A Chromosome-Level Genome Assembly for Yamadazyma tenuis ATCC 10573.

Pacific Biosciences (PacBio) long-read sequencing was used to generate a chromosome-level genome assembly for Yamadazyma tenuis strain ATCC 10573. The assembly featured 7 chromosomes that matched the electrophoretic karyotype and a 26.5-kb circular mitochondrial genome. The nuclear genome was 10.8 Mb, with a GC content of 43%, and 5,340 predicted genes.

Assessing Als3 Peptide-Binding Cavity and Amyloid-Forming Region Contributions to Candida albicans Invasion of Human Oropharyngeal Epithelial Cells.

Although it is widely recognized that disruption of ALS3 reduces the invasion of Candida albicans germ tubes into mammalian oral epithelial cells, the mechanism of this interaction was unexplored. C. albicans strains with structurally informed mutations to remove adhesive activity of the peptide-binding cavity (PBC) or aggregative activity mediated by the amyloid-forming region (AFR) were assessed for their ability to invade cultured human oropharyngeal epithelial cells. Initial assays utilized untreated fungal and epithelial cells. Subsequent work used epithelial cells treated with cytochalasin D and C. albicans cells treated with thimerosal to investigate invasion mediated by active penetration of germ tubes and epithelial cell induced endocytosis, respectively. Results demonstrated the importance of the PBC for the invasion process: loss of PBC function resulted in the same reduced-invasion phenotype as a C. albicans strain that did not produce Als3 on its surface. Invasion via active penetration was particularly compromised without PBC function. Loss of AFR function produced a wild-type phenotype in the untreated and thimerosal-treated invasion assays but increased invasion in cytochalasin D-treated epithelial cells. In previous work, reduced AFR-mediated Als3 aggregation increased C. albicans adhesion to cultured epithelial cell monolayers, presumably via increased PBC accessibility for ligand binding. Collectively, results presented here demonstrate that Als3 PBC-mediated adhesion is integral to its invasive function. These new data add to the mechanistic understanding of the role of Als3 in C. albicans invasion into mammalian oral epithelial cells.

Development and validation of monoclonal antibodies specific for Candida albicans Als2, Als9-1, and Als9-2.

Fungal agglutinin-like sequence (Als) cell-surface glycoproteins, best characterized in Candida albicans, mediate adhesive and aggregative interactions with host cells, other microbes, and abiotic surfaces. Monoclonal antibodies (MAbs) specific for each C. albicans Als protein are valuable reagents for gaining insight into Als protein localization and function. This manuscript describes development and validation of MAbs specific for C. albicans Als2, as well as for C. albicans Als9-1 and Als9-2, two protein variants produced from the ALS9 locus. Native C. albicans ALS9 expression levels were not sufficiently high to produce detectable Als9 protein on the wild-type cell surface so MAb validation required production of overexpression strains, each featuring one of the two ALS9 alleles. An anti-Als2 MAb was raised against an N-glycosylated form of the protein immunogen, as well as an Endoglycosidase H-treated immunogen. The MAb raised against the N-glycosylated immunogen proved superior and immunolabeled C. albicans yeast cells and germ tubes, and the surface of Candida dubliniensis and Candida tropicalis yeasts. Als2 was visible on C. albicans yeast cells recovered from a murine model of oral candidiasis, demonstrating Als2 production both in vivo and in vitro. These new MAbs add to the collection of anti-Als MAbs that are powerful tools to better understand the role of Als proteins in C. albicans biology and pathogenesis.

Development and Use of a Monoclonal Antibody Specific for the Candida albicans Cell-Surface Protein Hwp1.

The Candida albicans cell-surface protein Hwp1 functions in adhesion to the host and in biofilm formation. A peptide from the Gln-Pro-rich adhesive domain of Hwp1 was used to raise monoclonal antibody (MAb) 2-E8. MAb 2-E8 specificity for Hwp1 was demonstrated using a hwp1/hwp1 C. albicans isolate and strains that expressed at least one HWP1 allele. Immunofluorescence and atomic force microscopy experiments using MAb 2-E8 confirmed C. albicans germ-tube-specific detection of the Hwp1 protein. MAb 2-E8 also immunolabeled the tips of some Candida dubliniensis germ tubes grown under conditions that maximized HWP1 expression. The phylogeny of HWP1 and closely related genes suggested that the Gln-Pro-rich adhesive domain was unique to C. albicans and C. dubliniensis focusing the utility of MAb 2-E8 on these species. This new reagent can be used to address unanswered questions about Hwp1 and its interactions with other proteins in the context of C. albicans biology and pathogenesis.

