Clinical isolates of Candida auris with enhanced adherence and biofilm formation due to genomic amplification of ALS4

Abstract

Candida auris is an emerging multidrug-resistant fungal pathogen and a new global threat to human health. A unique morphological feature of this fungus is its multicellular aggregating phenotype, which has been thought to be associated with defects in cell division. In this study, we report a new aggregating form of two clinical C. auris isolates with increased biofilm forming capacity due to enhanced adherence of adjacent cells and surfaces. Unlike the previously reported aggregating morphology, this new aggregating multicellular form of C. auris can become unicellular after treatment with proteinase K or trypsin. Genomic analysis demonstrated that amplification of the subtelomeric adhesin gene ALS4 is the reason behind the strain’s enhanced adherence and biofilm forming capacities. Many clinical isolates of C. auris have variable copy numbers of ALS4, suggesting that this subtelomeric region exhibits instability. Global transcriptional profiling and quantitative real-time PCR assays indicated that genomic amplification of ALS4 results in a dramatic increase in overall levels of transcription. Compared to the previously characterized nonaggregative/yeast-form and aggregative-form strains of C. auris, this new Als4-mediated aggregative-form strain of C. auris displays several unique characteristics in terms of its biofilm formation, surface colonization, and virulence.

Candida aurisundergoes adhesin-dependent and -independent cellular aggregation

“…A different mechanism underpins antifungal-induced clustering, as this phenotype was unaffected als31Δ cells (Figs 3 & 8). This result corroborates the work by Bing et al (2003) where they showed that overexpression of Als31 induced the aggregation phenotype in non-aggregating strains and that clinical isolates lacking a fully functional Als31 do not aggregate [16].…”

“…Transcriptomic analysis demonstrated large differences in gene expression between strains of different clades, with 505 DEGs unique to UACa11 (clade I) and 653 DEGs unique to UACa20 (clade III). Of interest to us was that two different ALS genes were differentially expressed indicating that different Als-type adhesins might have distinct roles [16]. We produced an als31 null mutant with some difficulty despite trying multiple techniques, including CRISPR-Cas9-targeted mutation [13]; this might have been aggravated by copy-number variation at the locus [16].…”

“…Of interest to us was that two different ALS genes were differentially expressed indicating that different Als-type adhesins might have distinct roles [16]. We produced an als31 null mutant with some difficulty despite trying multiple techniques, including CRISPR-Cas9-targeted mutation [13]; this might have been aggravated by copy-number variation at the locus [16]. Our als31 mutant unfortunately is not a clean deletion, but the region removed does not contain differentially expressed genes identified in our RNA-seq data (Fig S4).…”

“…Cellular aggregation is a phenotype predominantly of clade III strains, but also aggregative clade I and clade II isolates are known [15][16][17]. The biological relevance and the genetic requirements for this aggregation morphology are not fully understood yet [18], but some potential factors have recently been identified [13,16]. Strains displaying the aggregation phenotype seem to be less virulent, more commonly associated with skin colonisation, have greater biofilm mass, and greater environmental persistence over non-aggregative isolates [15,17,19,20].…”

“…The copyright holder for this preprint (which this version posted April 21, 2023. ; https://doi.org/10.1101/2023.04. 21.537817 doi: bioRxiv preprint two types of aggregation, and has suggested that overexpression of an Als-family adhesin, caused by copy-number variation of the corresponding locus, is involved in this [16]. So far, a major limitation of studies exploring aggregation has been that aggregative strains are often compared to non-aggregative ones from different clades.…”