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.

The IV International Symposium on Fungal Stress and the XIII International Fungal Biology Conference.

For the first time, the International Symposium on Fungal Stress was joined by the XIII International Fungal Biology Conference. The International Symposium on Fungal Stress (ISFUS), always held in Brazil, is now in its fourth edition, as an event of recognized quality in the international community of mycological research. The event held in São José dos Campos, SP, Brazil, in September 2022, featured 33 renowned speakers from 12 countries, including: Austria, Brazil, France, Germany, Ghana, Hungary, México, Pakistan, Spain, Slovenia, USA, and UK. In addition to the scientific contribution of the event in bringing together national and international researchers and their work in a strategic area, it helps maintain and strengthen international cooperation for scientific development in Brazil.

Evolution of loss of heterozygosity patterns in hybrid genomes of Candida yeast pathogens.

BACKGROUND: Hybrids are chimeric organisms with highly plastic heterozygous genomes that may confer unique traits enabling the adaptation to new environments. However, most evolutionary theory frameworks predict that the high levels of genetic heterozygosity present in hybrids from divergent parents are likely to result in numerous deleterious epistatic interactions. Under this scenario, selection is expected to favor recombination events resulting in loss of heterozygosity (LOH) affecting genes involved in such negative interactions. Nevertheless, it is so far unknown whether this phenomenon actually drives genomic evolution in natural populations of hybrids. To determine the balance between selection and drift in the evolution of LOH patterns in natural yeast hybrids, we analyzed the genomic sequences from fifty-five hybrid strains of the pathogenic yeasts Candida orthopsilosis and Candida metapsilosis, which derived from at least six distinct natural hybridization events. RESULTS: We found that, although LOH patterns in independent hybrid clades share some level of convergence that would not be expected from random occurrence, there is an apparent lack of strong functional selection. Moreover, while mitosis is associated with a limited number of inter-homeologous chromosome recombinations in these genomes, induced DNA breaks seem to increase the LOH rate. We also found that LOH does not accumulate linearly with time in these hybrids. Furthermore, some C. orthopsilosis hybrids present LOH patterns compatible with footprints of meiotic recombination. These meiotic-like patterns are at odds with a lack of evidence of sexual recombination and with our inability to experimentally induce sporulation in these hybrids. CONCLUSIONS: Our results suggest that genetic drift is the prevailing force shaping LOH patterns in these hybrid genomes. Moreover, the observed LOH patterns suggest that these are likely not the result of continuous accumulation of sporadic events-as expected by mitotic repair of rare chromosomal breaks-but rather of acute episodes involving many LOH events in a short period of time.

Functionalized Mesoporous Silica Nanoparticles for Drug-Delivery to Multidrug-Resistant Cancer Cells.

Background: Multidrug resistance is a common reason behind the failure of chemotherapy. Even if the therapy is effective, serious adverse effects might develop due to the low specificity and selectivity of antineoplastic agents. Mesoporous silica nanoparticles (MSNs) are promising materials for tumor-targeting and drug-delivery due to their small size, relatively inert nature, and extremely large specific surfaces that can be functionalized by therapeutic and targeting entities. We aimed to create a fluorescently labeled MSN-based drug-delivery system and investigate their internalization and drug-releasing capability in drug-sensitive MCF-7 and P-glycoprotein-overexpressing multidrug-resistant MCF-7 KCR cancer cells. Methods and Results: To track the uptake and subcellular distribution of MSNs, particles with covalently coupled red fluorescent Rhodamine B (RhoB) were produced (RhoB@MSNs). Both MCF-7 and MCF-7 KCR cells accumulated a significant amount of RhoB@MSNs. The intracellular RhoB@MSN concentrations did not differ between sensitive and multidrug-resistant cells and were kept at the same level even after cessation of RhoB@MSN exposure. Although most RhoB@MSNs resided in the cytoplasm, significantly more RhoB@MSNs co-localized with lysosomes in multidrug-resistant cells compared to sensitive counterparts. To examine the drug-delivery capability of these particles, RhoB@Rho123@MSNs were established, where RhoB-functionalized nanoparticles carried green fluorescent Rhodamine 123 (Rho123) - a P-glycoprotein substrate - as cargo within mesopores. Significantly higher Rho123 fluorescence intensity was detected in RhoB@Rho123@MSN-treated multidrug-resistant cells than in free Rho123-exposed counterparts. The exceptional drug-delivery potential of MSNs was further verified using Mitomycin C (MMC)-loaded RhoB@MSNs (RhoB@MMC@MSNs). Exposures to RhoB@MMC@MSNs significantly decreased the viability not only of drug-sensitive but of multidrug-resistant cells and the elimination of MDR cells was significantly more robust than upon free MMC treatments. Conclusion: The efficient delivery of Rho123 and MMC to multidrug-resistant cells via MSNs, the amplified and presumably prolonged intracellular drug concentration, and the consequently enhanced cytotoxic effects envision the enormous potential of MSNs to defeat multidrug-resistant cancer.

