Variations in candidalysin amino acid sequence influence toxicity and host responses.

Candida albicans causes millions of mucosal infections in humans annually. Hyphal overgrowth on mucosal surfaces is frequently associated with tissue damage caused by candidalysin, a secreted peptide toxin that destabilizes the plasma membrane of host cells thereby promoting disease and immunopathology. Candidalysin was first identified in C. albicans strain SC5314, but recent investigations have revealed candidalysin "variants" of differing amino acid sequence in isolates of C. albicans, and the related species C. dubliniensis, and C tropicalis, suggesting that sequence variation among candidalysins may be widespread in natural populations of these Candida species. Here, we analyzed ECE1 gene sequences from 182 C. albicans isolates, 10 C. dubliniensis isolates, and 78 C. tropicalis isolates and identified 10, 3, and 2 candidalysin variants in these species, respectively. Application of candidalysin variants to epithelial cells revealed differences in the ability to cause cellular damage, changes in metabolic activity, calcium influx, MAPK signalling, and cytokine secretion, while biophysical analyses indicated that variants exhibited differences in their ability to interact with and permeabilize a membrane. This study identifies candidalysin variants with differences in biological activity that are present in medically relevant Candida species. IMPORTANCE: Fungal infections are a significant burden to health. Candidalysin is a toxin produced by Candida albicans that damages host tissues, facilitating infection. Previously, we demonstrated that candidalysins exist in the related species C. dubliniensis and C. tropicalis, thereby identifying these molecules as a toxin family. Recent genomic analyses have highlighted the presence of a small number of candidalysin "variant" toxins, which have different amino acid sequences to those originally identified. Here, we screened genome sequences of isolates of C. albicans, C. dubliniensis, and C. tropicalis and identified candidalysin variants in all three species. When applied to epithelial cells, candidalysin variants differed in their ability to cause damage, activate intracellular signaling pathways, and induce innate immune responses, while biophysical analysis revealed differences in the ability of candidalysin variants to interact with lipid bilayers. These findings suggest that intraspecies variation in candidalysin amino acid sequence may influence fungal pathogenicity.

Genome Analysis of a Newly Discovered Yeast Species, Hanseniaspora menglaensis.

Annual surveys of Irish soil samples identified three isolates, CBS 16921 (UCD88), CBS 18246 (UCD443), and CBS 18247 (UCD483), of an apiculate yeast species within the Hanseniaspora genus. The internal transcribed spacer (ITS) and D1/D2 region of the large subunit (LSU) rRNA sequences showed that these are isolates of the recently described species Hanseniaspora menglaensis, first isolated from Southwest China. No genome sequence for H. menglaensis is currently available. The genome sequences of the three Irish isolates were determined using short-read (Illumina) sequencing, and the sequence of one isolate (CBS 16921) was assembled to chromosome level using long-read sequencing (Oxford Nanopore Technologies). Phylogenomic analysis shows that H. menglaensis belongs to the fast-evolving lineage (FEL) of Hanseniaspora. Only one MAT idiomorph (encoding MATα1) was identified in all three sequenced H. menglaensis isolates, consistent with one mating type of a heterothallic species. Genome comparisons showed that there has been a rearrangement near MATα of FEL species compared to isolates from the slowly evolving lineage (SEL).

Evaluation of outbreak persistence caused by multidrug-resistant and echinocandin-resistant Candida parapsilosis using multidimensional experimental and epidemiological approaches.

Candida parapsilosis is known to cause severe and persistent outbreaks in clinical settings. Patients infected with multidrug-resistant C. parapsilosis (MDR Cp) isolates were identified in a large Turkish hospital from 2017-2020. We subsequently identified three additional patients infected with MDR Cp isolates in 2022 from the same hospital and two echinocandin-resistant (ECR) isolates from a single patient in another hospital. The increasing number of MDR and ECR isolates contradicts the general principle that the severe fitness cost associated with these phenotypes could prevent their dominance in clinical settings. Here, we employed a multidimensional approach to systematically assess the fitness costs of MDR and ECR C. parapsilosis isolates. Whole-genome sequencing revealed a novel MDR genotype infecting two patients in 2022. Despite severe in vitro defects, the levels and tolerances of the biofilms of our ECR and MDR isolates were generally comparable to those of susceptible wild-type isolates. Surprisingly, the MDR and ECR isolates showed major alterations in their cell wall components, and some of the MDR isolates consistently displayed increased tolerance to the fungicidal activities of primary human neutrophils and were more immunoevasive during exposure to primary human macrophages. Our systemic infection mouse model showed that MDR and ECR C. parapsilosis isolates had comparable fungal burden in most organs relative to susceptible isolates. Overall, we observed a notable increase in the genotypic diversity and frequency of MDR isolates and identified MDR and ECR isolates potentially capable of causing persistent outbreaks in the future.

Draft genome sequence of the yeast Schwanniomyces capriottii strain UCD805, isolated from soil in Ireland.

Schwanniomyces capriottii is a member of the Debaryomycetaceae family in the order Saccharomycetales. Here, we present the genome sequence of S. capriottii UCD805, which was isolated from soil in Dublin, Ireland. This genome is 12.2 Mb and was assembled into 14 scaffolds plus a mitochondrial genome scaffold.

Systematic Analysis of Copy Number Variations in the Pathogenic Yeast Candida parapsilosis Identifies a Gene Amplification in RTA3 That is Associated with Drug Resistance.

