Acquired Triazole Resistance Alters Pathogenicity-Associated Features in Candida auris in an Isolate-Dependent Manner.

Fluconazole resistance is commonly encountered in Candida auris, and the yeast frequently displays resistance to other standard drugs, which severely limits the number of effective therapeutic agents against this emerging pathogen. In this study, we aimed to investigate the effect of acquired azole resistance on the viability, stress response, and virulence of this species. Fluconazole-, posaconazole-, and voriconazole- resistant strains were generated from two susceptible C. auris clinical isolates (0381, 0387) and compared under various conditions. Several evolved strains became pan-azole-resistant, as well as echinocandin-cross-resistant. While being pan-azole-resistant, the 0381-derived posaconazole-evolved strain colonized brain tissue more efficiently than any other strain, suggesting that fitness cost is not necessarily a consequence of resistance development in C. auris. All 0387-derived evolved strains carried a loss of function mutation (R160S) in BCY1, an inhibitor of the PKA pathway. Sequencing data also revealed that posaconazole treatment can result in ERG3 mutation in C. auris. Despite using the same mechanisms to generate the evolved strains, both genotype and phenotype analysis highlighted that the development of resistance was unique for each strain. Our data suggest that C. auris triazole resistance development is a highly complex process, initiated by several pleiotropic factors.

Transcriptome and proteome profiling reveals complex adaptations of Candida parapsilosis cells assimilating hydroxyaromatic carbon sources.

Many fungal species utilize hydroxyderivatives of benzene and benzoic acid as carbon sources. The yeast Candida parapsilosis metabolizes these compounds via the 3-oxoadipate and gentisate pathways, whose components are encoded by two metabolic gene clusters. In this study, we determine the chromosome level assembly of the C. parapsilosis strain CLIB214 and use it for transcriptomic and proteomic investigation of cells cultivated on hydroxyaromatic substrates. We demonstrate that the genes coding for enzymes and plasma membrane transporters involved in the 3-oxoadipate and gentisate pathways are highly upregulated and their expression is controlled in a substrate-specific manner. However, regulatory proteins involved in this process are not known. Using the knockout mutants, we show that putative transcriptional factors encoded by the genes OTF1 and GTF1 located within these gene clusters function as transcriptional activators of the 3-oxoadipate and gentisate pathway, respectively. We also show that the activation of both pathways is accompanied by upregulation of genes for the enzymes involved in ß-oxidation of fatty acids, glyoxylate cycle, amino acid metabolism, and peroxisome biogenesis. Transcriptome and proteome profiles of the cells grown on 4-hydroxybenzoate and 3-hydroxybenzoate, which are metabolized via the 3-oxoadipate and gentisate pathway, respectively, reflect their different connection to central metabolism. Yet we find that the expression profiles differ also in the cells assimilating 4-hydroxybenzoate and hydroquinone, which are both metabolized in the same pathway. This finding is consistent with the phenotype of the Otf1p-lacking mutant, which exhibits impaired growth on hydroxybenzoates, but still utilizes hydroxybenzenes, thus indicating that additional, yet unidentified transcription factor could be involved in the 3-oxoadipate pathway regulation. Moreover, we propose that bicarbonate ions resulting from decarboxylation of hydroxybenzoates also contribute to differences in the cell responses to hydroxybenzoates and hydroxybenzenes. Finally, our phylogenetic analysis highlights evolutionary paths leading to metabolic adaptations of yeast cells assimilating hydroxyaromatic substrates.

The fungivorous amoeba Protostelium aurantium targets redox homeostasis and cell wall integrity during intracellular killing of Candida parapsilosis.

