Agglutinin-like Sequence Gene Research Articles

Molecular detection of agglutinin-like sequence 1 gene in Candida albicans that is isolated from diabetic foot patients

Objectives: Candida albicans is a microbe living within the natural human flora and is found in the upper respiratory tract, mouth, intestines, and vagina. C. albicans is able to cause infections that range from superficial infections of the skin to life-threatening systemic infections. Aim of Study: Detection of virulence gene agglutinin-like sequence (ALS) 1 by using molecular technology from clinical samples (C. albicans) that is isolated from ulcers of diabetic foot patients. Materials and Methods: This work was done on 235 patients who had diabetic foot patients admitted to the Specialized Center for Endocrinology and Diabetes (Baghdad Health Department/Rusafa) for the treatment of diabetic foot ulcers during November 2020 till March 2021. The collected samples of diabetic foot ulcers were cultured on different media (Sabouraud's dextrose agar with chloramphenicol for selective isolation and culturing of yeasts and HiCrome Candida Medium) for isolation of C. albicans fungus as well as automated biochemical test VITEK 2 system. The ALS1 virulence gene was detected by polymerase chain reaction using newly designed primers with a molecular size (419 bp). Results: Out of 235 Diabetic Foot Ulcer (DFU) cases, C. albicans were isolated in 20 (8.5%) patients (12 males and 8 females) of diabetic foot ulcers. In this study, the incidence of C. albicans infection at age [50–59 years] group was [40%], and increased at age group [60–69 years] to [55%], which represents the highest incidence of infection, then decreased in the age group [79–79 years] to [5%]). Seventy-five percent of the isolates were ALS1 gene positive. Conclusions: Diabetic people are more susceptible to infections due to their hyperglycemic environment and reduced immunity. The use of HiCrome Candida Identification Media with VITEK 2 system can help reduce the unnecessary steps of microorganism identification process. C. albicans infection is more common in males the females regarding diabetic foot ulceration. Majority of diabetic foot ulcers occur in older adults. ALS gene might be associated with diabetic foot ulceration.

Using Genomics to Shape the Definition of the Agglutinin-Like Sequence (ALS) Family in the Saccharomycetales.

The Candida albicans agglutinin-like sequence (ALS) family is studied because of its contribution to cell adhesion, fungal colonization, and polymicrobial biofilm formation. The goal of this work was to derive an accurate census and sequence for ALS genes in pathogenic yeasts and other closely related species, while probing the boundaries of the ALS family within the Order Saccharomycetales. Bioinformatic methods were combined with laboratory experimentation to characterize 47 novel ALS loci from 8 fungal species. AlphaFold predictions suggested the presence of a conserved N-terminal adhesive domain (NT-Als) structure in all Als proteins reported to date, as well as in S. cerevisiae alpha-agglutinin (Sag1). Lodderomyces elongisporus, Meyerozyma guilliermondii, and Scheffersomyces stipitis were notable because each species had genes with C. albicans ALS features, as well as at least one that encoded a Sag1-like protein. Detection of recombination events between the ALS family and gene families encoding other cell-surface proteins such as Iff/Hyr and Flo suggest widespread domain swapping with the potential to create cell-surface diversity among yeast species. Results from the analysis also revealed subtelomeric ALS genes, ALS pseudogenes, and the potential for yeast species to secrete their own soluble adhesion inhibitors. Information presented here supports the inclusion of SAG1 in the ALS family and yields many experimental hypotheses to pursue to further reveal the nature of the ALS family.

The Role of B-Cells and Antibodies against Candida Vaccine Antigens in Invasive Candidiasis.