ALS1 Deletion Increases the Proportion of Small Cells in a Candida albicans Culture Population: Hypothesizing a Novel Role for Als1.

Candida albicans Als1 is a large cell-surface glycoprotein most often discussed for its role in mediating ligand-binding and aggregative interactions. Relative to a wild-type control, deletion of ALS1 produced a strain that showed delayed germ-tube formation and delayed disease progression in a murine model of disseminated candidiasis. Populations of Δals1/Δals1 cultured cells had a higher proportion of smaller cells compared to wild-type or ALS1 reintegrant control cultures. The goal of this work was to investigate whether this difference in cell-size distributions was responsible for delayed germ-tube formation and delayed disease progression. Flow cytometry was used to select populations of wild-type and Δals1/Δals1 cells with varied cell-size distributions. Delayed germ-tube formation was demonstrated for small cells sorted from a wild-type (ALS1/ALS1) culture population. Large cells sorted from a Δals1/Δals1 culture formed germ tubes as quickly as the wild-type control demonstrating clearly that the Δals1/Δals1 germ-tube formation delays were attributable to cell size. In vivo, smaller-sized cells of the wild-type control showed fewer colony-forming units (cfu) per gram of kidney tissue and less-severe histopathology lesions compared to larger cells of the same strain. The Δals1/Δals1 strain showed reduced cfu/g of kidney tissue and less-severe lesions compared to the wild-type control. However, isolation and testing of the larger cells from the Δals1/Δals1 population increased cfu/g of tissue and showed increased lesion severity compared to the overall mutant cell population. In vivo hypha lengths from the large, sorted Δals1/Δals1 cells were comparable to those for the wild-type control strain. These results demonstrated that a large share of the Δals1/Δals1 in-vivo phenotype was attributable to cell size. Collectively, the data suggest a role for Als1 in C. albicans cell size homeostasis, a novel hypothesis for further exploration.

Peering Into Candida albicans Pir Protein Function and Comparative Genomics of the Pir Family.

The fungal cell wall, comprised primarily of protein and polymeric carbohydrate, maintains cell structure, provides protection from the environment, and is an important antifungal drug target. Pir proteins (proteins with internal repeats) are linked to cell wall ß-1,3-glucan and are best studied in Saccharomyces cerevisiae. Sequential deletion of S. cerevisiae PIR genes produces strains with increasingly notable cell wall damage. However, a true null mutant lacking all five S. cerevisiae PIR genes was never constructed. Because only two PIR genes (PIR1, PIR32) were annotated in the Candida albicans genome, the initial goal of this work was to construct a true Δpir/Δpir null strain in this species. Unexpectedly, the phenotype of the null strain was almost indistinguishable from its parent, leading to the search for other proteins with Pir function. Bioinformatic approaches revealed nine additional C. albicans proteins that share a conserved Pir functional motif (minimally DGQ). Examination of the protein sequences revealed another conserved motif (QFQFD) toward the C-terminal end of each protein. Sequence similarities and presence of the conserved motif(s) were used to identify a set of 75 proteins across 16 fungal species that are proposed here as Pir proteins. The Pir family is greatly expanded in C. albicans and C. dubliniensis compared to other species and the orthologs are known to have specialized function during chlamydospore formation. Predicted Pir structures showed a conserved core of antiparallel beta-sheets and sometimes-extensive loops that contain amino acids with the potential to form linkages to cell wall components. Pir phylogeny demonstrated emergence of specific ortholog groups among the fungal species. Variation in gene expression patterns was noted among the ortholog groups during growth in rich medium. PIR allelic variation was quite limited despite the presence of a repeated sequence in many loci. Results presented here demonstrate that the Pir family is larger than previously recognized and lead to new hypotheses to test to better understand Pir proteins and their role in the fungal cell wall.

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.

Effect of light exposure on growth rate of veterinary clinical dermatophyte isolates.