Characterization and functional analysis of zinc trafficking in the human fungal pathogen Candida parapsilosis.

The zinc restriction and zinc toxicity are part of host defence, called nutritional immunity. The crucial role of zinc homeostasis in microbial survival within a host is established, but little is known about these processes in the opportunistic human fungal pathogen Candida parapsilosis. Our in silico predictions suggested the presence of at least six potential zinc transporters (ZnTs) in C. parapsilosis-orthologues of ZRC1, ZRT3 and ZRT101-but an orthologue of PRA1 zincophore was not found. In addition, we detected a species-specific gene expansion of the novel ZnT ZRT2, as we identified three orthologue genes in the genome of C. parapsilosis. Based on predictions, we created homozygous mutant strains of the potential ZnTs and characterized them. Despite the apparent gene expansion of ZRT2 in C. parapsilosis, only CpZRT21 was essential for growth in a zinc-depleted acidic environment, in addition we found that CpZrc1 is essential for zinc detoxification and also protects the fungi against the elimination of murine macrophages. Significantly, we demonstrated that C. parapsilosis forms zincosomes in a Zrc1-independent manner and zinc detoxification is mediated by the vacuolar importer CpZrc1. Our study defines the functions of C. parapsilosis ZnTs, including a species-specific survival and zinc detoxification system.

A complete nicotinate degradation pathway in the microbial eukaryote Aspergillus nidulans.

Several strikingly different aerobic and anaerobic pathways of nicotinate breakdown are extant in bacteria. Here, through reverse genetics and analytical techniques we elucidated in Aspergillus nidulans, a complete eukaryotic nicotinate utilization pathway. The pathway extant in this fungus and other ascomycetes, is quite different from bacterial ones. All intermediate metabolites were identified. The cognate proteins, encoded by eleven genes (hxn) mapping in three clusters are co-regulated by a specific transcription factor. Several enzymatic steps have no prokaryotic equivalent and two metabolites, 3-hydroxypiperidine-2,6-dione and 5,6-dihydroxypiperidine-2-one, have not been identified previously in any organism, the latter being a novel chemical compound. Hydrolytic ring opening results in α-hydroxyglutaramate, a compound not detected in analogous prokaryotic pathways. Our earlier phylogenetic analysis of Hxn proteins together with this complete biochemical pathway illustrates convergent evolution of catabolic pathways between fungi and bacteria.

Lactobacillus acidophilus, L. plantarum, L. rhamnosus, and L. reuteri Cell-Free Supernatants Inhibit Candida parapsilosis Pathogenic Potential upon Infection of Vaginal Epithelial Cells Monolayer and in a Transwell Coculture System In Vitro.

