Two-Step Epimerization of Deoxynivalenol by Quinone-Dependent Dehydrogenase and Candida parapsilosis ACCC 20221.

Deoxynivalenol (DON), one of the main mycotoxins with enteric toxicity, genetic toxicity, and immunotoxicity, and is widely found in corn, barley, wheat, and rye. In order to achieve effective detoxification of DON, the least toxic 3-epi-DON (1/357th of the toxicity of DON) was chosen as the target for degradation. Quinone-dependent dehydrogenase (QDDH) reported from Devosia train D6-9 detoxifies DON by converting C3-OH to a ketone group with toxicity of less than 1/10 that of DON. In this study, the recombinant plasmid pPIC9K-QDDH was constructed and successfully expressed in Pichia pastoris GS115. Within 12 h, recombinant QDDH converted 78.46% of the 20 µg/mL DON to 3-keto-DON. Candida parapsilosis ACCC 20221 was screened for its activity in reducing 86.59% of 3-keto-DON within 48 h; its main products were identified as 3-epi-DON and DON. In addition, a two-step method was performed for epimerizing DON: 12 h catalysis by recombinant QDDH and 6 h transformation of the C. parapsilosis ACCC 20221 cell catalyst. The production rates of 3-keto-DON and 3-epi-DON were 51.59% and 32.57%, respectively, after manipulation. Through this study, effective detoxification of 84.16% of DON was achieved, with the products being mainly 3-keto-DON and 3-epi-DON.

The antifungal effect induced by itraconazole in Candida parapsilosis largely depends on the oxidative stress generated at the mitochondria.

In Candida parapsilosis, homozygous disruption of the two genes encoding trehalase activity increased the susceptibility to Itraconazole compared with the isogenic parental strain. The fungicidal effect of this azole can largely be counteracted by preincubating growing cells with rotenone and the protonophore 2,4-Dinitrophenol. In turn, measurement of endogenous reactive oxygen species formation by flow cytometry confirmed that Itraconazole clearly induced an internal oxidative stress, which can be significantly abolished in rotenone-exposed cells. Analysis of the antioxidant enzymatic activities of catalase and superoxide dismutase pointed to a moderate decrease of catalase in trehalase-deficient mutant cells compared to the wild type, with an additional increase upon addition of rotenone. These enzymatic changes were imperceptible in the case of superoxide dismutase. Alternative assays with Voriconazole led to a similar profile in the results regarding cell growth and antioxidant activities. Collectively, our data suggest that the antifungal action of Itraconazole on C. parapsilosis is dependent on a functional mitochondrial activity. They also suggest that the central metabolic pathways in pathogenic fungi should be considered as preferential antifungal targets in new research.

Candida parapsilosis cell wall proteins-CPAR2_404800 and CPAR2_404780-Are adhesins that bind to human epithelial and endothelial cells and extracellular matrix proteins.

One of the initial steps necessary for the development of Candida infections is the adherence to the host tissues and cells. Recent transcriptomic studies suggest that, in Candida parapsilosis-a fungal infectious agent that causes systemic candidiasis in immunosuppressed individuals-the adhesion is mediated by pathogen cell-exposed proteins belonging to the agglutinin-like sequence (Als) family. However, to date, the actual interactions of individual members of this family with human cells and extracellular matrix (ECM) have not been characterized in detail. In the current study, we focused attention on two of these C. parapsilosis Als proteins-CPAR2_404800 and CPAR2_404780-that were proteomically identified in the fungal cell wall of yeasts grown in the media suitable for culturing human epithelial and endothelial cells. Both proteins were extracted from the cell wall and purified, and using a microplate binding assay and a fluorescence microscopic analysis were shown to adhere to human cells of A431 (epithelial) and HMEC-1 (endothelial) lines. The human extracellular matrix components that are also plasma proteins-fibronectin and vitronectin-enhanced these interactions, and also could directly bind to CPAR2_404800 and CPAR2_404780 proteins, with a high affinity (KD in a range of 10-7 to 10-8 M) as determined by surface plasmon resonance measurements. Our findings highlight the role of proteins CPAR2_404800 and CPAR2_404780 in adhesion to host cells and proteins, contributing to the knowledge of the mechanisms of host-pathogen interactions during C. parapsilosis-caused infections.

The Pathogenic Yeast Candida parapsilosis Forms Pseudohyphae through Different Signaling Pathways Depending on the Available Carbon Source.