Systemic candidiasis is an invasive fungal infection caused by members of the genus Candida. The recent emergence of antifungal drug resistance and increased incidences of infections caused by non-albicans Candida species merit the need for developing immune therapies against Candida infections. Although the role of cellular immune responses in anti-Candida immunity is well established, less is known about the role of humoral immunity against systemic candidiasis. This review summarizes currently available information on humoral immune responses induced by several promising Candida vaccine candidates, which have been identified in the past few decades. The protective antibody and B-cell responses generated by polysaccharide antigens such as mannan, β-glucan, and laminarin, as well as protein antigens like agglutinin-like sequence gene (Als3), secreted aspartyl proteinase (Sap2), heat shock protein (Hsp90), hyphally-regulated protein (Hyr1), hyphal wall protein (Hwp1), enolase (Eno), phospholipase (PLB), pyruvate kinase (Pk), fructose bisphosphate aldolase (Fba1), superoxide dismutase gene (Sod5) and malate dehydrogenase (Mdh1), are outlined. As per studies reviewed, antibodies induced in response to leading Candida vaccine candidates contribute to protection against systemic candidiasis by utilizing a variety of mechanisms such as opsonization, complement fixation, neutralization, biofilm inhibition, direct candidacidal activity, etc. The contributions of B-cells in controlling fungal infections are also discussed. Promising results using anti-Candida monoclonal antibodies for passive antibody therapy reinforces the need for developing antibody-based therapeutics including anti-idiotypic antibodies, single-chain variable fragments, peptide mimotopes, and antibody-derived peptides. Future research involving combinatorial immunotherapies using humanized monoclonal antibodies along with antifungal drugs/cytokines may prove beneficial for treating invasive fungal infections.

Different expression levels of ALS and SAP genes contribute to recurrent vulvovaginal candidiasis by Candida albicans.

Aim: To study the behavior of Candida albicans in women with vulvovaginal candidiasis (VVC), recurrent VVC (RVVC) and asymptomatic (AS), regarding adhesion on HeLa cells and their ability to express secreted aspartic proteinases (SAP) genes, agglutinin-like sequence (ALS) genes and HWP1. Materials & methods: The adhesion of Candidaalbicans to HeLa cells was evaluated by colony-forming units, and the expressed genes were evaluated by qRT-PCR. Results: AS and VVC isolates showed greater ability to adhere HeLa cells when compared with RVVC isolate. Nevertheless, RVVC isolate exhibited upregulation of a large number of genes of ALS and SAP gene families and HWP1 gene. Conclusion: The results demonstrated that RVVC isolate expressed significantly important genes for invasion and yeast-host interactions.

Pursuing Advances in DNA Sequencing Technology to Solve a Complex Genomic Jigsaw Puzzle: The Agglutinin-Like Sequence (ALS) Genes of Candida tropicalis.

The agglutinin-like sequence (ALS) gene family encodes cell-surface adhesins that interact with host and abiotic surfaces, promoting colonization by opportunistic fungal pathogens such as Candida tropicalis. Studies of Als protein contribution to C. tropicalis adhesion would benefit from an accurate catalog of ALS gene sequences as well as insight into relative gene expression levels. Even in the genomics era, this information has been elusive: genome assemblies are often broken within ALS genes because of their extensive regions of highly conserved, repeated DNA sequences and because there are many similar ALS genes at different chromosomal locations. Here, we describe the benefit of long-read DNA sequencing technology to facilitate characterization of C. tropicalis ALS loci. Thirteen ALS loci in C. tropicalis strain MYA-3404 were deduced from a genome assembly constructed from Illumina MiSeq and Oxford Nanopore MinION data. Although the MinION data were valuable, PCR amplification and Sanger sequencing of ALS loci were still required to complete and verify the gene sequences. Each predicted Als protein featured an N-terminal binding domain, a central domain of tandemly repeated sequences, and a C-terminal domain rich in Ser and Thr. The presence of a secretory signal peptide and consensus sequence for addition of a glycosylphosphatidylinositol (GPI) anchor was consistent with predicted protein localization to the cell surface. TaqMan assays were designed to recognize each ALS gene, as well as both alleles at the divergent CtrALS3882 locus. C. tropicalis cells grown in five different in vitro conditions showed differential expression of various ALS genes. To place the C. tropicalis data into a larger context, TaqMan assays were also designed and validated for analysis of ALS gene expression in Candida albicans and Candida dubliniensis. These comparisons identified the subset of highly expressed C. tropicalis ALS genes that were predicted to encode proteins with the most abundant cell-surface presence, prioritizing them for subsequent functional analysis. Data presented here provide a solid foundation for future experimentation to deduce ALS family contributions to C. tropicalis adhesion and pathogenesis.