Predatory interactions among microbes are major evolutionary driving forces for biodiversity. The fungivorous amoeba Protostelium aurantium has a wide fungal food spectrum including foremost pathogenic members of the genus Candida. Here we show that upon phagocytic ingestion by the amoeba, Candida parapsilosis is confronted with an oxidative burst and undergoes lysis within minutes of processing in acidified phagolysosomes. On the fungal side, a functional genomic approach identified copper and redox homeostasis as primary targets of amoeba predation, with the highly expressed copper exporter gene CRP1 and the peroxiredoxin gene PRX1 contributing to survival when encountered with P. aurantium. The fungicidal activity was largely retained in intracellular vesicles of the amoebae. Following their isolation, the content of these vesicles induced immediate killing and lysis of C. parapsilosis in vitro. Proteomic analysis identified 56 vesicular proteins from P. aurantium. Although completely unknown proteins were dominant, many of them could be categorised as hydrolytic enzymes targeting the fungal cell wall, indicating that fungal cell wall structures are under selection pressure by predatory phagocytes in natural environments. TAKE AWAY: The amoeba Protostelium aurantium feeds on fungi, such as Candida parapsilosis. Ingested yeast cells are exposed to reactive oxygen species. A copper exporter and a peroxiredoxin contribute to fungal defence. Yeast cells undergo intracellular lysis. Lysis occurs via a cocktail of hydrolytic enzymes from intracellular vesicles.

OCT1 - a yeast mitochondrial thiolase involved in the 3-oxoadipate pathway.

The 3-oxoacyl-CoA thiolases catalyze the last step of the fatty acid ß-oxidation pathway. In yeasts and plants, this pathway takes place exclusively in peroxisomes, whereas in animals it occurs in both peroxisomes and mitochondria. In contrast to baker's yeast Saccharomyces cerevisiae, yeast species from the Debaryomycetaceae family also encode a thiolase with predicted mitochondrial localization. These yeasts are able to utilize a range of hydroxyaromatic compounds via the 3-oxoadipate pathway the last step of which is catalyzed by 3-oxoadipyl-CoA thiolase and presumably occurs in mitochondria. In this work, we studied Oct1p, an ortholog of this enzyme from Candida parapsilosis. We found that the cells grown on a 3-oxoadipate pathway substrate exhibit increased levels of the OCT1 mRNA. Deletion of both OCT1 alleles impairs the growth of C. parapsilosis cells on 3-oxoadipate pathway substrates and this defect can be rescued by expression of the OCT1 gene from a plasmid vector. Subcellular localization experiments and LC-MS/MS analysis of enriched organellar fraction-proteins confirmed the presence of Oct1p in mitochondria. Phylogenetic profiling of Oct1p revealed an intricate evolutionary pattern indicating multiple horizontal gene transfers among different fungal groups.

Oral Epithelial Cells Distinguish between Candida Species with High or Low Pathogenic Potential through MicroRNA Regulation.

Oral epithelial cells monitor microbiome composition and initiate immune response upon dysbiosis, as in the case of Candida imbalances. Candida species, such as C. albicans and C. parapsilosis, are the most prevalent yeasts in the oral cavity. Comparison of healthy oral epithelial cell responses revealed that while C. albicans infection robustly activated inflammation cascades, C. parapsilosis primarily activated various inflammation-independent pathways. In posttranscriptional regulatory processes, several miRNAs were altered by both species. For C. parapsilosis, the dose of yeast cells directly correlated with changes in transcriptomic responses with higher fungal burdens inducing significantly different and broader changes. MicroRNAs (miRNAs) associated with carbohydrate metabolism-, hypoxia-, and vascular development-related responses dominated with C. parapsilosis infection, whereas C. albicans altered miRNAs linked to inflammatory responses. Subsequent analyses of hypoxia-inducible factor 1α (HIF1-α) and hepatic stellate cell (HSC) activation pathways predicted target genes through which miRNA-dependent regulation of yeast-specific functions may occur, which also supported the observed species-specific responses. Our findings suggest that C. parapsilosis is recognized as a commensal at low doses by the oral epithelium; however, increased fungal burden activates different pathways, some of which overlap with the inflammatory processes robustly induced by C. albicans IMPORTANCE A relatively new topic within the field of immunology involves the role of miRNAs in innate as well as adaptive immune response regulation. In recent years, posttranscriptional regulation of host-pathogenic fungal interactions through miRNAs was also suggested. Our study reveals that the distinct nature of human oral epithelial cell responses toward C. parapsilosis and C. albicans is possibly due to species-specific fine-tuning of host miRNA regulatory processes. The findings of this study also shed new light on the nature of early host cell transcriptional responses to the presence of C. parapsilosis and highlight the species' potential inflammation-independent host activation processes. These findings contribute to our better understanding of how miRNA deregulation at the oral immunological barrier, in noncanonical immune cells, may discriminate between fungal species, particularly Candida species with high or low pathogenic potential.