Candida parapsilosis is an emerging fungal pathogen that primarily affects immunocompromised patients in hospitals. A significant risk factor is the use of implanted medical devices, which support the growth of biofilms composed of a mixture of individual yeast cells and chains of elongated pseudohyphal cells. The morphological switch between these two forms is triggered by cues from the environment, including nutrient availability and temperature. We examined how different nutrient sources affect the balance between yeast and pseudohyphae and found that cells grown in the presence of five- or six-carbon sugars form more pseudohyphae at 30°C than at 37°C. Conversely, cells grown on glycerol, a three-carbon polyalcohol, form more pseudohyphae at 37°C. Furthermore, we found that different regulators influence pseudohyphal growth on glucose at 30°C compared with those on glycerol at 37°C. In particular, cAMP signaling and the sirtuin deacetylase Hst1 were required for pseudohyphal growth on glycerol at 37°C but not on glucose at 30°C. Finally, we found that the carbon source on which C. parapsilosis is grown can influence its ability to establish an infection in a wax moth model. Overall, this study reveals that environmental conditions affect not only the extent of pseudohyphal growth but also which pathways and regulators govern pseudohyphal formation. IMPORTANCE Candida parapsilosis is one of the leading causes of hospital-acquired yeast infections and poses a significant risk to immunocompromised people. Two of its properties that contribute to infection are metabolic flexibility, to use a range of nutrients available in the host, and cellular dimorphism, to switch between round yeast cells and chains of elongated pseudohyphal cells. Uncovering the molecular mechanisms that regulate these processes could reveal new targets for antifungal drugs. We found that for C. parapsilosis, the balance between yeast and pseudohyphal cells depends on the nutrients available and the growth temperature. Moreover, these environmental changes can affect its ability to cause infections. Finally, we found that a potential sensor of the cell's metabolic state, the sirtuin Hst1, contributes to pseudohyphal growth for cells grown on glycerol. These findings indicate that the shape and virulence of C. parapsilosis likely vary depending on its location in the host.

Candida parapsilosis Mdr1B and Cdr1B Are Drivers of Mrr1-Mediated Clinical Fluconazole Resistance.

Candida parapsilosis is a common cause of invasive candidiasis worldwide and is the most commonly is7olated Candida species among pediatric and neonatal populations. Previous work has demonstrated that nonsynonymous mutations in the gene encoding the putative transcription factor CpMrr1 can influence fluconazole susceptibility. However, the direct contribution of these mutations and how they influence fluconazole resistance in clinical isolates are poorly understood. We identified 7 nonsynonymous CpMRR1 mutations in 12 isolates from within a collection of 35 fluconazole-resistant clinical isolates. The mutations leading to the A854V, R479K, and I283R substitutions were further examined and found to be activating mutations leading to increased fluconazole resistance. In addition to CpMDR1, we identified two other genes, one encoding a major facilitator superfamily (MFS) transporter (CpMDR1B, CPAR2_603010) and one encoding an ATP-binding cassette (ABC) transporter (CpCDR1B, CPAR2_304370), as being upregulated in isolates carrying CpMRR1-activating mutations. Overexpression of CpMDR1 in a susceptible strain and disruption in resistant clinical isolates that overexpress CpMDR1 had little to no effect on fluconazole susceptibility. Conversely, overexpression of either CpMDR1B or CpCDR1B increased resistance, and disruption in clinical isolates overexpressing these genes decreased fluconazole resistance. Our findings suggest that activating mutations in CpMRR1 represent important genetic determinants of fluconazole resistance in clinical isolates of C. parapsilosis, and unlike what is observed in Candida albicans, this is primarily driven by upregulation of both MFS (CpMdr1B) and ABC (CpCdr1B) transporters.

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.

Metabolic profile of Candida albicans and Candida parapsilosis interactions within dual-species biofilms.

Within the oral cavity, the ability of Candida species to adhere and form biofilms is well-recognized, especially when Candida albicans is considered. Lately, a knowledge gap has been identified regarding dual-species communication of Candida isolates, as a way to increase virulence, with evidences being collected to support the existence of interactions between C. albicans and Candida parapsilosis. The present work evaluated the synergistic effect of the two Candida species, and explored chemical interactions between cells, evaluating secreted extracellular alcohols and their relation with yeasts' growth and matrix composition. A total of four clinical strains of C. albicans and C. parapsilosis species, isolated from single infections of different patients or from co-infections of a same patient, were tested. It was found that dual-species biofilms negatively impacted the growth of C. parapsilosis and their biofilm matrix, in comparison with mono-species biofilms, and had minor effects on the biofilm biomass. Alcohol secretion revealed to be species- and strain-dependent. However, some dual-species cultures produced much higher amounts of some alcohols (E-nerolidol and E, E-Farnesol) than the respective single cultures, which proves the existence of a synergy between species. These results show evidence that interactions between Candida species affect the biofilm matrix, which is a key element of oral biofilms.

