Candida auris undergoes adhesin-dependent and -independent cellular aggregation.

Candida auris is a fungal pathogen of humans responsible for nosocomial infections with high mortality rates. High levels of resistance to antifungal drugs and environmental persistence mean these infections are difficult to treat and eradicate from a healthcare setting. Understanding the life cycle and the genetics of this fungus underpinning clinically relevant traits, such as antifungal resistance and virulence, is of the utmost importance to develop novel treatments and therapies. Epidemiological and genomic studies have identified five geographical clades (I-V), which display phenotypic and genomic differences. Aggregation of cells, a phenotype primarily of clade III strains, has been linked to reduced virulence in some infection models. The aggregation phenotype has thus been associated with conferring an advantage for (skin) colonisation rather than for systemic infection. However, strains with different clade affiliations were compared to infer the effects of different morphologies on virulence. This makes it difficult to distinguish morphology-dependent causes from clade-specific or even strain-specific genetic factors. Here, we identify two different types of aggregation: one induced by antifungal treatment which is a result of a cell separation defect; and a second which is controlled by growth conditions and only occurs in strains with the ability to aggregate. The latter aggregation type depends on an ALS-family adhesin which is differentially expressed during aggregation in an aggregative C. auris strain. Finally, we demonstrate that macrophages cannot clear aggregates, suggesting that aggregation might after all provide a benefit during systemic infection and could facilitate long-term persistence in the host.

Genetic Transformation of Candida auris via Homology-Directed Repair Using a Standard Lithium Acetate Protocol.

Reverse genetics is a particularly powerful tool in non-model organisms with known whole-genome sequences enabling the characterization of gene and, thus, protein function via a mutant phenotype. Reverse genetic approaches require genetic manipulation techniques which often need to be specifically developed for non-model organisms; this can be fraught with difficulties. Here, we describe a genetic transformation protocol for the recently emerged human pathogen Candida auris to target the integration of DNA constructs into genomic locations via homology-directed repair using long flanking homologous sequences (>1 kb). We detail the generation of DNA constructs for gene deletion with dominant drug resistance markers via fusion PCR, the transformation of these constructs into chemically competent C. auris cells, and the confirmation of correct integration by PCR. This strategy can be adapted to deliver DNA constructs other than deletion cassettes, including promoter exchanges and protein tags.

Labelling of Candida auris Cell Walls to Examine Levels of PAMP Exposure by Flow Cytometry and Fluorescence Microscopy.

Pathogen-associated molecular patterns (PAMPs) of the fungal cell wall are primary targets for the innate immune system of animals. Therefore, characterizing PAMP exposure of fungal pathogens helps to elucidate how they interact with their hosts at a molecular level. Fluorescent labelling can be used to monitor exposure of multiple fungal cell wall PAMPs in a single experiment. Here, we describe a protocol to simultaneously label chitin, mannan, and ß-1,3-glucan in Candida auris to study these PAMPs by fluorescence microscopy and allow high-throughput examination of their exposure by flow cytometry.

Gene conversion: a non-Mendelian process integral to meiotic recombination.

Meiosis is undoubtedly the mechanism that underpins Mendelian genetics. Meiosis is a specialised, reductional cell division which generates haploid gametes (reproductive cells) carrying a single chromosome complement from diploid progenitor cells harbouring two chromosome sets. Through this process, the hereditary material is shuffled and distributed into haploid gametes such that upon fertilisation, when two haploid gametes fuse, diploidy is restored in the zygote. During meiosis the transient physical connection of two homologous chromosomes (one originally inherited from each parent) each consisting of two sister chromatids and their subsequent segregation into four meiotic products (gametes), is what enables genetic marker assortment forming the core of Mendelian laws. The initiating events of meiotic recombination are DNA double-strand breaks (DSBs) which need to be repaired in a certain way to enable the homologous chromosomes to find each other. This is achieved by DSB ends searching for homologous repair templates and invading them. Ultimately, the repair of meiotic DSBs by homologous recombination physically connects homologous chromosomes through crossovers. These physical connections provided by crossovers enable faithful chromosome segregation. That being said, the DSB repair mechanism integral to meiotic recombination also produces genetic transmission distortions which manifest as postmeiotic segregation events and gene conversions. These processes are non-reciprocal genetic exchanges and thus non-Mendelian.