A Candida parapsilosis Overexpression Collection Reveals Genes Required for Pathogenesis.

Relative to the vast data regarding the virulence mechanisms of Candida albicans, there is limited knowledge on the emerging opportunistic human pathogen Candida parapsilosis. The aim of this study was to generate and characterize an overexpression mutant collection to identify and explore virulence factors in C. parapsilosis. With the obtained mutants, we investigated stress tolerance, morphology switch, biofilm formation, phagocytosis, and in vivo virulence in Galleria mellonella larvae and mouse models. In order to evaluate the results, we compared the data from the C. parapsilosis overexpression collection analysis to the results derived from previous deletion mutant library characterizations. Of the 37 overexpression C. parapsilosis mutants, we identified eight with altered phenotypes compared to the controls. This work is the first report to identify CPAR2_107240, CPAR2_108840, CPAR2_302400, CPAR2_406400, and CPAR2_602820 as contributors to C. parapsilosis virulence by regulating functions associated with host-pathogen interactions and biofilm formation. Our findings also confirmed the role of CPAR2_109520, CPAR2_200040, and CPAR2_500180 in pathogenesis. This study was the first attempt to use an overexpression strategy to systematically assess gene function in C. parapsilosis, and our results demonstrate that this approach is effective for such investigations.

Candida albicans Enhances the Progression of Oral Squamous Cell Carcinoma In Vitro and In Vivo.

Oral squamous cell carcinoma (OSCC) is associated with oral Candida albicans infection, although it is unclear whether the fungus promotes the genesis and progression of OSCC or whether cancer facilitates fungal growth. In this study, we investigated whether C. albicans can potentiate OSCC tumor development and progression. In vitro, the presence of live C. albicans, but not Candida parapsilosis, enhanced the progression of OSCC by stimulating the production of matrix metalloproteinases, oncometabolites, protumor signaling pathways, and overexpression of prognostic marker genes associated with metastatic events. C. albicans also upregulated oncogenes in nonmalignant cells. Using a newly established xenograft in vivo mouse model to investigate OSCC-C. albicans interactions, oral candidiasis enhanced the progression of OSCC through inflammation and induced the overexpression of metastatic genes and significant changes in markers of the epithelial-mesenchymal transition. Finally, using the 4-nitroquinoline 1-oxide (4NQO) murine model, we directly correlate these in vitro and short-term in vivo findings with the progression of oncogenesis over the long term. Taken together, these data indicate that C. albicans upregulates oncogenes, potentiates a premalignant phenotype, and is involved in early and late stages of malignant promotion and progression of oral cancer. IMPORTANCE Oral squamous cell carcinoma (OSCC) is a serious health issue worldwide that accounts for 2% to 4% of all cancer cases. Previous studies have revealed a higher yeast carriage and diversity in oral cancer patients than in healthy individuals. Furthermore, fungal colonization in the oral cavity bearing OSCC is higher on the neoplastic epithelial surface than on adjacent healthy surfaces, indicating a positive association between oral yeast carriage and epithelial carcinoma. In addition to this, there is strong evidence supporting the idea that Candida contributes to carcinogenesis events in the oral cavity. Here, we show that an increase in Candida albicans burden promotes an oncogenic phenotype in the oral cavity.

Triazole Evolution of Candida parapsilosis Results in Cross-Resistance to Other Antifungal Drugs, Influences Stress Responses, and Alters Virulence in an Antifungal Drug-Dependent Manner.