Phloretin inhibited the pathogenicity and virulence factors against Candida albicans

ABSTRACT Oral candidiasis is one of the most common types of fungal infection caused by Candida albicans (C. albicans). The present study aims to investigate the antifungal effects of phloretin (a dihydrochalcone flavonoid) against the C. albicans pathogenicity. In this work, we treated C. albicans SC5314 with 37.28, 74.55, or 149.10 μg/mL (equivalent to 0.5×, 1× or 2× MIC) phloretin in vitro. Besides, we established a mice model of oral candidiasis by a sublingual infection of C. albicans suspension (1 × 107 colony-forming unit/mL), and mice were treated with phloretin (3.73 or 7.46 mg/mL, which were equivalent to 50× or 100× MIC) twice a day starting on day one post-infection. The results showed that the MIC of phloretin against C. albicans was 74.55 μg/mL. Phloretin exerted antifungal activity by inhibiting the biofilm formation and suppressing the yeast-to-hyphae transition upon the downregulation of hypha-associated genes including enhanced adherence to polystyrene 1, the extent of cell elongation gene 1, hyphal wall protein 1 gene, and agglutinin-like sequence gene 3. Next, phloretin repressed the secretion of proteases and phospholipases via reducing the expression of protease-encoding genes secreted aspartyl proteases (SAP)1 and SAP2, as well as phospholipase B1. Subsequently, the in vivo antifungal activity of phloretin was testified by the reverse of the enhanced lesion severity, inflammatory infiltration, and the increased colony-forming unit counts caused by C. albicans of tongue tissues in oral candidiasis mice. In conclusion, phloretin suppressed the pathogenicity and virulence factors against C. albicans both in vivo and in vitro.

Characterization of the Candida orthopsilosis agglutinin-like sequence (ALS) genes.

Agglutinin like sequence (Als) cell-wall proteins play a key role in adhesion and virulence of Candida species. Compared to the well-characterized Candida albicans ALS genes, little is known about ALS genes in the Candida parapsilosis species complex. Three incomplete ALS genes were identified in the genome sequence for Candida orthopsilosis strain 90–125 (GenBank assembly ASM31587v1): CORT0C04210 (named CoALS4210), CORT0C04220 (CoALS4220) and CORT0B00800 (CoALS800). To complete the gene sequences, new data were derived from strain 90–125 using Illumina (short-read) and Oxford Nanopore (long-read) methods. Long-read sequencing analysis confirmed the presence of 3 ALS genes in C. orthopsilosis 90–125 and resolved the gaps located in repetitive regions of CoALS800 and CoALS4220. In the new genome assembly (GenBank PQBP00000000), the CoALS4210 sequence was slightly longer than in the original assembly. C. orthopsilosis Als proteins encoded features well-known in C. albicans Als proteins such as a secretory signal peptide, N-terminal domain with a peptide-binding cavity, amyloid-forming region, repeated sequences, and a C-terminal site for glycosylphosphatidylinositol anchor addition that, in yeast, suggest localization of the proteins in the cell wall. CoAls4210 and CoAls800 lacked the classic C. albicans Als tandem repeats, instead featuring short, imperfect repeats with consensus motifs such as SSSEPP and GSGN. Quantitative RT-PCR showed differential regulation of CoALS genes by growth stage in six genetically diverse C. orthopsilosis clinical isolates, which also exhibited length variation in the ALS alleles, and strain-specific gene expression patterns. Overall, long-read DNA sequencing methodology was instrumental in generating an accurate assembly of CoALS genes, thus revealing their unconventional features and first insights into their allelic variability within C. orthopsilosis clinical isolates.

Profiling of adhesive properties of the agglutinin-like sequence (ALS) protein family, a virulent attribute ofCandida albicans

Candida albicans is normally present in nearly all humans but can cause fatal diseases in immunocompromised patients. The agglutinin-like sequence (ALS) gene family of C. albicans has been suggested to be important for biofilm formation on medical devices. Here, we cloned all ALS genes and determined the binding properties of their gene products by cell surface engineering of Saccharomyces cerevisiae. Most of the ALS homologues had the ability to bind polypropylene, borosilicate glass and polyvinyl chloride, which are often used as materials in medical devices. However, ALS homologues were not able to bind polyurethane, polymethyl methacrylate, polytetrafluoroethylene or titanium. These findings will aid in the development of biofilm-resistant medical devices.