New tools for the new bug Candida auris.

Candida auris is a recently emerged yeast pathogen of humans causing severe hospital-acquired systemic infections. It is of the utmost importance to understand the genetic and cellular basis of its virulence and pathogenicity. In a recent study, Santana and O'Meara generated forward and reverse genetic tools to manipulate C. auris.

The Curious Case of Nonrepetitive Centromeric DNA Sequences in Candida auris and Related Species.

2009 saw the first description of Candida auris, a yeast pathogen of humans. C. auris has since grown into a global problem in intensive care settings, where it causes systemic infections in patients with underlying health issues. Recent whole-genome sequencing has discerned five C. auris clades with distinct phenotypic features which display genomic divergence on a DNA sequence and a chromosome structure level. In the absence of sexual reproduction in C. auris, the mechanism(s) behind the rapid genomic evolution of this emerging killer yeast has remained obscure. Yet, one important bit of information about chromosome organization was missing, the identification of the centromeres. In a recent study, Sanyal and coworkers (A. Narayanan, R. N. Vadnala, P. Ganguly, P. Selvakumar, et al., mBio 12:e00905-21, 2021, https://doi.org/10.1128/mBio.00905-21) filled this knowledge gap by mapping the centromeres in C. auris and its close relatives. This represents a major advance in the chromosome biology of the Candida/Clavispora clade.

What do we know about the biology of the emerging fungal pathogen of humans Candida auris?

Candida auris is a worrisome fungal pathogen of humans which emerged merely about a decade ago. Ever since then the scientific community worked hard to understand clinically relevant traits, such as virulence factors, antifungal resistance mechanisms, and its ability to adhere to human skin and medical devices. Whole-genome sequencing of clinical isolates and epidemiological studies outlining the path of nosocomial outbreaks have been the focus of research into this pathogenic and multidrug-resistant yeast since its first description in 2009. More recently, work was started by several laboratories to explore the biology of C. auris. Here, we review the insights of studies characterizing the mechanisms underpinning antifungal drug resistance, biofilm formation, morphogenetic switching, cell aggregation, virulence, and pathogenicity of C. auris. We conclude that, although some progress has been made, there is still a long journey ahead of us, before we fully understand this novel pathogen. Critically important is the development of molecular tools for C. auris to make this fungus genetically tractable and traceable. This will allow an in-depth molecular dissection of the life cycle of C. auris, of its characteristics while interacting with the human host, and the mechanisms it employs to avoid being killed by antifungals and the immune system.

Catalase T-Deficient Fission Yeast Meiocytes Show Resistance to Ionizing Radiation.

Environmental stress, reactive oxygen species (ROS), or ionizing radiation (IR) can induce adverse effects in organisms and their cells, including mutations and premature aging. DNA damage and its faulty repair can lead to cell death or promote cancer through the accumulation of mutations. Misrepair in germ cells is particularly dangerous as it may lead to alterations in developmental programs and genetic disease in the offspring. DNA damage pathways and radical defense mechanisms mediate resistance to genotoxic stresses. Here, we investigated, in the fission yeast Schizosaccharomyces pombe, the role of the H2O2-detoxifying enzyme cytosolic catalase T (Ctt1) and the Fe2+/Mn2+ symporter Pcl1 in protecting meiotic chromosome dynamics and gamete formation from radicals generated by ROS and IR. We found that wild-type and pcl1-deficient cells respond similarly to X ray doses of up to 300 Gy, while ctt1∆ meiocytes showed a moderate sensitivity to IR but a hypersensitivity to hydrogen peroxide with cells dying at >0.4 mM H2O2. Meiocytes deficient for pcl1, on the other hand, showed a resistance to hydrogen peroxide similar to that of the wild type, surviving doses >40 mM. In all, it appears that in the absence of the main H2O2-detoxifying pathway S. pombe meiocytes are able to survive significant doses of IR-induced radicals.

Intragenic meiotic recombination in Schizosaccharomyces pombe is sensitive to environmental temperature changes.