The number of invasive infections caused by Candida species is increasing worldwide. The incidence of candidiasis cases caused by non-albicans Candida species, such as Candida parapsilosis, is also increasing, and non-albicans Candida species are currently responsible for more invasive infections than C. albicans Additionally, while the development of azole resistance during invasive disease with C. albicans remains uncommon, azole-resistant C. parapsilosis strains are frequently isolated in the hospital setting. In this study, we applied direct selection to generate azole-adapted and azole-evolved C. parapsilosis strains in order to examine the effect of azole resistance development on fungal viability and pathogenesis progression. Depending on the drug applied, the different evolved strains developed distinct cross-resistance patterns: the fluconazole-evolved (FLUEVO) and voriconazole-evolved (VOREVO) strains gained resistance to fluconazole and voriconazole only, while posaconazole evolution resulted in cross-resistance to all azoles and the posaconazole-evolved (POSEVO) strains showed higher echinocandin MIC values than the FLUEVO and VOREVO strains. Whole-genome sequencing results identified the development of different resistance mechanisms in the evolved strains: the FLUEVO and VOREVO strains harbored amino acid substitutions in Mrr1p (A808T and N394Y, respectively), and the POSEVO strain harbored an amino acid change in Erg3p (D14Y). By revealing increased efflux pump activity in both the FLUEVO and the VOREVO strains, along with the altered sterol composition of the POSEVO strain, we now highlight the impact of the above-mentioned amino acid changes in C. parapsilosis azole resistance development. We further revealed that the virulence of this species was only slightly or partially affected by fluconazole and voriconazole adaptation, while it significantly decreased after posaconazole adaptation. Our results suggest that triazole adaptation can result in azole cross-resistance and that this process may also result in virulence alterations in C. parapsilosis, depending on the applied drug.IMPORTANCECandida parapsilosis causes life-threatening fungal infections. In the last 2 decades, the increasing number of azole-resistant C. parapsilosis clinical isolates has been attributable to the overuse and misuse of fluconazole, the first-line antifungal agent most commonly used in several countries. To date, the range of applicable antifungal drugs is limited. As a consequence, it is essential to understand the possible mechanisms of antifungal resistance development and their effect on virulence in order to optimize antifungal treatment strategies in the clinical setting. Our results revealed that the prolonged exposure to azoles resulted not only in azole resistance but also in cross-resistance development. Our data further indicate that resistance development may occur through different mechanisms that can also alter the virulence of C. parapsilosis These results highlight the consequences of prolonged drug usage and suggest the need for developing alternative antifungal treatment strategies in clinical practice.

Iron Metabolism, Pseudohypha Production, and Biofilm Formation through a Multicopper Oxidase in the Human-Pathogenic Fungus Candida parapsilosis.

Among all the essential micronutrients, iron plays an important role in mammalian biology. It is also essential for pathogens infecting mammalian hosts, including bacteria, fungi, and protozoans. As the availability of accessible iron is limited within the mammalian host, several human-pathogenic fungal pathogens, such as Candida albicans, Cryptococcus neoformans, Candida glabrata, and Aspergillus fumigatus, have developed various iron uptake mechanisms. Although Candida parapsilosis is the second or third most common non-albicans Candida species associated with systemic and superficial Candida infections in immunocompromised patients, the mechanisms of iron uptake and homoeostasis remain unknown in this fungus. In the current report, we show that a homologue of the multicopper oxidase gene FET3 is present in the genome of C. parapsilosis (CPAR2_603600) and plays a significant role in iron acquisition. We found that homozygous deletion mutants of CPAR2_603600 showed defects under low-iron conditions and were also sensitive to various stressors. Our results also revealed that the levels of pseudohypha formation and biofilm formation were reduced in the null mutants compared to the wild type. This phenotypic defect could be partially rescued by supplementation with excess iron in the growth medium. The expression levels of the orthologues of various iron metabolism-related genes were also altered in the mutants compared to the parental strain. In conclusion, our report describes the role of CPAR2_603600 in iron homoeostasis maintenance as well as morphology and biofilm formation regulation in this pathogenic fungus.IMPORTANCEC. parapsilosis is the second or third most common opportunistic human-pathogenic Candida species, being responsible for severe fungal infections among immunocompromised patients, especially low-birth-weight infants (0 to 2 years of age). Among the major virulence factors that pathogenic fungi possess is the ability to compete with the host for essential micronutrients, including iron. Accessible iron is required for the maintenance of several metabolic processes. In order to obtain accessible iron from the host, pathogenic fungi have developed several iron acquisition and metabolic mechanisms. Although C. parapsilosis is a frequent cause of invasive candidiasis, little is known about what iron metabolic processes this fungus possesses that could contribute to the species' virulent behavior. In this study, we identified the multicopper oxidase FET3 gene that regulates iron homeostasis maintenance and also plays important roles in the morphology of the fungus as well as in biofilm formation, two additional factors in fungal virulence.