Characterization of gene family that mediates the adhesion of biofilms formed by Candida tropicalis isolated from HIV and non-HIV patients

Background Candida tropicalis is an important cause of candidemia in immunocompromised patients. Biofilm formation helps the organism to establish infection. Agglutinin like sequence (ALS) genes encodes glycoproteins which are important adhesins. This study was done to detect the presence of ALS genes by PCR in C.tropicalis strains isolated from HIV and non-HIV patients in comparison with biofilm formation.

Frequency and expression of ALS and HWP1 genotypes in Candida albicans strains isolated from Mexican patients suffering from vaginal candidosis

To detect the frequency and expression of eight ALS (agglutinin-like sequence) genes and the HWP1 genotype in a group of Candida albicans strains isolated from Mexican women suffering from vaginal candidosis. A group of 264 women (age 15-57 years) with vaginal infections were evaluated. C. albicans was identified by PCR amplification of the rRNA internal transcribed spacer regions ITS1 and ITS2. The ALS and HWP1 genes were identified by conventional PCR, and their expression levels were determined by real-time PCR after growing C. albicans strains in reconstituted human vaginal epithelium (RHVE). C. albicans was identified in 50 women (18.9%). The genotypic frequencies were ALS1 100%, ALS2 60%, ALS3 36%, ALS4 54%, ALS5 70%, ALS6 56%, ALS7 64%, ALS9 66% and HWP1 92%. The most frequently expressed genes in the strains harbouring all of the genes were ALS4 (100%), ALS1 (87.5%), ALS2 (87.5%), ALS3 (87.5%), ALS5 (87.5%), ALS7 (87.5%) and HWP1 (75.0%). Nineteen per cent of the vaginal infections were caused by C. albicans, and a high proportion of the strains carried genes encoding proteins involved in adhesion to epithelia. The ALS and HWP1 genes were expressed in RHVE, suggesting that the Als and Hwp1 proteins play an important role in the pathogenesis of the infection.

Evaluation of the Role of Candida albicans Agglutinin-Like Sequence (Als) Proteins in Human Oral Epithelial Cell Interactions

The fungus C. albicans uses adhesins to interact with human epithelial surfaces in the processes of colonization and pathogenesis. The C. albicans ALS (agglutinin-like sequence) gene family encodes eight large cell-surface glycoproteins (Als1-Als7 and Als9) that have adhesive function. This study utilized C. albicans Δals mutant strains to investigate the role of the Als family in oral epithelial cell adhesion and damage, cytokine induction and activation of a MAPK-based (MKP1/c-Fos) signaling pathway that discriminates between yeast and hyphae. Of the eight Δals mutants tested, only the Δals3 strain showed significant reductions in oral epithelial cell adhesion and damage, and cytokine production. High fungal:epithelial cell multiplicities of infection were able to rescue the cell damage and cytokine production phenotypes, demonstrating the importance of fungal burden in mucosal infections. Despite its adhesion, damage and cytokine induction phenotypes, the Δals3 strain induced MKP1 phosphorylation and c-Fos production to a similar extent as control cells. Our data demonstrate that Als3 is involved directly in epithelial adhesion but indirectly in cell damage and cytokine induction, and is not the factor targeted by oral epithelial cells to discriminate between the yeast and hyphal form of C. albicans.