Vulvovaginal candidiasis (VVC) is a common clinical condition with symptoms and signs of vaginal inflammation in the presence of Candida species. At least one episode of VVC is experienced in up to 75% of women in the reproductive age group during their lifetime, and 5% to 8% of such women suffer from the chronic form. Most cases of VVC are still caused by C. albicans. However, the incidence of VVC cases by non-albicans Candida (NAC) species, such as C. parapsilosis, is continuously increasing. Despite the prevalence of VVC from NAC, little is known about these species and almost nothing about the mechanisms that trigger the VVC. Lactobacillus spp. are the most widely before represented microorganisms in the vaginal microbiota of healthy women. Here, cell-free supernatants (CFS) obtained from L. acidophilus, L. plantarum, L. rhamnosus, and L. reuteri were assessed for their effect on C. parapsilosis virulence traits. Moreover, we assessed if such an effect persisted even after the removal of the CFS (CFS preincubation effect). Moreover, a transwell coculture system was employed by which the relevant antifungal effect was shown to be attributable to the compounds released by lactobacilli. Our results suggest that lactobacilli can work (i) by reducing C. parapsilosis virulence traits, as indicated by the reduced fungal proliferation, viability, and metabolic activity, and (ii) by improving epithelial resistance to the fungus. Overall, these data suggest that, in the context of the vaginal microbiota, the lactobacilli may play a role in preventing the onset of mucosal C. parapsilosis infection. IMPORTANCE The incidence of VVC by non-albicans Candida (NAC) species, such as C. parapsilosis, is increasing. Treatment failure is common in NAC-VVC because some species are resistant or poorly susceptible to the antifungal agents normally employed. Research on C. parapsilosis's pathogenic mechanisms and alternative treatments are still lacking. C. albicans triggers the VVC by producing hyphae, which favor the loss of epithelial tolerance. Differently, C. parapsilosis only produces pseudohyphae. Hence, different virulence factors may trigger the VVC. Likewise, the therapeutic options could also involve different fungal targets. Substantial in vitro and in vivo studies on the pathogenicity mechanisms of C. parapsilosis are lacking. The data presented here ascribe a novel beneficial role to different Lactobacillus spp., whose CFS provides a postbiotic-like activity against C. parapsilosis. Further studies are needed to unravel the mechanisms involved in the bioactivities of such compounds, to better understand the role of single postbiotics in the CFS.

The effect of antifungal resistance development on the virulence of Candida species.

In recent years, the relevance of diseases associated with fungal pathogens increased worldwide. Members of the Candida genus are responsible for the greatest number of fungal bloodstream infections every year. Epidemiological data consistently indicate a modest shift toward non-albicans species, albeit Candidaalbicans is still the most recognizable species within the genus. As a result, the number of clinically relevant pathogens has increased, and, despite their distinct pathogenicity features, the applicable antifungal agents remained the same. For bloodstream infections, only three classes of drugs are routinely used, namely polyenes, azoles and echinocandins. Antifungal resistance toward all three antifungal drug classes frequently occurs in clinical settings. Compared with the broad range of literature on virulence and antifungal resistance of Candida species separately, only a small portion of studies examined the effect of resistance on virulence. These studies found that resistance to polyenes and echinocandins concluded in significant decrease in the virulence in different Candida species. Meanwhile, in some cases, resistance to azole type antifungals resulted in increased virulence depending on the species and isolates. These findings underline the importance of studies aiming to dissect the connections of virulence and resistance in Candida species.

Kynurenic Acid and Its Analogue SZR-72 Ameliorate the Severity of Experimental Acute Necrotizing Pancreatitis.