Multicopper Oxidases in Saccharomyces cerevisiae and Human Pathogenic Fungi.

Multicopper oxidases (MCOs) are produced by microscopic and macroscopic fungal species and are involved in various physiological processes such as morphogenesis, lignin degradation, and defense mechanisms to stress inducing environmental conditions as well as fungal virulence. This review will summarize our current understanding regarding the functions of MCOs present in Saccharomyces cerevisiae and in different human fungal pathogens. Of the two main MCO groups, the first group of MCOs is involved in iron homoeostasis and the second includes laccases. This review will also discuss their role in the pathogenesis of human fungal pathogens.

Mechanisms of Pathogenic Candida Species to Evade the Host Complement Attack.

Candida species are common colonizers of the human skin, vagina, and the gut. As human commensals, Candida species do not cause any notable damage in healthy individuals; however, in certain conditions they can initiate a wide range of diseases such as chronic disseminated candidiasis, endocarditis, vaginitis, meningitis, and endophthalmitis. The incidence of Candida caused infections has increased worldwide, with mortality rates exceeding 70% in certain patient populations. C. albicans, C. glabrata, C. tropicalis, C. parapsilosis, and C. krusei are responsible for more than 90% of Candida-related infections. Interestingly, the host immune response against these closely related fungi varies. As part of the innate immune system, complement proteins play a crucial role in host defense, protecting the host by lysing pathogens or by increasing their phagocytosis by phagocytes through opsonization. This review summarizes interactions of host complement proteins with pathogenic Candida species, including C. albicans and non-albicans Candida species such as C. parapsilosis. We will also highlight the various ways of complement activation, describe the antifungal effects of complement cascades and explore the mechanisms adopted by members of pathogenic Candida species for evading complement attack.

Cellular Immune Response Involving Multinucleated Giant Hemocytes with Two-Step Genome Amplification in the Drosophilid Zaprionus indianus.

Previously, a novel cell type, the multinucleated giant hemocyte (MGH) was identified in the ananassae subgroup of Drosophilidae. These cells share several features with mammalian multinucleated giant cells, a syncytium of macrophages formed during granulomatous inflammation. We were able to show that MGHs also differentiate in Zaprionus indianus, an invasive species belonging to the vittiger subgroup of the family, highly resistant to a large number of parasitoid wasp species. We have classified the MGHs of Z. indianusas giant hemocytes belonging to a class of cells which also include elongated blood cells carrying a single nucleus and anuclear structures. They are involved in encapsulating parasites, originate from the lymph gland, can develop by cell fusion, and generally carry many nuclei, while possessing an elaborated system of canals and sinuses, resulting in a spongiform appearance. Their nuclei are all transcriptionally active and show accretion of genetic material. Multinucleation and accumulation of the genetic material in the giant hemocytes represents a two-stage amplification of the genome, while their spongy ultrastructure substantially increases the contact surface with the extracellular space. These features may furnish the giant hemocytes with a considerable metabolic advantage, hence contributing to the mechanism of the effective immune response.