Changes in environmental temperature influence cellular processes and their dynamics, and thus affect the life cycle of organisms that are unable to control their cell/body temperature. Meiotic recombination is the cellular process essential for producing healthy haploid gametes by providing physical links (chiasmata) between homologous chromosomes to guide their accurate segregation. Additionally, meiotic recombination-initiated by programmed DNA double-strand breaks (DSBs)-can generate genetic diversity and, therefore, is a driving force of evolution. Environmental temperature influencing meiotic recombination outcome thus may be a crucial determinant of reproductive success and genetic diversity. Indeed, meiotic recombination frequency in fungi, plants and invertebrates changes with temperature. In most organisms, these temperature-induced changes in meiotic recombination seem to be mediated through the meiosis-specific chromosome axis organization, the synaptonemal complex in particular. The fission yeast Schizosaccharomyces pombe does not possess a synaptonemal complex. Thus, we tested how environmental temperature modulates meiotic recombination frequency in the absence of a fully-fledged synaptonemal complex. We show that intragenic recombination (gene conversion) positively correlates with temperature within a certain range, especially at meiotic recombination hotspots. In contrast, crossover recombination, which manifests itself as chiasmata, is less affected. Based on our observations, we suggest that, in addition to changes in DSB frequency, DSB processing could be another temperature-sensitive step causing temperature-induced recombination rate alterations.

Pseudohyphal Growth of the Emerging Pathogen Candida auris Is Triggered by Genotoxic Stress through the S Phase Checkpoint.

The morphogenetic switching between yeast cells and filaments (true hyphae and pseudohyphae) is a key cellular feature required for full virulence in many polymorphic fungal pathogens, such as Candida albicans In the recently emerged yeast pathogen Candida auris, occasional elongation of cells has been reported. However, environmental conditions and genetic triggers for filament formation have remained elusive. Here, we report that induction of DNA damage and perturbation of replication forks by treatment with genotoxins, such as hydroxyurea, methyl methanesulfonate, and the clinically relevant fungistatic 5-fluorocytosine, cause filamentation in C. auris The filaments formed were characteristic of pseudohyphae and not parallel-sided true hyphae. Pseudohyphal growth is apparently signaled through the S phase checkpoint and, interestingly, is Tup1 independent in C. auris Intriguingly, the morphogenetic switching capability is strain specific in C. auris, highlighting the heterogenous nature of the species as a whole.IMPORTANCECandida auris is a newly emerged fungal pathogen of humans. This species was first reported in 2009 when it was identified in an ear infection of a patient in Japan. However, despite intense interest in this organism as an often multidrug-resistant fungus, there is little knowledge about its cellular biology. During infection of human patients, fungi are able to change cell shape from ellipsoidal yeast cells to elongated filaments to adapt to various conditions within the host organism. There are different types of filaments, which are triggered by reactions to different cues. Candida auris fails to form filaments when exposed to triggers that stimulate yeast filament morphogenesis in other fungi. Here, we show that it does form filaments when its DNA is damaged. These conditions might arise when Candida auris cells interact with host immune cells or during growth in certain host tissues (kidney or bladder) or during treatment with antifungal drugs.

Surface grafted agents with various molecular lengths and photochemically active benzophenone moieties.

Homologues of benzophenone silane, a covalently graftable, photochemically active surface functionalizing agent, are investigated as surface functionalization agents for both small particles and planar substrates. In these homologues, a chlorosilane functional group and a photochemically active benzophenone oxo moiety are separated with an aliphatic spacer of varying length. The species obtained are first investigated by surface grafting on substrates (Si wafers, glass plates, and indium tin oxide coated glass plates). Si wafer samples are investigated with ellipsometry clearly indicating monolayer formation. The monolayer thickness can be controlled by the size of the aliphatic spacer group and also by the doping concentration of the solution used in the spin-casting step. The functionalized surfaces are further investigated by measuring the contact angle of a suitable organic fluid, a nematic liquid crystal. Photo exposure of these samples results in a drastically varied contact angle: The surface grafted species are still photochemically active and photo exposure leads to the addition of a nearby organic molecule (from the liquid crystalline phase) to each activated surface agent molecule. The synthesized species are then investigated as (covalently binding) surfactants in the wet planetary ball milling process aimed to fabricate solid-liquid dispersions (of Fe doped lithium niobate particles). It was found that the use of species with higher molecular length results in dispersions of small particles, functionalized with photochemically active surface agents. Indeed, they show better performance than conventional surfactants.