Biochemical features and early adhesion of marine Candida parapsilosis strains on high-density polyethylene.

AIMS: Plastic debris are constantly released into oceans where, due to weathering processes, they suffer fragmentation into micro- and nanoplastics. Diverse microbes often colonize these persisting fragments, contributing to their degradation. However, there are scarce reports regarding the biofilm formation of eukaryotic decomposing microorganisms on plastics. Here, we evaluated five yeast isolates from deep-sea sediment for catabolic properties and early adhesion ability on high-density polyethylene (HDPE). METHODS AND RESULTS: We assessed yeast catabolic features and adhesion ability on HDPE fragments subjected to abiotic weathering. Adhered cells were evaluated through Crystal Violet Assay, Scanning Electron Microscopy, Atomic Force Microscopy and Infrared Spectroscopy. Isolates were identified as Candida parapsilosis and exhibited wide catabolic capacity. Two isolates showed high adhesion ability on HDPE, consistently higher than the reference C. parapsilosis strain, despite an increase in fragment roughness due to weathering. Isolate Y5 displayed the most efficient colonization, with production of polysaccharides and lipids after 48 h of incubation. CONCLUSION: This work provides insights on catabolic metabolism and initial yeast-HDPE interactions of marine C. parapsilosis strains. SIGNIFICANCE AND IMPACT OF THE STUDY: Our findings represent an essential contribution to the characterization of early interactions between deep-sea undescribed yeast strains and plastic pollutants found in oceans.

Signaling through Syk or CARD9 Mediates Species-Specific Anti-Candida Protection in Bone Marrow Chimeric Mice.

The spleen tyrosine kinase (Syk) and the downstream adaptor protein CARD9 are crucial signaling molecules in antimicrobial immunity. Candida parapsilosis is an emerging fungal pathogen with a high incidence in neonates, while Candida albicans is the most common agent of candidiasis. While signaling through Syk/CARD9 promotes protective host mechanisms in response to C. albicans, its function in immunity against C. parapsilosis remains unclear. Here, we generated Syk-/- and CARD9-/- bone marrow chimeric mice to study the role of Syk/CARD9 signaling in immune responses to C. parapsilosis compared to C. albicans. We demonstrate various functions of this pathway (e.g., phagocytosis, phagosome acidification, and killing) in Candida-challenged, bone marrow-derived macrophages with differential involvement of Syk and CARD9 along with species-specific differences in cytokine production. We report that Syk-/- or CARD9-/- chimeras rapidly display high susceptibility to C. albicans, while C. parapsilosis infection exacerbates over a prolonged period in these animals. Thus, our results establish that Syk and CARD9 contribute to systemic resistance to C. parapsilosis and C. albicans differently. Additionally, we confirm prior studies but also detail new insights into the fundamental roles of both proteins in immunity against C. albicans. Our data further suggest that Syk has a more prominent influence on anti-Candida immunity than CARD9. Therefore, this study reinforces the Syk/CARD9 pathway as a potential target for anti-Candida immune therapy. IMPORTANCE While C. albicans remains the most clinically significant Candida species, C. parapsilosis is an emerging pathogen with increased affinity to neonates. Syk/CARD9 signaling is crucial in immunity to C. albicans, but its role in in vivo responses to other pathogenic Candida species is largely unexplored. We used mice with hematopoietic systems deficient in Syk or CARD9 to comparatively study the function of these proteins in anti-Candida immunity. We demonstrate that Syk/CARD9 signaling has a protective role against C. parapsilosis differently than against C. albicans. Thus, this study is the first to reveal that Syk can exert immune responses during systemic Candida infections species specifically. Additionally, Syk-dependent immunity to a nonalbicans Candida species in an in vivo murine model has not been reported previously. We highlight that the contribution of Syk and CARD9 to fungal infections are not identical and underline this pathway as a promising immune-therapeutic target to fight Candida infections.

Co-inoculation of yeasts starters: A strategy to improve quality of low altitude Arabica coffee.

