimicrobials were added. Confluent epithelial cell monolayers were grown in six-well tissue culture plates as described for the endothelial cell assay (Zhao et al., 2004
). C. albicans strains were cultured in RPMI 1640 to form germ tubes prior to the adhesion assay as previously described (Zhao et al., 2004
) and 500 cells with germ tubes added to each assay well. Following incubation, the monolayer was washed once with 5 ml Dulbecco's phosphate-buffered saline (no calcium or magnesium). Agar overlay of each well, viable counts of C. albicans c.f.u. and calculation of percentage adherence were carried out as described before (Zhao et al., 2004
). In a typical assay, wild-type CAI12 showed approximately 150 to 200 adherent c.f.u. per well. Six assay-wells were used for each strain in three separate experiments. The statistical significance of results was evaluated using a mixed-model analysis of variance (PROC MIXED in SAS). Separation of means was performed using the LSMEANS option. Specific comparisons between strains or combinations of strains were calculated using the ESTIMATE function.
ALS3 genotyping.
Genomic DNA was extracted from the C. albicans strains using a previously described method (Hoyer et al., 1995). The size of the tandem repeat domain in each ALS allele was determined by PCR using two independent primer pairs. Use of two primer pairs provided an additional control for the accuracy of the results. Each primer pair contained one that annealed 5' and another 3' of the tandem repeat domain. The first primer pair was ALS3GenoF (5'-ACC TTA CCA TTC GAT CCT AAC C-3') and ALS3GenoR (5'-GAT GGG GAT TGT GAA GTG G-3'). The second primer pair was ALS3GenoF2 (5'-CCA CAA CAC ATA CTA ATC CAA CTG A-3') and ALS3GenoR2 (5'-TGT AGA CCA CAA AGT TGT ATG GTT G-3'). Taq polymerase (Invitrogen) was used with both primer pairs. Reactions with the first primer pair (ALS3GenoF and ALS3GenoR) used Invitrogen Taq polymerase buffer with 1 mM MgCl2. Reactions were heated for 5 min at 94 °C followed by 35 cycles of 94 °C (30 s), 57 °C (30 s) 72 °C (3 min). A final 72 °C (7 min) extension completed the reaction. The second primer pair (ALS3GenoF2 and ALS3GenoR2) was used under similar conditions except for a difference in buffer (10 mM Tris/HCl, pH 8·8, 25 mM KCl, 1·5 mM MgCl2) and annealing temperature (65 °C). PCR products were separated on 3·5 % polyacrylamide-Tris/borate/EDTA (TBE) gels and stained with ethidium bromide. Amplicon sizes were determined by comparison with the 1 kb ladder (Invitrogen) and with ALS3 amplification products from strain SC5314 genomic DNA. In this strain, allele size was determined by DNA sequencing. Alleles have either 9 (GenBank accession no. AY223551) or 12 (AY223552) copies of the 108 bp tandem repeat in the central domain. The two pairs of ALS3 genotyping PCR primers were verified against Southern blots of genomic DNA from a variety of C. albicans strains (J. A. Nuessen & L. L. Hoyer, unpublished data). Genomic DNA was digested with either HindIII/NciI or FokI/ApaLI and hybridized with an ALS3-specific fragment from the 3' end of the gene that was contained within the restriction sites. The allelic tandem-repeat copy number and allelic combination derived for each strain was the same when determined using Southern blots or the PCR primer pairs described above.
RT-PCR reactions.
RT-PCR analysis used the method described by Green et al. (2004). C. albicans cells were grown overnight in YPD medium (per litre: 10 g yeast extract, 10 g peptone, 20 g glucose), washed in PBS, counted with a haemocytometer and released into prewarmed RPMI 1640 medium at a density of 5x106 cells ml1. These cultures were grown for 1 h at 37 °C and 200 r.p.m. shaking; at this point, germ tubes were visible microscopically. Cells were harvested by filtration, flash-frozen in dry ice/ethanol and stored at 80 °C. RNA was extracted using a hot phenol method (Collart & Oliviero, 1993
), DNase treated (Ambion) and quantified spectrophotometrically. Synthesis of cDNA used 2 µg total RNA and the SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen), primed with random hexamers. One-tenth volume was added to PCR reactions using the ALS3GenoF and ALS3GenoR primers and amplified as described above. Equal quantities of RNA without addition of reverse transcriptase were run to ensure that the observed products were not amplified from residual genomic DNA.