Functional Characterization of Secreted Aspartyl Proteases in Candida parapsilosis.

Candida parapsilosis is an emerging non-albicans Candida species that largely affects low-birth-weight infants and immunocompromised patients. Fungal pathogenesis is promoted by the dynamic expression of diverse virulence factors, with secreted proteolytic enzymes being linked to the establishment and progression of disease. Although secreted aspartyl proteases (Sap) are critical for Candida albicans pathogenicity, their role in C. parapsilosis is poorly elucidated. In the present study, we aimed to examine the contribution of C. parapsilosisSAPP genes SAPP1, SAPP2, and SAPP3 to the virulence of the species. Our results indicate that SAPP1 and SAPP2, but not SAPP3, influence adhesion, host cell damage, phagosome-lysosome maturation, phagocytosis, killing capacity, and cytokine secretion by human peripheral blood-derived macrophages. Purified Sapp1p and Sapp2p were also shown to efficiently cleave host complement component 3b (C3b) and C4b proteins and complement regulator factor H. Additionally, Sapp2p was able to cleave factor H-related protein 5 (FHR-5). Altogether, these data demonstrate the diverse, significant contributions that SAPP1 and SAPP2 make to the establishment and progression of disease by C. parapsilosis through enabling the attachment of the yeast cells to mammalian cells and modulating macrophage biology and disruption of the complement cascade.IMPORTANCE Aspartyl proteases are present in various organisms and, among virulent species, are considered major virulence factors. Host tissue and cell damage, hijacking of immune responses, and hiding from innate immune cells are the most common behaviors of fungal secreted proteases enabling pathogen survival and invasion. C. parapsilosis, an opportunistic human-pathogenic fungus mainly threatening low-birth weight neonates and children, possesses three SAPP protein-encoding genes that could contribute to the invasiveness of the species. Our results suggest that SAPP1 and SAPP2, but not SAPP3, influence host evasion by regulating cell damage, phagocytosis, phagosome-lysosome maturation, killing, and cytokine secretion. Furthermore, SAPP1 and SAPP2 also effectively contribute to complement evasion.

Candida parapsilosis: from Genes to the Bedside.

Patients with suppressed immunity are at the highest risk for hospital-acquired infections. Among these, invasive candidiasis is the most prevalent systemic fungal nosocomial infection. Over recent decades, the combined prevalence of non-albicans Candida species outranked Candida albicans infections in several geographical regions worldwide, highlighting the need to understand their pathobiology in order to develop effective treatment and to prevent future outbreaks. Candida parapsilosis is the second or third most frequently isolated Candida species from patients. Besides being highly prevalent, its biology differs markedly from that of C. albicans, which may be associated with C. parapsilosis' increased incidence. Differences in virulence, regulatory and antifungal drug resistance mechanisms, and the patient groups at risk indicate that conclusions drawn from C. albicans pathobiology cannot be simply extrapolated to C. parapsilosis Such species-specific characteristics may also influence their recognition and elimination by the host and the efficacy of antifungal drugs. Due to the availability of high-throughput, state-of-the-art experimental tools and molecular genetic methods adapted to C. parapsilosis, genome and transcriptome studies are now available that greatly contribute to our understanding of what makes this species a threat. In this review, we summarize 10 years of findings on C. parapsilosis pathogenesis, including the species' genetic properties, transcriptome studies, host responses, and molecular mechanisms of virulence. Antifungal susceptibility studies and clinician perspectives are discussed. We also present regional incidence reports in order to provide an updated worldwide epidemiology summary.

Methylation Status of the Adeno-Associated Virus Type 2 (AAV2).