The study aimed to improve the quality of dry-processed coffee grown at low altitudes through yeast inoculation, using three species (Saccharomyces cerevisiae CCMA 0543, Torulaspora delbrueckii CCMA 0684, and Candida parapsilosis CCMA 0544) singly and with co-inoculation for fermentation. Important chemical compounds and groups were analyzed by liquid and gas chromatography and Fourier-transform infrared spectroscopy (FTIR). The inoculated coffees with yeast populations around 106 cell/g obtained the highest scores, and the co-inoculation with C. parapsilosis CCMA 0544 and T. delbrueckii CCMA 0684 had the highest score in the sensory analysis (85). Different descriptors were observed in each treatment, and body, flavor, balance, and aftertaste are strongly related to C. parapsilosis CCMA 0544. The fermentation process improved the quality of low-altitude coffees, and the combination of non-Saccharomyces yeasts (C. parapsilosis CCMA 0544 and T. delbrueckii CCMA 0684) was the most indicated as starter cultures.

Transcription Profiles Associated with Inducible Adhesion in Candida parapsilosis.

Candida parapsilosis has emerged as a frequent cause of invasive candidiasis with increasing evidence of unique biological features relative to C. albicans As it adapts to conditions within a mammalian host, rapid changes in gene expression are necessary to facilitate colonization and persistence in this environment. Adhesion of the organism to biological surfaces is a key first step in this process and is the focus of this study. Building on previous observations showing the importance of a member of the ALS gene family in C. parapsilosis adhesion, three clinical isolates were cultured under two conditions that mimic the mammalian host and promote adhesion, incubation at 37°C in tissue culture medium 199 or in human plasma. Transcriptional profiles using RNA-seq were obtained in these adhesion-inducing conditions and compared to profiles following growth in yeast media that suppress adhesion to identify gene expression profiles associated with adhesion. Overall gene expression profiles among the three strains were similar in both adhesion-inducing conditions and distinct from adhesion-suppressing conditions. Pairwise analysis among the three growth conditions identified 133 genes that were differentially expressed at a cutoff of ±4-fold, with the most upregulated genes significantly enriched in iron acquisition and transmembrane transport, while the most downregulated genes were enriched in oxidation-reduction processes. Gene family enrichment analysis identified gene families with diverse functions that may have an important role in this important step for colonization and disease.IMPORTANCE Invasive Candida infections are frequent complications of the immunocompromised and are associated with substantive morbidity and mortality. Although C. albicans is the best-studied species, emerging infections by non-albicans Candida species have led to increased efforts to understand aspects of their pathogenesis that are unique from C. albicansC. parapsilosis is a frequent cause of invasive infections, particularly among premature infants. Recent efforts have identified important virulence mechanisms that have features distinct from C. albicansC. parapsilosis can exist outside a host environment and therefore requires rapid modifications when it encounters a mammalian host to prevent its clearance. An important first step in the process is adhesion to host surfaces. This work takes a global, nonbiased approach to investigate broad changes in gene expression that accompany efficient adhesion. As such, biological pathways and individual protein targets are identified that may be amenable to manipulation to reduce colonization and disease from this organism.

Characteristics of Extracellular Vesicles Released by the Pathogenic Yeast-Like Fungi Candida glabrata, Candida parapsilosis and Candida tropicalis.

Candida spp. yeast-like fungi are opportunistic pathogens in humans and have been recently found to release extracellular vesicles (EVs) that are involved in many vital biological processes in fungal cells. These include communication between microorganisms and host-pathogen interactions during infection. The production of EVs and their content have been significantly characterized in the most common candidal species Candida albicans, including the identification of numerous virulence factors and cytoplasmic proteins in the EV cargo. We have here conducted the isolation and proteomic characterization of EVs produced by the clinically important non-albicans Candida species C. glabrata, C. tropicalis and C. parapsilosis. With the use of ultracentrifugation of the cell-free culture supernatant, the candidal EVs were collected and found to be a heterogeneous population of particles for each species with sizes ranging from 60-280 nm. The proteinaceous contents of these vesicles were analyzed using LC-MS/MS, with particular attention paid to surface-expressed proteins that would come into immediate and direct contact with host cells. We thereby identified 42 extracellular and surface-connected proteins from C. glabrata, 33 from C. parapsilosis, and 34 from C. tropicalis, including membrane-associated transporters, glycoproteins and enzymes involved in the organization of the fungal cell wall, as well as several cytoplasmic proteins, including alcohol dehydrogenase, enolase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase and pyruvate kinase, for which the vesicular transport is a possible mechanism underlying their non-classical secretion.

Highly enantioselective synthesis of (R)-1,3-butanediol via deracemization of the corresponding racemate by a whole-cell stereoinverting cascade system.