Real-time PCR expression profiling of genes encoding potential virulence factors in Candida albicans biofilms: identification of model-dependent and -independent gene expression

BackgroundCandida albicans infections are often associated with biofilm formation. Previous work demonstrated that the expression of HWP1 (hyphal wall protein) and of genes belonging to the ALS (agglutinin-like sequence), SAP (secreted aspartyl protease), PLB (phospholipase B) and LIP (lipase) gene families is associated with biofilm growth on mucosal surfaces. We investigated using real-time PCR whether genes encoding potential virulence factors are also highly expressed in biofilms associated with abiotic surfaces. For this, C. albicans biofilms were grown on silicone in microtiter plates (MTP) or in the Centres for Disease Control (CDC) reactor, on polyurethane in an in vivo subcutaneous catheter rat (SCR) model, and on mucosal surfaces in the reconstituted human epithelium (RHE) model.ResultsHWP1 and genes belonging to the ALS, SAP, PLB and LIP gene families were constitutively expressed in C. albicans biofilms. ALS1-5 were upregulated in all model systems, while ALS9 was mostly downregulated. ALS6 and HWP1 were overexpressed in all models except in the RHE and MTP, respectively. The expression levels of SAP1 were more pronounced in both in vitro models, while those of SAP2, SAP4 and SAP6 were higher in the in vivo model. Furthermore, SAP5 was highly upregulated in the in vivo and RHE models. For SAP9 and SAP10 similar gene expression levels were observed in all model systems. PLB genes were not considerably upregulated in biofilms, while LIP1-3, LIP5-7 and LIP9-10 were highly overexpressed in both in vitro models. Furthermore, an elevated lipase activity was detected in supernatans of biofilms grown in the MTP and RHE model.ConclusionsOur findings show that HWP1 and most of the genes belonging to the ALS, SAP and LIP gene families are upregulated in C. albicans biofilms. Comparison of the fold expression between the various model systems revealed similar expression levels for some genes, while for others model-dependent expression levels were observed. This suggests that data obtained in one biofilm model cannot be extrapolated to other model systems. Therefore, the need to use multiple model systems when studying the expression of genes encoding potential virulence factors in C. albicans biofilms is highlighted.

Discovering the secrets of theCandida albicansagglutinin-like sequence (ALS) gene family – a sticky pursuit

The agglutinin-like sequence (ALS) family of Candida albicans includes eight genes that encode large cell-surface glycoproteins. The high degree of sequence relatedness between the ALS genes and the tremendous allelic variability often present in the same C. albicans strain complicated definition and characterization of the gene family. The main hypothesis driving ALS family research is that the genes encode adhesins, primarily involved in host-pathogen interactions. Although adhesive function has been demonstrated for several Als proteins, the challenge of studying putative adhesins in a highly adhesive organism like C. albicans has led to varying ideas about how best to pursue such investigations, and results that are sometimes contradictory. Recent analysis of alsdelta/alsdelta strains suggested roles for Als proteins outside of adhesion to host surfaces, and a broader scope of Als protein function than commonly believed. The availability and use of experimental methodologies to study C. albicans at the genomic level, and the ALS family en masse, have advanced knowledge of these genes and emphasized their importance in C. albicans biology and pathogenesis.

Unequal contribution of ALS9 alleles to adhesion between Candida albicans and human vascular endothelial cells

The Candida albicans ALS (agglutinin-like sequence) family includes eight genes (ALS1 to ALS7, and ALS9) that share a common general organization, consisting of a relatively conserved 5' domain, a central domain of tandemly repeated sequence units, and a 3' domain of relatively variable length and sequence. To test the hypothesis that the cell-surface glycoproteins encoded by the ALS genes mediate contact between the fungal cell and host surfaces, a set of C. albicans mutant strains was systematically constructed, each lacking one of the ALS sequences. Phenotypes of the mutant strains were evaluated, primarily using adhesion assays. ALS9 is unique within the ALS family due to extensive allelic sequence variation within the 5' domain that may result in functional differences between proteins encoded by ALS9-1 and ALS9-2. Deletion of ALS9 significantly reduces C. albicans adhesion to human vascular endothelial cell monolayers. The mutation was complemented by reintegration of a wild-type copy of ALS9-2, but not ALS9-1, suggesting allelic functional differences. Complementation of the mutation with a gene fusion between the 5' domain of ALS9-2 and the tandem repeats and 3' domain of ALS9-1 also restored wild-type adhesion levels. Analysis of the als9Delta/als9Delta 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 als9Delta/als9Delta 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.