Statistical methods.
The distributions of the number of tandem repeat copies in the different clades and the distributions of the difference in the number of tandem repeat copies per strain in the different clades were evaluated using the non-parametric KolmogorovSmirnov test. This approach was selected because the data were not distributed normally. The non-randomness of allelic combinations was calculated using Fisher's Exact test. Allele frequencies were used to calculate the expected frequencies of the diploid genotypes under the null hypothesis that combinations of alleles were random. The observed and expected numbers were then compared using Fisher's Exact test. Fst statistics were assessed using the MULTILOCUS 1.3 software package, available at http://www.agapow.net/software/multilocus/ (Agapow & Burt, 2001). The program calculates Weir's formulation of Wright's Fst (Weir, 1996
). The calculated statistic,
, is an approximation of the Fst. Fst statistics are used in population genetics studies as an estimate of the level of subdivision among populations to test their differentiation. An Fst of 0 means that there is no subdivision (the populations do not show any statistical difference), whereas an Fst of 1 means that populations are completely isolated genetically (the populations have nothing in common). In our study, Fst statistics were used to test if ALS3 gene frequencies reflected the genetic divergence shown by Ca3 fingerprinting among C. albicans clades and to determine whether clade differences in the study were a function of the geographic origin of the strains analysed.
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RESULTS |
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Range of alleles based on number of tandem repeat copies
A collection of strains was randomly selected to represent the five major C. albicans clades (Soll & Pujol, 2003). This collection included 51 strains from clade I, 47 strains from clade II, 40 from clade III, 22 from clade E and 36 from clade SA. The proportions of the different ALS3 alleles in the general C. albicans population were assessed by PCR using primers that annealed 5' and 3' of the tandem repeat domain to determine the number of tandem repeat copies in each allele. The number of copies of the tandem repeat sequence was calculated for each of the two alleles in the 196 strains, resulting in 392 analysed ALS3 alleles. The range of tandem repeat copy number was between 6 and 19 (Fig. 3
, Table 1
). The alleles did not fall into a Poisson distribution. The majority of the alleles (78 %) had either 10 copies (28 %), 11 copies (22 %) or 13 copies (28 %) of the repeat sequence. The remaining 22 % of the alleles represented all other categories (Fig. 3
, Table 1
). Alleles that contained 12 or 14 copies of the tandem repeat sequence were under-represented compared to the other categories (Fig. 3
). Overall, only 10 % of the total alleles had 9 tandem repeat copies or fewer, while 82 % of the alleles had between 10 and 13 repeat copies. Larger repeat copy numbers (>14) were less common, representing only 8 % of the total alleles.
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Statistical comparisons between the clades were also made, based on the relative distribution of tandem repeat copies per allele (Table 1). These comparisons revealed that the relative distribution of tandem repeat copies per allele differed significantly between every pair of clades except II and E. Therefore, although clades II and III appeared quite similar with respect to the most prevalent alleles, they were still significantly different with respect to the relative distribution of allelic sizes.
Clade differences in allelic pairing
The pairing of ALS3 alleles in strain SC5314 matched one allele with strong adhesive function (12 tandem repeat copies) with another that did not contribute substantially to adhesion in the cell monolayer assays (9 tandem repeat copies; Fig. 2). Because of this allelic pairing, genotyping data were analysed to determine how ALS3 alleles are paired in each clade. In the 196 strains analysed, the majority of strains (84 %) were heterozygous for ALS3 alleles; this trend was also present within each clade (Table 2
). However, in some clades, such as clade I, almost all strains were heterozygous for ALS3 alleles, while in other clades such as E, over one-third of the strains were homozygous. The homozygous strains in clade E encoded 11 copies of the tandem repeat in each ALS3 allele, consistent with allelic frequencies summarized in Table 1
.
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Fst statistics were calculated to determine if ALS3 allelic representation in each clade reflected the genetic divergence shown by Ca3 fingerprinting. Results of this analysis showed strong ALS3 genetic differentiation between each pair of clades (Table 2). Fst statistics were also used to determine whether ALS3 allelic composition of the strains was a function of the geographical origin of the strain. For example, 22 of the 47 clade II strains were from South Africa. The
value for comparison of the clade II South African population to the clade II non-South African strains was 0·0081 (P=0·65) showing that the two populations were not differentiated. These data further demonstrated the divergence in ALS3 alleles observed between clades.