BACKGROUND: Veterinary textbooks and literature suggest that exposure to light is inhibitory to growth of clinical dermatophyte isolates. HYPOTHESIS/OBJECTIVES: We hypothesized that this idea was derived from experiments that examined the effect of high doses of ultraviolet and visible light exposure on dermatophyte growth, and that exposure to typical room lighting would not adversely affect dermatophyte growth rate. METHODS AND MATERIALS: Isolates of common veterinary dermatophytes (three each of Microsporum canis, Nannizia gypsea and Trichophyton benhamiae) were exposed to typical fluorescent room lighting, incubated in a closed drawer, or exposed at close range to fluorescent wide-spectrum light. Dermatophytes were grown on Sabouraud Dextrose Agar (SAB) and Dermatophyte Test Medium (DTM). Colony diameter was measured and growth rate (expressed as colony diameter increase mm/day) calculated at the linear portion of the culture growth curve. Statistical analyses compared growth rates across the various incubation conditions and among dermatophyte isolates. RESULTS: There was little difference in growth rate between cultures incubated under typical fluorescent room lighting and those placed in the dark. Exposure to the close-range light increased growth rate as a consequence of the elevated incubation temperature created by the lamp. Significant differences in growth rate were noted among strains of the same dermatophyte species. Dermatophytes grew more rapidly on SAB than DTM agar. CONCLUSIONS AND CLINICAL RELEVANCE: Exposure to typical lighting conditions in a clinical environment does not inhibit growth of dermatophyte colonies. Veterinary clinicians may conduct routine dermatophyte cultures without incubating them in the dark.

Microbial Production of Retinyl Palmitate and Its Application as a Cosmeceutical.

Chemically synthesized retinyl palmitate has been widely used in the cosmetic and biotechnology industry. In this study, we aimed to demonstrate the microbial production of retinyl palmitate and the benefits of microbial retinyl palmitate in skin physiology. A heterologous retinyl palmitate biosynthesis pathway was reconstructed in metabolically engineered Escherichia coli using synthetic expression modules from Pantoea agglomerans, Salinibacter ruber, and Homo sapiens. High production of retinyl palmitate (69.96 ± 2.64 mg/L) was obtained using a fed-batch fermentation process. Moreover, application of purified microbial retinyl palmitate to human foreskin HS68 fibroblasts led to increased cellular retinoic acid-binding protein 2 (CRABP2) mRNA level [1.7-fold (p = 0.001) at 100 µg/mL], acceleration of cell proliferation, and enhancement of procollagen synthesis [111% (p < 0.05) at 100 µg/mL], strongly indicating an anti-ageing-related effect of this substance. These results would pave the way for large-scale production of retinyl palmitate in microbial systems and represent the first evidence for the application of microbial retinyl palmitate as a cosmeceutical.

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.

Agglutinin-Like Sequence (ALS) Genes in the Candida parapsilosis Species Complex: Blurring the Boundaries Between Gene Families That Encode Cell-Wall Proteins.

The agglutinin-like sequence (Als) proteins are best-characterized in Candida albicans and known for their role in adhesion of the fungal cell to host and abiotic surfaces. ALS sequences are often misassembled in whole-genome sequence data because each species has multiple ALS loci that contain similar sequences, most notably tandem copies of highly conserved repeated sequences. The Candida parapsilosis species complex includes Candida parapsilosis, Candida orthopsilosis, and Candida metapsilosis, three distinct but closely related species. Using publicly available genome resources, de novo genome assemblies, and laboratory experimentation including Sanger sequencing, five ALS genes were characterized in C. parapsilosis strain CDC317, three in C. orthopsilosis strain 90-125, and four in C. metapsilosis strain ATCC 96143. The newly characterized ALS genes shared similar features with the well-known C. albicans ALS family, but also displayed unique attributes such as novel short, imperfect repeat sequences that were found in other genes encoding fungal cell-wall proteins. Evidence of recombination between ALS sequences and other genes was most obvious in CmALS2265, which had the 5' end of an ALS gene and the repeated sequences and 3' end from the IFF/HYR family. Together, these results blur the boundaries between the fungal cell-wall families that were defined in C. albicans. TaqMan assays were used to quantify relative expression for each ALS gene. Some measurements were complicated by the assay location within the ALS gene. Considerable variation was noted in relative gene expression for isolates of the same species. Overall, however, there was a trend toward higher relative gene expression in saturated cultures rather than younger cultures. This work provides a complete description of the ALS genes in the C. parapsilosis species complex and a toolkit that promotes further investigations into the role of the Als proteins in host-fungal interactions.