Analysis of ALS5 and ALS6 allelic variability in a geographically diverse collection of Candida albicans isolates

The Candida albicans ALS (agglutinin-like sequence) gene family encodes eight cell-surface glycoproteins, some of which function in adhesion to host surfaces. ALS genes have a central tandem repeat-encoding domain comprised entirely of head-to-tail copies of a conserved 108-bp sequence. The number of copies of the tandemly repeated sequence varies between C. albicans strains and often between alleles within the same strain. Because ALS alleles can encode different-sized proteins that may have different functional characteristics, defining the range of allelic variability is important. Genomic DNA from C. albicans strains representing the major genetic clades was PCR amplified to determine the number of tandemly repeated sequence copies within the ALS5 and ALS6 central domain. ALS5 alleles had 2–10 tandem repeat sequence copies (mean = 4.82 copies) while ALS6 alleles had 2–8 copies (mean = 4.00 copies). Despite this variability, tandem repeat copy number was stable in C. albicans strains passaged for 3000 generations. Prevalent alleles and allelic distributions varied among the clades for ALS5 and ALS6. Overall, ALS6 exhibited less variability than ALS5. ALS5 deletions can occur naturally in C. albicans via direct repeats flanking the ALS5 locus. Deletion of both ALS5 alleles was associated particularly with clades III and SA. ALS5 exhibited allelic polymorphisms in the coding region 5′ of the tandem repeats; some alleles resembled ALS1, suggesting recombination between these contiguous loci. Natural deletion of ALS5 and the sequence variation within its coding region suggest relaxed selective pressure on this locus, and that Als5p function may be dispensable in C. albicans or redundant within the Als family.

Analysis of the Candida albicans Als2p and Als4p adhesins suggests the potential for compensatory function within the Als family.

The ALS (agglutinin-like sequence) gene family encodes eight large cell-surface glycoproteins. The work presented here focuses on Als2p and Als4p, and is part of a larger effort to deduce the function of each Als protein. Both ALS4 alleles were deleted from the Candida albicans genome and the phenotype of the mutant strain (als4Delta/als4Delta; named 2034) studied. Loss of Als4p slowed germ tube formation of cells grown in RPMI 1640 medium and resulted in decreased adhesion of C. albicans to vascular endothelial cells. Loss of Als4p did not affect adhesion to buccal epithelial cells, biofilm formation in a catheter model, or adhesion to or destruction of oral reconstituted human epithelium (RHE). Although deletion of one ALS2 allele was achieved readily, a strain lacking the second allele was not identified despite screening thousands of transformants. The remaining ALS2 allele was placed under control of the C. albicans MAL2 promoter to create an als2Delta/PMAL2-ALS2 strain (named 2342). Real-time RT-PCR analysis of strain 2342 grown in glucose-containing medium (non-inducing conditions) showed that although ALS2 transcript levels were greatly reduced compared to wild-type cells, some ALS2 transcript remained. The decreased ALS2 expression levels were sufficient to slow germ tube formation in RPMI 1640 and Lee medium, reduce adhesion to vascular endothelial cells and to RHE, decrease RHE destruction, and impair biofilm formation. Growth of strain 2342 in maltose-containing medium (inducing conditions) restored the wild-type phenotype in all assays. Real-time RT-PCR analysis demonstrated that in maltose-containing medium, strain 2342 overexpressed ALS2 compared to wild-type cells; however no overexpression phenotype was apparent. Microarray analysis revealed little transcriptional response to ALS4 deletion, but showed twofold up-regulation of orf19.4765 in the glucose-medium-grown als2Delta/PMAL2-ALS2 strain. orf19.4765 encodes a protein with features of a glycosylated cell wall protein with similarity to Saccharomyces cerevisiae Ccw12p, although initial analysis suggested functional differences between the two proteins. Real-time RT-PCR measurement of ALS2 and ALS4 transcript copy number showed a 2.8-fold increase in ALS2 expression in the als4Delta/als4Delta strain and a 3.2-fold increase in ALS4 expression in the als2Delta/PMAL2-ALS2 strain, suggesting the potential for compensatory function between these related proteins.

RT-PCR detection of Candida albicans ALS gene expression in the reconstituted human epithelium (RHE) model of oral candidiasis and in model biofilms.