BACKGROUND: Deracemization, the transformation of the racemate into a single stereoisomeric product in 100% theoretical yield, is an appealing but challenging option for the asymmetric synthesis of optically pure chiral compounds as important pharmaceutical intermediates. To enhance the synthesis of (R)-1,3-butanediol from the corresponding low-cost racemate with minimal substrate waste, we designed a stereoinverting cascade deracemization route and constructed the cascade reaction for the total conversion of racemic 1,3-butanediol into its (R)-enantiomer. This cascade reaction consisted of the absolutely enantioselective oxidation of (S)-1,3-butanediol by Candida parapsilosis QC-76 and the subsequent asymmetric reduction of the intermediate 4-hydroxy-2-butanone to (R)-1,3-butanediol by Pichia kudriavzevii QC-1. RESULTS: The key reaction conditions including choice of cosubstrate, pH, temperature, and rotation speed were optimized systematically and determined as follows: adding acetone as the cosubstrate at pH 8.0, a temperature of 30 °C, and rotation speed of 250 rpm for the first oxidation process; in the next reduction process, the optimal conditions were: adding glucose as the cosubstrate at pH 8.0, a temperature of 35 °C, and rotation speed of 200 rpm. By investigating the feasibility of the step-by-step method with one-pot experiment as a natural extension for performing the oxidation-reduction cascade, the step-by-step approach exhibited high efficiency for this cascade process from racemate to (R)-1,3-butanediol. Under optimal conditions, 20 g/L of the racemate transformed into 16.67 g/L of (R)-1,3-butanediol with 99.5% enantiomeric excess by the oxidation-reduction cascade system in a 200-mL bioreactor. CONCLUSIONS: The step-by-step cascade reaction efficiently produced (R)-1,3-butanediol from the racemate by biosynthesis and shows promising application prospects.

Machaerium acutifolium lectin alters membrane structure and induces ROS production in Candida parapsilosis.

Lectins are a group of widely distributed and structurally heterogeneous proteins of nonimmune origin. These proteins have the ability to interact with glycans present on cell surfaces and elicit diverse biological activities. Machaerium acutifolium lectin (MaL) is an N-acetyl-D-glucosamine-binding lectin that exhibits antinociceptive activity via transient receptor potential cation channel subfamily V member 1 (TRPV1). Lectins that have the ability to recognize and interact with N-acetyl-D-glucosamine residues are potential candidates for studies of fungicidal activity. In this work, we show that MaL has antifungal activity against Candida species, and we describe its mode of action towards Candida parapsilosis. MaL inhibited the growth of C. albicans and C. parapsilosis. However, MaL was more potent against C. parapsilosis. The candidacidal mode of action of MaL on C. parapsilosis involves enhanced cell permeabilization, alteration of the plasma membrane proton-pumping ATPase function (H+-ATPase), induction of oxidative stress, and DNA damage. MaL also exhibited antibiofilm activity and noncytotoxicity to Vero cells. These results indicate that MaL is a promising candidate for the future development of a new, natural, and safe drug for the treatment of infections caused by C. parapsilosis.

Fermentation in fine cocoa type Scavina: Change in standard quality as the effect of use of starters yeast in fermentation.

In the present work we aimed to demonstrate the influence of inoculum starter in support high quality fermentation. Cocoa fermentations were performed in wooden boxes and eight yeasts strains were used in separated fermentations of fine cocoa, type Scavina, as starter inoculum. Temperature, pH, titirable acidity, reducing sugar and free amino acids were evaluated during or after fermentation. The influence of starters yeasts on the decrease of acidity, sugar concentration and free amino acids was significant. The strains Candida parapsilosis, Torulaspora delbrueckii and Pichia kluyveri showed greater changes in the reducing sugar and free amino acids in fermented cocoa beans. These results indicate the ability of yeast used as inoculum starter to modify the end condition and further enhance the quality of fine cocoa beans.

Combined mutagenesis and metabolic regulation to enhance D-arabitol production from Candida parapsilosis.

D-Arabitol is an important pentitol that is widely used in the food, pharmaceutical and chemical industries. It is mainly produced by yeasts during the biotransformation of glucose. To obtain strains with high D-arabitol production, Candida parapsilosis was mutated using atmospheric and room temperature plasma (ARTP). Among the screened mutants, mutant A6 had the highest yield at 32.92 g/L, a 53.98% increase compared with the original strain (21.38 g/L). Furthermore, metabolic regulators were added to the medium to improve D-arabitol production. Pyrithioxin dihydrochloride increased D-arabitol production by 34.4% by regulating glucose-6-phosphate dehydrogenase, and 4-methylpyrazole increased D-arabitol production by 77.4% compared with the control group by inhibiting alcohol dehydrogenase activity. Amphotericin B and Triton X-100 increased D-arabitol production by 23.8% and 42.2% by improving the membrane permeability and dissolved oxygen content, respectively. This study may provide important implications for obtaining high-yield D-arabitol strains.