SERS immunoassay based on the capture and concentration of antigen-assembled gold nanoparticles.

A simple, rapid, and sensitive immunoassay has been developed based on antigen-mediated aggregation of gold nanoparticles (AuNP) and surface-enhanced Raman spectroscopy (SERS). Central to this platform is the extrinsic Raman label (ERL), which consists of a gold nanoparticle modified with a mixed monolayer of a Raman active molecule and an antibody. ERLs are mixed with sample, and antigen induces the aggregation of the ERLs. A membrane filter is then used to isolate and concentrate the ERL aggregates for SERS analysis. Preliminary work to establish proof-of-principle of the platform technology utilized mouse IgG as a model antigen. The effects of membrane pore diameter and AuNP size on the analytical performance of the assay were systematically investigated, and it was determined that a pore diameter of 200 nm and AuNP diameter of 80 nm provide maximum sensitivity while minimizing signal from blank samples. Optimization of the assay provided a detection limit of 1.9 ng/mL, 20-fold better than the detection limit achieved by an ELISA employing the same antibody-antigen system. Furthermore, this assay required only 60 min compared to 24h for the ELISA. To validate this assay, mouse serum was directly analyzed to accurately quantify IgG. Collectively, these results demonstrate the potential advantages of this technology over current diagnostic tests for protein biomarkers with respect to time, simplicity, and detection limits. Thus, this approach provides a framework for prospective development of new and more powerful tools that can be designed for point-of-care diagnostic or point-of-need detection.

Metabolic engineering of the Stevia rebaudiana ent-kaurene biosynthetic pathway in recombinant Escherichia coli.

The ent-kaurene is a dedicated precursor pool and is responsible for synthesizing natural sweeteners such as steviol glycosides. In this study, to produce ent-kaurene in Escherichia coli, we modularly constructed and expressed two ent-kaurene genes encoding ent-copalyl diphosphate synthase (CPPS) and ent-kaurene synthase (KS) from Stevia rebaudiana known as a typical plant producing steviol glycoside. The CPPS and KS from S. rebaudiana were functionally expressed in a heterologous host E. coli. Furthermore, in order to enhance ent-kaurene production in E. coli, six geranylgeranyl diphosphate synthases (GGPPS) from various microorganisms and eight strains of E. coli as host were compared by measuring ent-kaurene production. The highest ent-kaurene production of approximately 41.1mg/L was demonstrated in E. coli strain MG1655 co-expressing synthetic CPPS-KS module and GGPPS from Rhodobacter sphaeroides. The ent-kaurene production was further increased up to 179.6 mg/L by overexpression of the three key enzymes for isoprenoid precursor, 1-deoxyxylulose-5-phosphate synthase (DXS), farnesyl diphosphate synthase (IspA) and isopentenyl diphosphate isomerase (IDI) from E. coli. Finally, the highest titer of ent-kaurene (578 mg/L) with a specific yield of ent-kaurene of 143.5mg/g dry cell weight was obtained by culturing E. coli strain MG1655 co-expressing the ent-kaurene module, DXS, IDI and IspA in 1L bioreactor containing 20 g/L glycerol.

A proposed mechanism for the interaction between the Candida albicans Als3 adhesin and streptococcal cell wall proteins.

C. albicans binds various bacteria, including the oral commensal Streptococcus gordonii. Published reports documented the role of C. albicans Als3 and S. gordonii SspB in this interaction, and the importance of the Als N-terminal domain (NT-Als) in C. albicans adhesion. Here, we demonstrate that Als1 also binds S. gordonii. We also describe use of the NT-Als crystal structure to design mutations that precisely disrupt peptide-binding cavity (PBC) or amyloid-forming region (AFR) function in Als3. C. albicans displaying Als3 PBC mutant proteins showed significantly reduced binding to S. gordonii; mutation of the AFR did not affect the interaction. These observations present an enigma: the Als PBC binds free C termini of ligands, but the SspB C terminus is covalently linked to peptidoglycan and thus unavailable as a ligand. These observations and the predicted SspB elongated structure suggest that partial proteolysis of streptococcal cell wall proteins is necessary for recognition by Als adhesins.

