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.

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.

Evolution of pathogenicity and sexual reproduction in eight Candida genomes.

Candida species are the most common cause of opportunistic fungal infection worldwide. Here we report the genome sequences of six Candida species and compare these and related pathogens and non-pathogens. There are significant expansions of cell wall, secreted and transporter gene families in pathogenic species, suggesting adaptations associated with virulence. Large genomic tracts are homozygous in three diploid species, possibly resulting from recent recombination events. Surprisingly, key components of the mating and meiosis pathways are missing from several species. These include major differences at the mating-type loci (MTL); Lodderomyces elongisporus lacks MTL, and components of the a1/2 cell identity determinant were lost in other species, raising questions about how mating and cell types are controlled. Analysis of the CUG leucine-to-serine genetic-code change reveals that 99% of ancestral CUG codons were erased and new ones arose elsewhere. Lastly, we revise the Candida albicans gene catalogue, identifying many new genes.

Structural basis for the broad specificity to host-cell ligands by the pathogenic fungus Candida albicans.

Candida albicans is the most prevalent fungal pathogen in humans and a major source of life-threatening nosocomial infections. The Als (agglutinin-like sequence) glycoproteins are an important virulence factor for this fungus and have been associated with binding of host-cell surface proteins and small peptides of random sequence, the formation of biofilms and amyloid fibers. High-resolution structures of N-terminal Als adhesins (NT-Als; up to 314 amino acids) show that ligand recognition relies on a motif capable of binding flexible C termini of peptides in extended conformation. Central to this mechanism is an invariant lysine that recognizes the C-terminal carboxylate of ligands at the end of a deep-binding cavity. In addition to several protein-peptide interactions, a network of water molecules runs parallel to one side of the ligand and contributes to the recognition of diverse peptide sequences. These data establish NT-Als adhesins as a separate family of peptide-binding proteins and an unexpected adhesion system for primary, widespread protein-protein interactions at the Candida/host-cell interface.

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.

Use of a Candida albicans SC5314 PacBio HiFi reads dataset to close gaps in the reference genome assembly, reveal a subtelomeric gene family, and produce accurate phased allelic sequences.

Candida albicans SC5314 is the most-often used strain for molecular manipulation of the species. The SC5314 reference genome sequence is the result of considerable effort from many scientists and has advanced research into fungal biology and pathogenesis. Although the resource is highly developed and presented in a phased diploid format, the sequence includes gaps and does not extend to the telomeres on its eight chromosome pairs. Accurate SC5314 genome assembly is complicated by the presence of extensive repeated sequences and considerable allelic length variation at some loci. Advances in genome sequencing technology provide the tools to obtain highly accurate long-read data that span even the most-difficult-to-assemble genome regions. Here, we describe derivation of a PacBio HiFi data set and creation of a collapsed haploid telomere-to-telomere assembly of the SC5314 genome (ASM3268872v1) that revealed previously unknown features of the strain. ASM3268872v1 subtelomeric distances were up to 19 kb larger than in the reference genome and revealed a family of highly conserved DNA helicase-encoding genes at 10 of the 16 chromosome ends. We also describe alignments of individual HiFi reads to deduce accurate diploid sequences for the most notoriously difficult-to-assemble C. albicans genes: the agglutinin-like sequence (ALS) gene family. We provide a tutorial that demonstrates how the HiFi reads can be visualized to explore any region of interest. Availability of the HiFi reads data set and the ASM3268872v1 comparative guide assembly will streamline research efforts because accurate diploid sequences can be derived using simple in silico methods rather than time-consuming laboratory-bench approaches.

An improved Lodderomyces elongisporus NRRL YB-4239 genome assembly substantiated by its electrophoretic karyotype.

Pacific Biosciences long-read sequencing was used to improve the genome assembly for Lodderomyces elongisporus strain NRRL YB-4239 (ATCC 11503). The new assembly included eight chromosomes that were substantiated by the electrophoretic karyotype. The nuclear genome was 16.1 Mb (37.2% GC) with 5,740 genes predicted.

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.

Genome sequence of Emydomyces testavorans type strain 16-2883 (ATCC TSD-145), an onygenalean fungus associated with freshwater aquatic turtle shell lesions.