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.

Enhanced Efflux Pump Expression in Candida Mutants Results in Decreased Manogepix Susceptibility.

Manogepix is a broad-spectrum antifungal agent that inhibits glycosylphosphatidylinositol (GPI) anchor biosynthesis. Using whole-genome sequencing, we characterized two efflux-mediated mechanisms in the fungal pathogens Candida albicans and Candida parapsilosis that resulted in decreased manogepix susceptibility. In C. albicans, a gain-of-function mutation in the transcription factor gene ZCF29 activated expression of ATP-binding cassette transporter genes CDR11 and SNQ2 In C. parapsilosis, a mitochondrial deletion activated expression of the major facilitator superfamily transporter gene MDR1.

Candida biofilm production is associated with higher mortality in patients with candidaemia.

BACKGROUND: Candidaemia is a common life-threatening disease among hospitalised patients, but the effect of the Candida biofilm-forming ability on the clinical outcome remains controversial. OBJECTIVE: The aim was to determine the impact of biofilms, specifically focusing on biofilm mass and metabolic activity, on the mortality in candidaemia. PATIENTS/METHODS: The clinical data of patients (n = 127) treated at the University of Debrecen, Clinical Centre, between January 2013 and December 2018, were investigated retrospectively. Biofilm formation was assessed using the crystal violet and XTT assays, measuring the biofilm mass and metabolic activity, respectively. Isolates were classified as low, intermediate and high biofilm producers both regarding biofilm mass and metabolic activity. The susceptibility of one-day-old biofilms to fluconazole, amphotericin B, anidulafungin, caspofungin and micafungin was evaluated and compared to planktonic susceptibility. RESULTS: Intermediate/high biofilm mass was associated with significantly higher mortality (61%). All Candida tropicalis, Candida parapsilosis and Candida glabrata isolates originating from fatal infections were intermediate/high biofilm producers, whereas this ratio was 85% for Candida albicans. Solid malignancy was associated with intermediate/high biofilm producers (P = .043). The mortality was significantly higher in infections caused by Candida strains producing biofilms with intermediate/high metabolic activity (62% vs. 33%, P = .010). The ratio of concomitant bacteraemia was higher for isolates forming biofilms with low metabolic activity (53% vs 28%, P = .015). CONCLUSIONS: This study provides evidence that the Candida biofilms especially with intermediate/high metabolic activity are related to higher mortality in candidaemia.

Moonlighting proteins are variably exposed at the cell surfaces of Candida glabrata, Candida parapsilosis and Candida tropicalis under certain growth conditions.

BACKGROUND: Adaptability to different environmental conditions is an essential characteristic of pathogenic microorganisms as it facilitates their invasion of host organisms. The most external component of pathogenic yeast-like fungi from the Candida genus is the multilayered cell wall. This structure is composed mainly of complex polysaccharides and proteins that can undergo dynamic changes to adapt to the environmental conditions of colonized niches. RESULTS: We utilized cell surface shaving with trypsin and a shotgun proteomic approach to reveal the surface-exposed proteins of three important non-albicans Candida species-C. glabrata, C. parapsilosis and C. tropicalis. These proteinaceous components were identified after the growth of the fungal cells in various culture media, including artificial saliva, artificial urine and vagina-simulative medium under aerobic conditions and anaerobically in rich YPD medium. Several known proteins involved in cell wall maintenance and fungal pathogenesis were identified at the cell surface as were a number of atypical cell wall components-pyruvate decarboxylase (Pdc11), enolase (Eno1) and glyceraldehyde-3-phosphate dehydrogenase (Tdh3) which are so-called 'moonlighting' proteins. Notably, many of these proteins showed significant upregulation at the cell surface in growth media mimicking the conditions of infection compared to defined synthetic medium. CONCLUSIONS: Moonlighting proteins are expressed under diverse conditions at the cell walls of the C. glabrata, C. parapsilosis and C. tropicalis fungal pathogens. This indicates a possible universal surface-associated role of these factors in the physiology of these fungi and in the pathology of the infections they cause.