An RT-PCR assay was developed to analyse expression patterns of genes in the Candida albicans ALS (agglutinin-like sequence) family. Inoculation of a reconstituted human buccal epithelium (RHE) model of mucocutaneous candidiasis with strain SC5314 showed destruction of the epithelial layer by C. albicans and also formation of an upper fungal layer that had characteristics similar to a biofilm. RT-PCR analysis of total RNA samples extracted from C. albicans-inoculated buccal RHE showed that ALS1, ALS2, ALS3, ALS4, ALS5 and ALS9 were consistently detected over time as destruction of the RHE progressed. Detection of transcripts from ALS7, and particularly from ALS6, was more sporadic, but not associated with a strictly temporal pattern. The expression pattern of ALS genes in C. albicans cultures used to inoculate the RHE was similar to that observed in the RHE model, suggesting that contact of C. albicans with buccal RHE does little to alter ALS gene expression. RT-PCR analysis of RNA samples extracted from model denture and catheter biofilms showed similar gene expression patterns to the buccal RHE specimens. Results from the RT-PCR analysis of biofilm RNA specimens were consistent between various C. albicans strains during biofilm development and were comparable to gene expression patterns in planktonic cells. The RT-PCR assay described here will be useful for analysis of human clinical specimens and samples from other disease models. The method will provide further insight into the role of ALS genes and their encoded proteins in the diverse interactions between C. albicans and its host.

Sixty alleles of the ALS7 open reading frame in Candida albicans: ALS7 is a hypermutable contingency locus.

The ALS (agglutinin-like sequence) gene family encodes proteins that play a role in adherence of the yeast Candida albicans to endothelial and epithelial cells. The proteins are proposed as virulence factors for this important fungal pathogen of humans. We analyzed 66 C. albicans strains, representing a worldwide collection of 266 infection-causing isolates, and discovered 60 alleles of the ALS7 open reading frame (ORF). Differences between alleles were largely caused by rearrangements of repeat elements in the so-called tandem repeat domain (21 different types occurred) and the VASES region (19 different types). C. albicans is diploid, and combinations of ALS7 alleles generated 49 different genotypes. ALS7 expression was detected in samples isolated directly from five oral candidosis patients. ORFs in the opposite direction contained within the ALS7 ORF were also transcribed in all strains tested. Isolates representing a more pathogenic general-purpose genotype (GPG) cluster of strains tended to have more tandem repeats than other strains. Two types of VASES regions were largely exclusive to GPG strains; the remaining types were largely exclusive to noncluster strains. Our results provide evidence that ALS7 is a hypermutable contingency locus and important for the success of C. albicans as an opportunistic pathogen of humans.

Characterization of agglutinin-like sequence genes from non-albicans Candida and phylogenetic analysis of the ALS family.

The ALS (agglutinin-like sequence) gene family of Candida albicans encodes cell-surface glycoproteins implicated in adhesion of the organism to host surfaces. Southern blot analysis with ALS-specific probes suggested the presence of ALS gene families in C. dubliniensis and C. tropicalis; three partial ALS genes were isolated from each organism. Northern blot analysis demonstrated that mechanisms governing expression of ALS genes in C. albicans and C. dubliniensis are different. Western blots with an anti-Als serum showed that cross-reactive proteins are linked by beta 1,6-glucan in the cell wall of each non-albicans Candida, suggesting similar cell wall architecture and conserved processing of Als proteins in these organisms. Although an ALS family is present in each organism, phylogenetic analysis of the C. albicans, C. dubliniensis, and C. tropicalis ALS genes indicated that, within each species, sequence diversification is extensive and unique ALS sequences have arisen. Phylogenetic analysis of the ALS and SAP (secreted aspartyl proteinase) families show that the ALS family is younger than the SAP family. ALS genes in C. albicans, C. dubliniensis, and C. tropicalis tend to be located on chromosomes that also encode genes from the SAP family, yet the two families have unexpectedly different evolutionary histories. Homologous recombination between the tandem repeat sequences present in ALS genes could explain the different histories for co-localized genes in a predominantly clonal organism like C. albicans.