Emydomyces testavorans is an onygenalean keratinophilic fungus associated with shell and skin lesions in freshwater aquatic turtles. The genome sequence presented here includes five contigs (ranging in size from 2.8 to 9.8 Mb; 31.8 Mb total; 40% GC) and a 92.2-kb mitochondrial genome. The nuclear genome predicted 7,550 genes.

Staphylococcus aureus adherence to Candida albicans hyphae is mediated by the hyphal adhesin Als3p.

The bacterium Staphylococcus (St.) aureus and the opportunistic fungus Candida albicans are currently among the leading nosocomial pathogens, often co-infecting critically ill patients, with high morbidity and mortality. Previous investigations have demonstrated preferential adherence of St. aureus to C. albicans hyphae during mixed biofilm growth. In this study, we aimed to characterize the mechanism behind this observed interaction. C. albicans adhesin-deficient mutant strains were screened by microscopy to identify the specific receptor on C. albicans hyphae recognized by St. aureus. Furthermore, an immunoassay was developed to validate and quantify staphylococcal binding to fungal biofilms. The findings from these experiments implicated the C. albicans adhesin agglutinin-like sequence 3 (Als3p) in playing a major role in the adherence process. This association was quantitatively established using atomic force microscopy, in which the adhesion force between single cells of the two species was significantly reduced for a C. albicans mutant strain lacking als3. Confocal microscopy further confirmed these observations, as St. aureus overlaid with a purified recombinant Als3 N-terminal domain fragment (rAls3p) exhibited robust binding. Importantly, a strain of Saccharomyces cerevisiae heterologously expressing Als3p was utilized to further confirm this adhesin as a receptor for St. aureus. Although the parental strain does not bind bacteria, expression of Als3p on the cell surface conferred upon the yeast the ability to strongly bind St. aureus. To elucidate the implications of these in vitro findings in a clinically relevant setting, an ex vivo murine model of co-infection was designed using murine tongue explants. Fluorescent microscopic images revealed extensive hyphal penetration of the epithelium typical of C. albicans mucosal infection. Interestingly, St. aureus bacterial cells were only seen within the epithelial tissue when associated with the invasive hyphae. This differed from tongues infected with St. aureus alone or in conjunction with the als3 mutant strain of C. albicans, where bacterial presence was limited to the outer layers of the oral tissue. Collectively, the findings generated from this study identified a key role for C. albicans Als3p in mediating this clinically relevant fungal-bacterial interaction.

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.

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.

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.

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.

Candida albicans evades NK cell elimination via binding of Agglutinin-Like Sequence proteins to the checkpoint receptor TIGIT.

Candida albicans is the most common fungal pathogen and a prevalent cause of deadly bloodstream infections. Better understanding of the immune response against it, and the ways by which it evades immunity, are crucial for developing new therapeutics against it. Natural Killer (NK) cells are innate lymphocytes best known for their role against viruses and tumors. In recent years it became clear that NK cells also play an important role in anti-fungal immunity. Here we show that while NK cells recognize and eliminate C. albicans, the fungal cells inhibit NK cells by manipulating the immune checkpoint receptor TIGIT (T cell immunoreceptor with Ig and ITIM domains) in both humans and mice. We identify the responsible fungal ligands as members of the Als (Agglutinin-Like Sequences) protein family. Furthermore, we show that blocking this interaction using immunotherapy with a TIGIT-blocking antibody can re-establish anti-Candida immunity and serve as a potential therapeutic tool.

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.

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.

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.

Heterogeneity of vaginal microbial communities within individuals.

Recent culture-independent studies have revealed that a healthy vaginal ecosystem harbors a surprisingly complex assemblage of microorganisms. However, the spatial distribution and composition of vaginal microbial populations have not been investigated using molecular methods. Here, we evaluated site-specific microbial composition within the vaginal ecosystem and examined the influence of sampling technique in detection of the vaginal microbiota. 16S rRNA gene clone libraries were prepared from samples obtained from different locations (cervix, fornix, outer vaginal canal) and by different methods (swabbing, scraping, lavaging) from the vaginal tracts of eight clinically healthy, asymptomatic women. The data reveal that the vaginal microbiota is not homogenous throughout the vaginal tract but differs significantly within an individual with regard to anatomical site and sampling method used. Thus, this study illuminates the complex structure of the vaginal ecosystem and calls for the consideration of microenvironments when sampling vaginal microbiota as a clinical predictor of vaginal health.