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

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.

A case of Candida auris candidemia in Xiamen, China, and a comparative analysis of clinical isolates in China.

The recently emerged fungal pathogen Candida auris often displays resistance to one or more antifungal drugs. Its infections have been identified in at least 40 countries on six continents to date. Here we report a case of C. auris candidemia in a patient in Xiamen, a city in south China. We also review currently reported cases of C. auris infection in China and compare the genetic and biological features of C. auris strains isolated from this country. Our phylogenetic analysis indicates that there are at least two C. auris genetic clades present in China (the South African clade and the south Asian clade) that display opposite mating type loci (one is MTL a and the other is MTLα). We also found that there are several distinct features among the clinical isolates studied, including the expression of virulence factors, antifungal susceptibilities, and cellular morphologies, and that these features could be associated with the mating-type of the isolate. For example, C. auris MTL a isolates generally secreted higher levels of secreted aspartyl proteases (Saps) at ambient environmental temperatures. Taken together, this study demonstrates that C. auris clinical isolates from China exhibit diversity in both biological and genetic features.

A biological and genomic comparison of a drug-resistant and a drug-susceptible strain of Candida auris isolated from Beijing, China.

The fungal pathogen Candida auris has emerged as a new threat to human health. We previously reported the first isolate of C. auris (BJCA001) in China, which belongs to the South Asian clade (I) and was susceptible to all antifungals tested. In this study, we report the isolation of a drug-resistant C. auris strain (BJCA002) from the same city (Beijing). Strain BJCA002 belongs to the South African clade (III) and is resistant to fluconazole and amphotericin B based on the tentative MIC breakpoints. Taking advantage of the two isolates with distinct antifungal susceptibility and genetic origins, we performed a biological and genomic comparative study. Besides antifungal susceptibility, strains BJCA001 and BJCA002 showed differences in multiple aspects including morphologies, expression of virulence factors, virulence, mating type, and genomic sequence and organization. Notably, strain BJCA002 was less virulent than BJCA001 in both the Galleria mellonella and mouse systemic infection models. Genomic analysis demonstrated that strain BJCA002 but not BJCA001 had multiple mutations in drug resistance-associated genes, including a hot-spot mutation of ERG11 (VF125AL, namely V125A and F126L) and some missense mutations in CDR1, MDR1, and TAC1. Notably, strain BJCA001 carried 64 copies of the Zorro3 retrotransposon, whereas BJCA002 had only 3 copies in the genome. Taken together, our findings not only reveal the genetic and phenotypic diversities of the two isolates from Beijing, China, but also shed new light on the genetic basis of the antifungal resistance and virulence of C. auris.

Filamentous growth is a general feature of Candida auris clinical isolates.

A striking feature of pathogenic Candida species is morphological plasticity that facilitates environmental adaptation and host infection. Candida auris is an emerging multidrug-resistant fungal pathogen first described in Japan in 2009. In this study, we demonstrate that clinical isolates of C. auris have multiple colony and cellular morphologies including the yeast, filamentous, aggregated, and elongated forms. This phenotypic diversity has been observed in eight clinical isolates of C. auris representing four major genetic clades, suggesting that it could be a general characteristic. We further demonstrate that different cell types of C. auris exhibit distinct antifungal resistance and virulence properties in a Galleria mellonella infection model. Our findings imply that morphological diversity is an important biological feature of C. auris and could be a contributor to its emergence and rapid prevalence worldwide. LAY SUMMARY: Candida auris is an emerging multidrug-resistant fungal pathogen. Morphological analyses indicate that filamentation is a general feature of clinical isolates of C. auris. This ability is associated with antifungal resistance and virulence.

Discovery of the Diploid Form of the Emerging Fungal Pathogen Candida auris.