DNA sequence differences are determinants of meiotic recombination outcome.

Meiotic recombination is essential for producing healthy gametes, and also generates genetic diversity. DNA double-strand break (DSB) formation is the initiating step of meiotic recombination, producing, among other outcomes, crossovers between homologous chromosomes (homologs), which provide physical links to guide accurate chromosome segregation. The parameters influencing DSB position and repair are thus crucial determinants of reproductive success and genetic diversity. Using Schizosaccharomyces pombe, we show that the distance between sequence polymorphisms across homologs has a strong impact on meiotic recombination rate. The closer the sequence polymorphisms are to each other across the homologs the fewer recombination events were observed. In the immediate vicinity of DSBs, sequence polymorphisms affect the frequency of intragenic recombination events (gene conversions). Additionally, and unexpectedly, the crossover rate of flanking markers tens of kilobases away from the sequence polymorphisms was affected by their relative position to each other amongst the progeny having undergone intragenic recombination. A major regulator of this distance-dependent effect is the MutSα-MutLα complex consisting of Msh2, Msh6, Mlh1, and Pms1. Additionally, the DNA helicases Rqh1 and Fml1 shape recombination frequency, although the effects seen here are largely independent of the relative position of the sequence polymorphisms.

Fast copolymer network liquid crystals for tunable birefringence colors.

A fast-switching, tunable color filter was found in a copolymer network liquid crystal (LC), which was in situ generated in a conventional LC test cell with parallel aligned glass plates and investigated with polarized light. Polarization filters were used to convert the tunable optical phase retardance of the test cells to birefringence colors as is always possible in a LC test cell with carefully adjusted cell gap and effective birefringence. The cell gap of the samples could be adjusted to a value of 9 µm, which is not easily possible in a polymer LC composite without creating defects. In these samples, the typical pastel colors seen frequently in birefringent samples could be avoided. The transmittance spectra were recorded and converted to CIE 1931 color coordinates, which showed that the colors seen had a reasonable distance to the white point. The electro-optic switching times of the samples were investigated: Fast responses of t on +t off <5 ms were found, which is an impressive speed for tunable birefringence colors in LCs and LC composites. Upon increasing addressing voltages, a blueshift of the peak seen in the transmittance spectra was observed. The samples consisted of copolymer network LC, generated from a reactive mixture with mesogenic monomer and nonmesogenic comonomer. The tunable color was seen selectively in samples with dodecyl acrylate as comonomer. The experiments show how even a straightforward electro-optic experiment still can result in unexpended findings, which may expand the use of LC composites in nondisplay applications. The polymer morphology in samples with a larger cell gap was investigated with scanning electron microscopy, and interdefect distances of ≈40 µm were found. The appearance of defects in test cells with a cell gap of 9 µm could be avoided because the cell gap was much smaller than the measured interdefect distances in test cells with a larger cell gap.

ABC Transporter Genes Show Upregulated Expression in Drug-Resistant Clinical Isolates of Candida auris: A Genome-Wide Characterization of ATP-Binding Cassette (ABC) Transporter Genes.

ATP-binding cassette (ABC) superfamily members have a key role as nutrient importers and exporters in bacteria. However, their role as drug exporters in eukaryotes brought this superfamily member to even greater prominence. The capacity of ABC transporters to efflux a broad spectrum of xenobiotics represents one of the major mechanisms of clinical multidrug resistance in pathogenic fungi including Candida species. Candida auris, a newly emerged multidrug-resistant fungal pathogen of humans, has been responsible for multiple outbreaks of drug-resistant infections in hospitals around the globe. Our study has analyzed the entire complement of ABC superfamily transporters to assess whether these play a major role in drug resistance mechanisms of C. auris. Our bioinformatics analyses identified 28 putative ABC proteins encoded in the genome of the C. auris type-strain CBS 10913T; 20 of which contain transmembrane domains (TMDs). Quantitative real-time PCR confirmed the expression of all 20 TMD transporters, underlining their potential in contributing to the C. auris drug-resistant phenotype. Changes in transcript levels after short-term exposure of drugs and in drug-resistant C. auris isolates suggested their importance in the drug resistance phenotype of this pathogen. CAUR_02725 orthologous to CDR1, a major multidrug exporter in other yeasts, showed consistently higher expression in multidrug-resistant strains of C. auris. Homologs of other ABC transporter genes, such as CDR4, CDR6, and SNQ2, also displayed raised expression in a sub-set of clinical isolates. Together, our analysis supports the involvement of these transporters in multidrug resistance in C. auris.