The frequency of different allelic combinations in the entire strain collection was also examined. Some combinations were prevalent, such as the pairing of one allele containing 10 repeat copies with another containing 13 repeat copies (denoted 10/13). This allelic pair was found in 82 % of clade I strains and was also the most frequent pair in clade III (35 %). In contrast, the 10/13 combination was almost absent in strains from clades II, E and SA (Table 2). In these clades, the 11/13 combination (for II and E) or the 10/15 combination (for SA) predominated.
Differences in allelic pairing are, in part, a result of the differences in prevalent alleles between the clades. However, the combinations observed are unlikely to be random. To assess the non-randomness of allelic combinations, we tested the total collection and then individual clades for the expected proportion of the 10/10 and 13/13 combinations, using Fisher's Exact test. The entire pool of 196 analysed strains was found to have a deficit in the 10/10 combination (P=0·046) and in the 13/13 combination (P=0·031). When clades were analysed, group I alone showed these deficits (P<0·001). No clade I isolates with a 10/10 combination were found among the 51 strains tested, even though the expected number was 9·9. Only two clade I isolates with a 13/13 combination were found, even though the expected number was 11·3. In contrast, non-clade I strains contained the expected number of 10/10 and 13/13 isolates, and, therefore, no deficit. We then tested if this type of deficit was restricted to clade I or was true for other alleles in other clades. Analysis of the 11/13 allelic pairing did not reveal non-random distribution of this allelic pair either in the whole population or in specific clades.
A qualitative survey of ALS3 allelic combinations suggested that in individual strains, short and long alleles were often paired in the same strain. To test whether this observation was significant, we grouped the shorter alleles together (fewer than 12 repeat copies) and considered them to represent a single allele that we called short. We also grouped the longer alleles together (12 or more repeat copies) and considered them to represent a single allele called long. Twelve repeat copies were chosen as the boundary based on the results of the functional analysis (Fig. 2) and the distribution of alleles (Fig. 3
), which showed a dramatic lack of alleles with 12 repeat copies compared to those with 10, 11 or 13. We found a statistically significant excess of strains with a short and long allelic pair in the total population (P<0·001). The excess was still significant (P<0·001) when the 51 clade I strains were removed from the analysis. This result demonstrated that the significance of long/short allelic pairing was not solely due to the preponderance of 10/13 strains in clade I, but was more of a general phenomenon characteristic of the whole C. albicans population.
Clades are similar for mean number of tandem repeat copies per allele
Even though differences existed in predominant alleles and in the relative distribution of alleles, the mean number of repeats per allele was similar among the clades. The range was 10·8±1·6 to 11·7±2·5, a difference of 0·9 repeats (Table 1). The mean number of repeats for the ALS3 alleles of two clades (II, E) was the same as the mean for the alleles of all clades, and the mean number for the ALS3 alleles of two more clades (I, SA) differed from the mean by only 0·3 repeats. The small differences observed in the mean number of repeats per allele was remarkable when one considers the dramatic differences demonstrated between clades in the prevalent ALS3 alleles, and in the distribution of tandem repeat copy numbers, the difference in the number of repeats between the alleles of each isolate and the non-random combinations of alleles in individual strains.
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DISCUSSION |
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Currently, the functional model for Als proteins is that an adhesive N-terminal domain is projected away from the cell surface by the highly glycosylated tandem repeat and C-terminal domains. This relationship between structure and function has been demonstrated for the Epa1p protein of Candida glabrata (Frieman et al., 2002). With this model, the longer ALS3 alleles would produce proteins that projected the N-terminal domain farther from the C. albicans cell surface, presumably lending an advantage in contacting host ligands. However, results from our study showed that C. albicans strains tend to be heterozygous for ALS3 alleles, with strains commonly encoding a longer and shorter allele. The prevalence of this allelic combination suggests that it is advantageous to the organism and may be selected for in nature. The role of the shorter Als3p protein in adhesion is not immediately clear, since changes in sequenced alleles appear to be limited to the tandem repeat domain and C-terminal domain, with the amino acid sequence of the N-terminal domain the same for the encoded proteins. Unless adhesive capacity resides in a domain in addition to the N-terminal domain, adhesion of the proteins produced from shorter alleles will not be less avid than that from larger alleles, but may only result in a less accessible protein on the C. albicans cell surface. In the strains assayed, expression of the two alleles appears to be under the same control, and differential allelic expression has yet to be observed (Hoyer et al., 1998
). These results suggest that the role of the protein encoded by the shorter allele may be distinct from adhesion and lead to the possibility that strains containing two shorter alleles may be less adherent and possibly less virulent than those with the long/short allele combination. This hypothesis remains to be tested.