A piglet model for studying Candida albicans colonization of the human oro-gastrointestinal tract.

Pigs from a variety of sources were surveyed for oro-gastrointestinal (oro-GIT) carriage of Candida albicans. Candida albicans-positive animals were readily located, but we also identified C. albicans-free pigs. We hypothesized that pigs could be stably colonized with a C. albicans strain of choice, simply by feeding yeast cells. Piglets were farrowed routinely and remained with the sow for 4 days to acquire a normal microbiota. Piglets were then placed in an artificial rearing environment and fed sow milk replacer. Piglets were inoculated orally with one of three different C. albicans strains. Piglets were weighed daily, and culture swabs were collected to detect C. albicans orally, rectally and in the piglet's environment. Stable C. albicans colonization over the course of the study did not affect piglet growth. Necropsy revealed mucosally associated C. albicans throughout the oro-GIT with the highest abundance in the esophagus. Uninoculated control piglets remained C. albicans-negative. These data establish the piglet as a model to study C. albicans colonization of the human oro-GIT. Similarities between oro-GIT colonization in humans and pigs, as well as the ease of working with the piglet model, suggest its adaptability for use among investigators interested in understanding C. albicans-host commensal interactions.

The peptide-binding cavity is essential for Als3-mediated adhesion of Candida albicans to human cells.

The adhesive phenotype of Candida albicans contributes to its ability to colonize the host and cause disease. Als proteins are one of the most widely studied C. albicans virulence attributes; deletion of ALS3 produces the greatest reduction in adhesive function. Although adhesive activity is thought to reside within the N-terminal domain of Als proteins (NT-Als), the molecular mechanism of adhesion remains unclear. We designed mutations in NT-Als3 that test the contribution of the peptide-binding cavity (PBC) to C. albicans adhesion and assessed the adhesive properties of other NT-Als3 features in the absence of a functional PBC. Structural analysis of purified loss-of-PBC-function mutant proteins showed that the mutations did not alter the overall structure or surface properties of NT-Als3. The mutations were incorporated into full-length ALS3 and integrated into the ALS3 locus of a deletion mutant, under control of the native ALS3 promoter. The PBC mutant phenotype was evaluated in assays using monolayers of human pharyngeal epithelial and umbilical vein endothelial cells, and freshly collected human buccal epithelial cells in suspension. Loss of PBC function resulted in an adhesion phenotype that was indistinguishable from the Δals3/Δals3 strain. The adhesive contribution of the Als3 amyloid-forming-region (AFR) was also tested using these methods. C. albicans strains producing cell surface Als3 in which the amyloidogenic potential was destroyed showed little contribution of the AFR to adhesion, instead suggesting an aggregative function for the AFR. Collectively, these results demonstrate the essential and principal role of the PBC in Als3 adhesion.

Frequent isolation of Arthroderma benhamiae from dogs with dermatophytosis.

BACKGROUND: Molecular analysis methods have led to many changes in the taxonomy of dermatophyte species. HYPOTHESIS/OBJECTIVES: We hypothesized that fungi displaying morphology consistent with a traditional identification of 'Trichophyton mentagrophytes' represent multiple species, consistent with the new taxonomy. METHODS: Fungal specimens (n = 20) were collected directly from animals with dermatophytosis, were among those submitted for diagnostic analysis or were part of historical teaching collections. Primers that amplified a portion of the 28S ribosomal RNA gene and primers specific for a fragment from the internal transcribed spacer region were used for PCR amplification of genomic DNA. The DNA sequences from the amplified products were compared with databases to identify the isolates. RESULTS: Of the 80% (n = 16) of the fungal isolates identified as Arthroderma benhamiae, eight were collected from dogs. One isolate was identified as Arthroderma vanbreuseghemii, two were Trichophyton erinacei and one was Nannizziopsis (Chrysosporium) guarroi, which was probably present as a saprophyte. CONCLUSIONS AND CLINICAL IMPORTANCE: Frequent isolation of A. benhamiae from dogs suggests a greater host range for this fungus than reflected in the current literature. Our data also suggest the potential for geographical restriction of strain types within the species. Efforts to identify fungal isolates using molecular techniques create a better understanding of diversity and epidemiology of the dermatophytes.

A monoclonal antibody specific for Candida albicans Als4 demonstrates overlapping localization of Als family proteins on the fungal cell surface and highlights differences between Als localization in vitro and in vivo.