The new multidrug-resistant pathogen Candida auris was first described in 2009 in Japan and has emerged in many countries worldwide. This human fungal pathogen has long been considered a haploid fungus. Here, we report the discovery of the diploid form and spontaneous ploidy shifts in clinical isolates of C. auris. Haploid and diploid cells of C. auris differ in several aspects including growth rates, virulence, and global gene expression profiles. For example, diploid cells exhibit a slower growth rate than haploid cells in in vitro culture media; however, they are more virulent than haploid cells in a mouse systemic infection model. Global transcriptional expression analysis demonstrates that both haploid and diploid cells express a set of ploidy-enriched genes, which are involved in the regulation of metabolism, cell wall maintenance, translation and DNA replication, and other important biological processes. Antifungal susceptibility testing shows that haploid and diploid cells exhibit similar responses when treated with a number of antifungals. Taken together, haploid and diploid cells may have different fitness responses to diverse niches, and ploidy changes could be an adaptive strategy of C. auris to environmental changes. Our findings shed new light on the biology and pathogenesis of this emerging fungal pathogen.

Environment-induced same-sex mating in the yeast Candida albicans through the Hsf1-Hsp90 pathway.

While sexual reproduction is pervasive in eukaryotic cells, the strategies employed by fungal species to achieve and complete sexual cycles is highly diverse and complex. Many fungi, including Saccharomyces cerevisiae and Schizosaccharomyces pombe, are homothallic (able to mate with their own mitotic descendants) because of homothallic switching (HO) endonuclease-mediated mating-type switching. Under laboratory conditions, the human fungal pathogen Candida albicans can undergo both heterothallic and homothallic (opposite- and same-sex) mating. However, both mating modes require the presence of cells with two opposite mating types (MTLa/a and α/α) in close proximity. Given the predominant clonal feature of this yeast in the human host, both opposite- and same-sex mating would be rare in nature. In this study, we report that glucose starvation and oxidative stress, common environmental stresses encountered by the pathogen, induce the development of mating projections and efficiently permit same-sex mating in C. albicans with an "a" mating type (MTLa/a). This induction bypasses the requirement for the presence of cells with an opposite mating type and allows efficient sexual mating between cells derived from a single progenitor. Glucose starvation causes an increase in intracellular oxidative species, overwhelming the Heat Shock transcription Factor 1 (Hsf1)- and Heat shock protein (Hsp)90-mediated stress-response pathway. We further demonstrate that Candida TransActivating protein 4 (Cta4) and Cell Wall Transcription factor 1 (Cwt1), downstream effectors of the Hsf1-Hsp90 pathway, regulate same-sex mating in C. albicans through the transcriptional control of the master regulator of a-type mating, MTLa2, and the pheromone precursor-encoding gene Mating α factor precursor (MFα). Our results suggest that mating could occur much more frequently in nature than was originally appreciated and that same-sex mating could be an important mode of sexual reproduction in C. albicans.

Integration of the tricarboxylic acid (TCA) cycle with cAMP signaling and Sfl2 pathways in the regulation of CO2 sensing and hyphal development in Candida albicans.

Morphological transitions and metabolic regulation are critical for the human fungal pathogen Candida albicans to adapt to the changing host environment. In this study, we generated a library of central metabolic pathway mutants in the tricarboxylic acid (TCA) cycle, and investigated the functional consequences of these gene deletions on C. albicans biology. Inactivation of the TCA cycle impairs the ability of C. albicans to utilize non-fermentable carbon sources and dramatically attenuates cell growth rates under several culture conditions. By integrating the Ras1-cAMP signaling pathway and the heat shock factor-type transcription regulator Sfl2, we found that the TCA cycle plays fundamental roles in the regulation of CO2 sensing and hyphal development. The TCA cycle and cAMP signaling pathways coordinately regulate hyphal growth through the molecular linkers ATP and CO2. Inactivation of the TCA cycle leads to lowered intracellular ATP and cAMP levels and thus affects the activation of the Ras1-regulated cAMP signaling pathway. In turn, the Ras1-cAMP signaling pathway controls the TCA cycle through both Efg1- and Sfl2-mediated transcriptional regulation in response to elevated CO2 levels. The protein kinase A (PKA) catalytic subunit Tpk1, but not Tpk2, may play a major role in this regulation. Sfl2 specifically binds to several TCA cycle and hypha-associated genes under high CO2 conditions. Global transcriptional profiling experiments indicate that Sfl2 is indeed required for the gene expression changes occurring in response to these elevated CO2 levels. Our study reveals the regulatory role of the TCA cycle in CO2 sensing and hyphal development and establishes a novel link between the TCA cycle and Ras1-cAMP signaling pathways.