AUTHOR=Oh Soon-Hwan , Isenhower Allyson , Rodriguez-Bobadilla Rubi , Smith Brooke , Jones Jillian , Hubka Vit , Fields Christopher , Hernandez Alvaro , Hoyer Lois L. 

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

JOURNAL=Frontiers in Microbiology

VOLUME=11

YEAR=2021

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2020.594531

DOI=10.3389/fmicb.2020.594531

ISSN=1664-302X

ABSTRACT=<p>The agglutinin-like sequence (<italic>ALS</italic>) gene family encodes cell-surface adhesins that interact with host and abiotic surfaces, promoting colonization by opportunistic fungal pathogens such as <italic>Candida tropicalis</italic>. Studies of Als protein contribution to <italic>C. tropicalis</italic> adhesion would benefit from an accurate catalog of <italic>ALS</italic> 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 <italic>ALS</italic> genes because of their extensive regions of highly conserved, repeated DNA sequences and because there are many similar <italic>ALS</italic> genes at different chromosomal locations. Here, we describe the benefit of long-read DNA sequencing technology to facilitate characterization of <italic>C. tropicalis ALS</italic> loci. Thirteen <italic>ALS</italic> loci in <italic>C. tropicalis</italic> 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 <italic>ALS</italic> 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 <italic>ALS</italic> gene, as well as both alleles at the divergent <italic>CtrALS3882</italic> locus. <italic>C. tropicalis</italic> cells grown in five different <italic>in vitro</italic> conditions showed differential expression of various <italic>ALS</italic> genes. To place the <italic>C. tropicalis</italic> data into a larger context, TaqMan assays were also designed and validated for analysis of <italic>ALS</italic> gene expression in <italic>Candida albicans</italic> and <italic>Candida dubliniensis</italic>. These comparisons identified the subset of highly expressed <italic>C. tropicalis ALS</italic> 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 <italic>ALS</italic> family contributions to <italic>C. tropicalis</italic> adhesion and pathogenesis.</p>