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.

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 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 β-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.