To analyze the methylation status of wild-type adeno-associated virus type 2 (AAV2), bisulfite PCR sequencing (BPS) of the packaged viral genome and its integrated form was performed and 262 of the total 266 CG dinucleotides (CpG) were mapped. In virion-packaged DNA, the ratio of the methylated cytosines ranged between 0⁻1.7%. In contrast, the chromosomally integrated AAV2 genome was hypermethylated with an average of 76% methylation per CpG site. The methylation level showed local minimums around the four known AAV2 promoters. To study the effect of methylation on viral rescue and replication, the replication initiation capability of CpG methylated and non-CpG methylated AAV DNA was compared. The in vitro hypermethylation of the viral genome does not inhibit its rescue and replication from a plasmid transfected into cells. This insensitivity of the viral replicative machinery to methylation may permit the rescue of the integrated heavily methylated AAV genome from the host's chromosomes.

Eicosanoid production by Candida parapsilosis and other pathogenic yeasts.

Eicosanoids are bioactive lipid mediators generated in almost all mammalian cells from the oxidation of arachidonic acid and other related twenty-carbon polyunsaturated fatty acids (PUFA). Eicosanoids regulate various physiological functions, including cellular homoeostasis and modulation of inflammatory responses in mammals. The mode of action of these lipid mediators depend on their binding to different G-protein coupled receptors. The three main enzymatic pathways associated with their production are the COX pathway, LOX pathway and cytochrome P450 pathway. Interestingly, investigations have also revealed that several human pathogenic fungi are capable of producing these bioactive lipid mediators; however, the exact biosynthetic pathways and their function in pathogenicity are not yet extensively characterized. The aim of the current review is to summarize the recent discoveries pertaining to eicosanoid production by human pathogenic yeasts with a special focus on the opportunistic human fungal pathogen Candida parapsilosis.

Echinocandin-Induced Microevolution of Candida parapsilosis Influences Virulence and Abiotic Stress Tolerance.

Candida species are a major cause of life-threatening bloodstream infections worldwide. Although Candida albicans is responsible for the vast majority of infections, the clinical relevance of other Candida species has also emerged over the last twenty years. This shift might be due in part to changes in clinical guidelines, as echinocandins became the first line of therapeutics for the treatment. Candida parapsilosis is an emerging non-albicans Candida species that exhibits lower susceptibility levels to these drugs. Candida species frequently display resistance to echinocandins, and the mechanism for this is well-known in C. albicans and Candida glabrata, where it is mediated by amino acid substitutions at defined locations of the ß-1,3-glucan synthase, Fks1p. In C. parapsilosis isolates, Fks1p harbors an intrinsic amino acid change at position 660 of the hot spot 1 (HS1) region, which is thought to be responsible for the high MIC values. Less is known about acquired substitutions in this species. In this study, we used directed evolution experiments to generate C. parapsilosis strains with acquired resistance to caspofungin, anidulafungin, and micafungin. We showed that cross-resistance was dependent on the type of echinocandin used to generate the evolved strains. During their characterization, all mutant strains showed attenuated virulence in vivo and also displayed alterations in the exposure of inner cell wall components. The evolved strains harbored 251 amino acid changes, including three in the HS1, HS2, and HS3 regions of Fks1p. Altogether, our results demonstrate a direct connection between acquired antifungal resistance and virulence of C. parapsilosisIMPORTANCECandida parapsilosis is an opportunistic fungal pathogen with the ability to cause infections in immunocompromised patients. Echinocandins are the currently recommended first line of treatment for all Candida species. Resistance of Candida albicans to this drug type is well characterized. C. parapsilosis strains have the lowest in vitro susceptibility to echinocandins; however, patients with such infections typically respond well to echinocandin therapy. There is little knowledge of acquired resistance in C. parapsilosis and its consequences on other characteristics such as virulence properties. In this study, we aimed to dissect how acquired echinocandin resistance influences the pathogenicity of C. parapsilosis and to develop explanations for why echinocandins are clinically effective in the setting of acquired resistance.

Eicosanoid biosynthesis influences the virulence of Candida parapsilosis.

