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

Abstract

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.

The secretory Candida effector Sce1 licenses fungal virulence by masking the immunogenic β‐1,3‐glucan and promoting apoptosis of the host cells

“…In contrast, most of the other Pir family proteins in S. cerevisiae contain multiple PIR motifs to the left of the Kex2 cleavage site (Figure S6A ). Deletion of the CaPir1 showed a distinguishable phenotype from its parent 55 . Single deletion of CSP1 or CSP2 in Candida dubliniensis did not reveal any discernible difference in chlamydospore production 40 .…”

Cell wall-resident PIR proteins show an inverted architecture inNeurospora crassa, but keep their role as wall stabilizers

“…Their role is to provide strength and stability to the cell wall either through the formation of covalent bonds between the conserved Q residue of PIR domains and β-1,3-glucans, or the formation of disulfide bonds between the cysteines of the Cys-rich domain and already present CWPs [14,15,22]. Although PIRs have been inferred in filamentous fungi [34], previous phylogenetic, cellular, and biochemical analyses have only been performed on yeast PIRs [25,30]. Here, a comprehensive search of PIR-domains containing proteins in the proteomes of 840 species, representatives of the fungal tree of life, was undertaken to identify the whole diversity of such proteins, but also to better understand their evolutionary history and biological role.…”

“…To explore the diversity of PIRs beyond the already characterized PIRs from yeasts [11,30,58], the yeast PIR-domain (PF00399) was used as a query to explore the diversity of PIRs in the tree of life. Despite systematic searches in different databases (Ensembl All, UniProtKB, and SwissProt), only sequences from fungi were retrieved.…”

“…PIRs have been widely studied in yeasts: S. cerevisiae [10,11,18], Zigosaccharomyces rouxi [12] , Kluyveromyces lactis [29], Candida albicans [30,31], Yarrowia lipolytica [17] , Pichia pastoris [32] and Candida glabrata [33] . They have only been bioinformatically inferred for four filamentous fungi: Magnaporte grisea, Gibberella zeae, Blumeria graminis and Neurospora crassa [34].…”

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

“…Genomic information streamlines gene deletion experiments by facilitating creation and targeting of disruption constructs. However, in the diploid C. albicans , these efforts are sometimes complicated by allelic variation ( Kim et al., 2022 ).…”