We analyzed the genomes of 170 C. parapsilosis isolates and identified multiple copy number variations (CNVs). We identified two genes, RTA3 (CPAR2_104610) and ARR3 (CPAR2_601050), each of which was the target of multiple independent amplification events. Phylogenetic analysis shows that most of these amplifications originated only once. For ARR3, which encodes a putative arsenate transporter, 8 distinct CNVs were identified, ranging in size from 2.3 kb to 10.5 kb with 3 to 23 copies. For RTA3, 16 distinct CNVs were identified, ranging in size from 0.3 kb to 4.5 kb with 2 to ~50 copies. One unusual amplification resulted in a DUP-TRP/INV-DUP structure similar to some human CNVs. RTA3 encodes a putative phosphatidylcholine (PC) floppase which is known to regulate the inward translocation of PC in Candida albicans. We found that an increased copy number of RTA3 correlated with resistance to miltefosine, an alkylphosphocholine drug that affects PC metabolism. Additionally, we conducted an adaptive laboratory evolution experiment in which two C. parapsilosis isolates were cultured in increasing concentrations of miltefosine. Two genes, CPAR2_303950 and CPAR2_102700, coding for putative PC flippases homologous to S. cerevisiae DNF1 gained homozygous protein-disrupting mutations in the evolved strains. Overall, our results show that C. parapsilosis can gain resistance to miltefosine, a drug that has recently been granted orphan drug designation approval by the United States Food and Drug Administration for the treatment of invasive candidiasis, through both CNVs or loss-of-function alleles in one of the flippase genes. IMPORTANCE Copy number variations (CNVs) are an important source of genomic diversity that have been associated with drug resistance. We identify two unusual CNVs in the human fungal pathogen Candida parapsilosis. Both target a single gene (RTA3 or ARR3), and they have occurred multiple times in multiple isolates. The copy number of RTA3, a putative floppase that controls the inward translocation of lipids in the cell membrane, correlates with resistance to miltefosine, a derivative of phosphatidylcholine (PC) that was originally developed as an anticancer drug. In 2021, miltefosine was designated an orphan drug by the United States Food and Drug Administration for the treatment of invasive candidiasis. Importantly, we find that resistance to miltefosine is also caused by mutations in flippases, which control the outward movement of lipids, and that many C. parapsilosis isolates are prone to easily acquiring an increased resistance to miltefosine.

Clinical azole cross-resistance in Candida parapsilosis is related to a novel MRR1 gain-of-function mutation.

OBJECTIVES: Hereby, we describe the molecular mechanisms underlying the acquisition of azole resistance by a Candida parapsilosis isolate following fluconazole treatment due to candiduria. METHODS: A set of three consecutive C. parapsilosis isolates were recovered from the urine samples of a patient with candiduria. Whole-genome sequencing and antifungal susceptibility assays were performed. The expression of MRR1, MDR1, ERG11 and CDR1B (CPAR2_304370) was quantified by RT-qPCR. RESULTS: The initial isolate CPS-A was susceptible to all three azoles tested (fluconazole, voriconazole and posaconazole); isolate CPS-B, collected after the second cycle of treatment, exhibited a susceptible-dose-dependent phenotype to fluconazole and isolate CPS-C, recovered after the third cycle, exhibited a cross-resistance profile to fluconazole and voriconazole. Whole-genome sequencing revealed a putative resistance mechanism in isolate CPS-C, associated with a G1810A nucleotide substitution, leading to a G604R change in the Mrr1p transcription factor. Introducing this mutation into the susceptible CPS-A isolate (MRR1RI) resulted in resistance to fluconazole and voriconazole, as well as up-regulation of MRR1 and MDR1. Interestingly, the susceptible-dose-dependent phenotype exhibited by isolate CPS-B was associated with an increased copy number of the CDR1B gene. The expression of CDR1B was increased in both isolates CPS-B and CPS-C and in the MRR1RI strain, harbouring the gain-of-function mutation. CONCLUSIONS: Our results describe clinical azole cross-resistance acquisition in C. parapsilosis due to a G1810A (G604R) gain-of-function mutation, resulting in MRR1 hyperactivation and consequently, MDR1 efflux pump overexpression. We also associated amplification of the CDR1B gene with decreased fluconazole susceptibility and showed that it is a putative target of the MRR1 gain-of-function mutation.

Genomic diversity and meiotic recombination among isolates of the biotech yeast Komagataella phaffii (Pichia pastoris).

BACKGROUND: Komagataella phaffii is a yeast widely used in the pharmaceutical and biotechnology industries, and is one of the two species that were previously called Pichia pastoris. However, almost all laboratory work on K. phaffii has utilized strains derived from a single natural isolate, CBS7435. There is little information about the sequence diversity of K. phaffii or the genetic properties of this species. RESULTS: We sequenced the genomes of all the known isolates of K. phaffii. We made a genetic cross between derivatives of two isolates that differ at 44,000 single nucleotide polymorphism sites, and used this cross to analyze the rate and landscape of meiotic recombination. We conducted tetrad analysis by making use of the property that K. phaffii haploids do not mate in rich media, which enabled us to isolate and sequence the four types of haploid cell that are present in the colony that forms when a tetra-type ascus germinates. CONCLUSIONS: We found that only four distinct natural isolates of K. phaffii exist in public yeast culture collections. The meiotic recombination rate in K. phaffii is approximately 3.5 times lower than in Saccharomyces cerevisiae, with an average of 25 crossovers per meiosis. Recombination is suppressed, and genetic diversity among natural isolates is low, in a region around centromeres that is much larger than the centromeres themselves. Our work lays a foundation for future quantitative trait locus analysis in K. phaffii.

Draft Genome Sequence of the Birch Tree Fungal Pathogen Taphrina betulina UCD315.

Taphrina betulina is the ascomycete yeast that causes the formation of witches' brooms in birch trees. Here, we report the first draft genome sequence of T. betulina, from strain UCD315, isolated from soil in Ireland. The genome is haploid and 12.5 Mb long.