Lipid mediators, derived from arachidonic acid metabolism, play an important role in immune regulation. The functions of bioactive eicosanoids range from modulating cytokine signaling and inflammasome formation to anti-inflammatory and pro-resolving activities. Human pathogenic fungi such as Candida albicans, Candida parapsilosis, Cryptococcus neoformans and Aspergillus fumigatus have been shown to produce such lipid mediators, associated with their virulence. To date, investigations into the molecular mechanisms of fungal eicosanoid biosynthesis in different species have revealed that several genes are associated with prostaglandin production. However, these routes remain uncharacterized in C. parapsilosis with early results suggesting it uses pathways distinct from those found in C. albicans. Therefore, we aimed to identify and characterize C. parapsilosis genes involved in eicosanoid biosynthesis. Following arachidonic acid treatment of C. parapsilosis cells, we identified several genes interfering with prostaglandin production. Out of the identified genes, homologues of a multi copper oxidase (FET3), an Acyl-CoA thiolase (POT1) and an Acyl-CoA oxidase (POX1-3) were found to play a significant role in prostaglandin synthesis. Furthermore, all three genes were confirmed to enhance C. parapsilosis pathogenicity, as the corresponding deletion mutants were cleared more efficiently by human macrophages and induced higher levels of pro-inflammatory cytokines. In addition, the mutants were less virulent than the wild-type strain in a mouse model of systemic infection. Taken together, we identified three genes that regulate eicosanoid biosynthesis in C. parapsilosis and impact the fungus' virulence.

Investigation of Candida parapsilosis virulence regulatory factors during host-pathogen interaction.

Invasive candidiasis is among the most life-threatening infections in patients in intensive care units. Although Candida albicans is the leading cause of candidaemia, the incidence of Candida parapsilosis infections is also rising, particularly among the neonates. Due to differences in their biology, these species employ different antifungal resistance and virulence mechanisms and also induce dissimilar immune responses. Previously, it has been suggested that core virulence effecting transcription regulators could be attractive ligands for future antifungal drugs. Although the virulence regulatory mechanisms of C. albicans are well studied, less is known about similar mechanisms in C. parapsilosis. In order to search for potential targets for future antifungal drugs against this species, we analyzed the fungal transcriptome during host-pathogen interaction using an in vitro infection model. Selected genes with high expression levels were further examined through their respective null mutant strains, under conditions that mimic the host environment or influence pathogenicity. As a result, we identified several mutants with relevant pathogenicity affecting phenotypes. During the study we highlight three potentially tractable signaling regulators that influence C. parapsilosis pathogenicity in distinct mechanisms. During infection, CPAR2_100540 is responsible for nutrient acquisition, CPAR2_200390 for cell wall assembly and morphology switching and CPAR2_303700 for fungal viability.

Eukaryotic transporters for hydroxyderivatives of benzoic acid.

Several yeast species catabolize hydroxyderivatives of benzoic acid. However, the nature of carriers responsible for transport of these compounds across the plasma membrane is currently unknown. In this study, we analyzed a family of genes coding for permeases belonging to the major facilitator superfamily (MFS) in the pathogenic yeast Candida parapsilosis. Our results revealed that these transporters are functionally equivalent to bacterial aromatic acid: H+ symporters (AAHS) such as GenK, MhbT and PcaK. We demonstrate that the genes HBT1 and HBT2 encoding putative transporters are highly upregulated in C. parapsilosis cells assimilating hydroxybenzoate substrates and the corresponding proteins reside in the plasma membrane. Phenotypic analyses of knockout mutants and hydroxybenzoate uptake assays provide compelling evidence that the permeases Hbt1 and Hbt2 transport the substrates that are metabolized via the gentisate (3-hydroxybenzoate, gentisate) and 3-oxoadipate pathway (4-hydroxybenzoate, 2,4-dihydroxybenzoate and protocatechuate), respectively. Our data support the hypothesis that the carriers belong to the AAHS family of MFS transporters. Phylogenetic analyses revealed that the orthologs of Hbt permeases are widespread in the subphylum Pezizomycotina, but have a sparse distribution among Saccharomycotina lineages. Moreover, these analyses shed additional light on the evolution of biochemical pathways involved in the catabolic degradation of hydroxyaromatic compounds.