The tendency to find a longer and shorter allele paired in C. albicans strains suggests selection of distinct alleles in the same strain. This result contrasts with the study of ALS7 allelic variation that showed that alleles in the same strain are more similar to each other than expected by chance (Zhang et al., 2003). From these comparisons emerges the conclusion that distinct selective pressures are placed on the various ALS genes. Comparison of DNA sequences and length of the tandem repeat domain for ALS3 and other ALS genes also supports this conclusion of differing selective pressures within the ALS family. For example, despite the range of changes in tandem repeat copy number between ALS3 alleles, the sequence of the N-terminal domain of the encoded protein remains constant, suggesting that there is selective pressure to maintain it. This result differs from that for Als5p, in which amino acid sequence differences within the N-terminal domain are more frequent than those noted for other C. albicans proteins (Hoyer & Hecht, 2001
; Zhao et al., 2003
). However, for Als5p, there appears to be strong pressure to maintain the number of tandem repeat copies within the central domain in a range much more narrow than for Als3p (R. Jajko & L. L. Hoyer, unpublished observations). Large differences in amino acid sequence within the N-terminal domain of Als9p have also been found (Zhao et al., 2003
), and other ALS genes have a far more broad variation in repeat copy number within the central domain (Lott et al., 1999
; Zhang et al., 2003
; J. A. Nuessen & L. L. Hoyer, unpublished data). Analysis of each gene across the various clades will define these relationships and provide conclusions regarding Als proteins that can be generalized across the C. albicans strain population.
Our analysis of allelism of ALS3 based on differences in the tandem repeat copy number in the central domain also highlighted differences between the five major genetic clades of C. albicans (Soll & Pujol, 2003). The clade-specific differences observed were not a function of the geographical origin of the strains examined. We found that although ALS3 alleles contained between 6 and 19 tandem repeat copies, the majority of alleles (78 %) had 10, 11 or 13 copies, suggesting that these lengths were selected either based on function or as a result of the mechanism of reorganization (e.g. unequal crossing over) (Pujol et al., 1999
).
The distribution of alleles differed significantly amongst clades. For instance, while the great majority of clade I isolates carried 10 and 13 tandem repeat alleles, the majority of clade E isolates carried the 11 tandem repeat allele, the majority of clade II isolates carried the 11 and 13 tandem repeat alleles and the majority of clade SA isolates carried the 10 and 15 tandem repeat alleles. What is significant about these results is not the identity of the prevalent alleles, but rather the fact that each clade differed so markedly in allelic prevalence. These differences between clades suggest that while there is some degree of uniformity of prevalent alleles within clades, there is far less uniformity amongst clades. Recently, Pujol et al. (2004) discovered that natural 5-fluorocytosine (flucytosine)-resistant strains were members of a single clade and the great majority of clade I isolates were less susceptible to 5-fluorocytosine than members of all other clades. Resistance and decreased susceptibility were due to a single allele with a mutation in one nucleotide (Dodgson et al., 2004
). The former were homozygous and the latter heterozygous for the allele. The authors interpreted these results to suggest that while recombination occurred between strains within a clade, it was far rarer between strains of different clades. Clade-specific differences in the prevalence of particular ALS3 alleles could be explained similarly. Alternatively, different selective pressures on isolates from different clades may determine the most common clade-specific ALS3 alleles, or clade-specific differences in the mechanisms determining tandem repeat number may exist. Functional analysis of additional ALS3 alleles from a wider population of C. albicans isolates will further our understanding of the importance of tandem repeat copy number and the presence of heterozygous ALS3 alleles within the same strain. The genotyping of C. albicans strains presented here prioritizes alleles for these additional studies. Similar analyses for other ALS genes will indicate how broadly applicable these themes are across the ALS family.
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ACKNOWLEDGEMENTS |
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Received 6 October 2004;
revised 30 November 2004;
accepted 2 December 2004.
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