Clarifying the concept of chronic kidney disease for non-nephrologists.

Chronic kidney disease (CKD) expands the prior concept of chronic renal insufficiency by including patients with relatively preserved renal function, as assessed by the estimated glomerular filtration rate (eGFR), as even these early CKD stages are associated with an increased risk for all-cause death and cardiovascular death, CKD progression and acute kidney injury. A decreased eGFR (<60 mL/min/1.73 m2) is by itself diagnostic of CKD when persisting for >3 months. However, when eGFR is ≥60 mL/min/1.73 m2, an additional criterion is required to diagnose CKD. In a recent clinical trial published in The New England Journal of Medicine, all 6190 participants were reported to have CKD: 47% had Stages 1 and 2 CKD and 53% had Stage 3 CKD. This illustrates a widespread misunderstanding of the concept of CKD. Moreover, CKD categories in this study were assigned based on the estimated creatinine clearance. Since both estimated creatinine clearance and creatinine clearance overestimate eGFR, this illustrates another frequent misunderstanding: equating GFR with creatinine clearance. In this commentary, we clarify the concept of CKD and of CKD categories for non-nephrologists. Assigning a diagnosis of CKD to a patient with normal renal function and absence of other evidence of CKD may have negative consequences for the individual (e.g. insurance and others) as well as for the medical community at large by creating confusion about the concept.

Rapid and extensive karyotype diversification in haploid clinical Candida auris isolates.

Candida auris is a newly emerged pathogenic microbe, having been identified as a medically relevant fungus as recently as 2009. It is one of the most drug-resistant yeast species known to date and its emergence and population structure are unusual. Because of its recent emergence, we are largely ignorant about fundamental aspects of its general biology, life cycle, and population dynamics. Here, we report the karyotype variability of 26 C. auris strains representing the four main clades. We demonstrate that all strains are haploid and have a highly plastic karyotype containing five to seven chromosomes, which can undergo marked alterations within a short time frame when the fungus is put under genotoxic, heat, or osmotic stress. No simple correlation was found between karyotype pattern, drug resistance, and clade affiliation indicating that karyotype heterogeneity is rapidly evolving. As with other Candida species, these marked karyotype differences between isolates are likely to have an important impact on pathogenic traits of C. auris.

Immediate visualization of recombination events and chromosome segregation defects in fission yeast meiosis.

Schizosaccharomyces pombe, also known as fission yeast, is an established model for studying chromosome biological processes. Over the years, research employing fission yeast has made important contributions to our knowledge about chromosome segregation during meiosis, as well as meiotic recombination and its regulation. Quantification of meiotic recombination frequency is not a straightforward undertaking, either requiring viable progeny for a genetic plating assay, or relying on laborious Southern blot analysis of recombination intermediates. Neither of these methods lends itself to high-throughput screens to identify novel meiotic factors. Here, we establish visual assays novel to Sz. pombe for characterizing chromosome segregation and meiotic recombination phenotypes. Genes expressing red, yellow, and/or cyan fluorophores from spore-autonomous promoters have been integrated into the fission yeast genomes, either close to the centromere of chromosome 1 to monitor chromosome segregation, or on the arm of chromosome 3 to form a genetic interval at which recombination frequency can be determined. The visual recombination assay allows straightforward and immediate assessment of the genetic outcome of a single meiosis by epi-fluorescence microscopy without requiring tetrad dissection. We also demonstrate that the recombination frequency analysis can be automatized by utilizing imaging flow cytometry to enable high-throughput screens. These assays have several advantages over traditional methods for analyzing meiotic phenotypes.