The Candida albicans agglutinin-like sequence (ALS) family encodes large cell surface glycoproteins that function in adhesion of the fungus to host and abiotic surfaces. Monoclonal antibodies (mAbs) specific for each Als protein were developed to study Als localization on the C. albicans surface. An anti-Als4 mAb demonstrated that Als4 covers the surface of yeast cells, with a greater abundance of Als4 on cells grown at 30 °C compared to 37 °C. On germ tubes, Als4 is localized in a restricted area proximal to the mother yeast. Immunolabeling with several anti-Als mAbs showed overlapping localization of Als1 and Als4 on yeast cells and Als1, Als3 and Als4 on germ tubes. Overlapping localization of Als proteins was also observed on yeast and hyphae recovered from mouse models of disseminated and oral candidiasis. Differences between Als localization in vivo and in vitro suggested changes in regulation of Als production in the host compared to the culture flask. Characterization with the anti-Als mAbs reveals the simultaneous presence and differences in relative abundance of Als proteins, creating an accurate image of Als representation and localization that can be used to guide conclusions regarding individual and collective Als protein function.

ALS51, a newly discovered gene in the Candida albicans ALS family, created by intergenic recombination: analysis of the gene and protein, and implications for evolution of microbial gene families.

The Candida albicans ALS family has eight genetic loci, each encoding a large glycoprotein. Als protein function is discussed most frequently in terms of adhesion to host and abiotic surfaces. Analyses of C. albicans strain WO-1 indicated variation within the ALS1 locus compared with other isolates such as SC5314. Investigation revealed a recombination between the contiguous ALS5 and ALS1 loci to generate a new coding region, named ALS51, because it encodes the 5' domain of ALS5 fused in-frame to the tandem repeat region and 3' domain of ALS1. ALS51 was detected in 11 isolates (4.6%) from a collection of 239 C. albicans strains of diverse origin and clade assignment. The 12 ALS51-positive strains identified in this study represented three different ALS family genotypes with respect to the presence and copy number of ALS51, ALS5 and ALS1. ALS51 transcription was detected by real-time reverse-transcription-PCR in WO-1. Although the cell-surface abundance of Als51 on WO-1 and Als5 on SC5314 was too low to visualize by indirect immunofluorescence using an anti-Als5 monoclonal antibody, both proteins were observed on Western blots of ß-1,6-glucanase-digested C. albicans cell walls. Characterization of ALS51 illustrates one of the recombination mechanisms that generate diversity within C. albicans gene families.

Heterogeneous distribution of Candida albicans cell-surface antigens demonstrated with an Als1-specific monoclonal antibody.

Despite an abundance of data describing expression of genes in the Candida albicans ALS (agglutinin-like sequence) gene family, little is known about the production of Als proteins on individual cells, their spatial localization or stability. Als proteins are most commonly discussed with respect to function in adhesion of C. albicans to host and abiotic surfaces. Development of a mAb specific for Als1, one of the eight large glycoproteins encoded by the ALS family, provided the opportunity to detect Als1 during growth of yeast and hyphae, both in vitro and in vivo, and to demonstrate the utility of the mAb in blocking C. albicans adhesion to host cells. Although most C. albicans yeast cells in a saturated culture are Als1-negative by indirect immunofluorescence, Als1 is detected on the surface of nearly all cells shortly after transfer into fresh growth medium. Als1 covers the yeast cell surface, with the exception of bud scars. Daughters of the inoculum cells, and sometimes granddaughters, also have detectable Als1, but Als1 is not detectable on cells from subsequent generations. On germ tubes and hyphae, most Als1 is localized proximal to the mother yeast. Once deposited on yeasts or hyphae, Als1 persists long after the culture has reached saturation. Growth stage-dependent production of Als1, coupled with its persistence on the cell surface, results in a heterogeneous population of cells within a C. albicans culture. Anti-Als1 immunolabelling patterns vary depending on the source of the C. albicans cells, with obvious differences between cells recovered from culture and those from a murine model of disseminated candidiasis. Results from this work highlight the temporal parallels for ALS1 expression and Als1 production in yeasts and germ tubes, the specialized spatial localization and persistence of Als1 on the C. albicans cell surface, and the differences in Als1 localization that occur in vitro and in vivo.