The pathophysiology of acute pancreatitis (AP) is not well understood, and the disease does not have specific therapy. Tryptophan metabolite L-kynurenic acid (KYNA) and its synthetic analogue SZR-72 are antagonists of the N-methyl-D-aspartate receptor (NMDAR) and have immune modulatory roles in several inflammatory diseases. Our aims were to investigate the effects of KYNA and SZR-72 on experimental AP and to reveal their possible mode of action. AP was induced by intraperitoneal (i.p.) injection of L-ornithine-HCl (LO) in SPRD rats. Animals were pretreated with 75-300 mg/kg KYNA or SZR-72. Control animals were injected with physiological saline instead of LO, KYNA and/or SZR-72. Laboratory and histological parameters, as well as pancreatic and systemic circulation were measured to evaluate AP severity. Pancreatic heat shock protein-72 and IL-1ß were measured by western blot and ELISA, respectively. Pancreatic expression of NMDAR1 was investigated by RT-PCR and immunohistochemistry. Viability of isolated pancreatic acinar cells in response to LO, KYNA, SZR-72 and/or NMDA administration was assessed by propidium-iodide assay. The effects of LO and/or SZR-72 on neutrophil granulocyte function was also studied. Almost all investigated laboratory and histological parameters of AP were significantly reduced by administration of 300 mg/kg KYNA or SZR-72, whereas the 150 mg/kg or 75 mg/kg doses were less or not effective, respectively. The decreased pancreatic microcirculation was also improved in the AP groups treated with 300 mg/kg KYNA or SZR-72. Interestingly, pancreatic heat shock protein-72 expression was significantly increased by administration of SZR-72, KYNA and/or LO. mRNA and protein expression of NMDAR1 was detected in pancreatic tissue. LO treatment caused acinar cell toxicity which was reversed by 250 µM KYNA or SZR-72. Treatment of acini with NMDA (25, 250, 2000 µM) did not influence the effects of KYNA or SZR-72. Moreover, SZR-72 reduced LO-induced H2O2 production of neutrophil granulocytes. KYNA and SZR-72 have dose-dependent protective effects on LO-induced AP or acinar toxicity which seem to be independent of pancreatic NMDA receptors. Furthermore, SZR-72 treatment suppressed AP-induced activation of neutrophil granulocytes. This study suggests that administration of KYNA and its derivative could be beneficial in AP.

Virulence Factors and in-Host Selection on Phenotypes in Infectious Probiotic Yeast Isolates (Saccharomyces 'boulardii').

Saccharomyces yeast probiotics (S. 'boulardii') have long been applied in the treatment of several gastrointestinal conditions. Despite their widespread use, they are rare opportunistic pathogens responsible for a high proportion of Saccharomyces mycosis cases. The potential virulence attributes of S. 'boulardii' as well as its interactions with the human immune system have been studied, however, no information is available on how these yeasts may change due to in-host evolution. To fill this gap, we compared the general phenotypic characteristics, cell morphology, virulence factors, epithelial and immunological interactions, and pathogenicity of four probiotic product samples, two mycosis, and eight non-mycosis samples of S. 'boulardii'. We assessed the characteristics related to major steps of yeast infections. Mycosis and non-mycosis isolates both displayed novel characters when compared to the product isolates, but in the case of most virulence factors and in pathogenicity, differences were negligible or, surprisingly, the yeasts from products showed elevated levels. No isolates inflicted considerable damage to the epithelial model or bore the hallmarks of immune evasion. Our results show that strains in probiotic products possess characteristics that enable them to act as pathogens upon permissive conditions, and their entry into the bloodstream is not due to active mechanisms but depends on the host. Survival in the host is dependent on yeast phenotypic characteristics which may change in many ways once they start evolving in the host. These facts call attention to the shortcomings of virulence phenotyping in yeast research, and the need for a more thorough assessment of probiotic use.

Deciphering of Candida parapsilosis induced immune response in Drosophila melanogaster.

Candida infections are the most prevalent cause of serious human mycoses and are the third most common pathogens isolated from bloodstream infections in hospitalized patients. C. parapsilosis is a member of the non-albicans spp., which have a predilection for causing life-threatening disease in neonates and hospitalized pediatric patients. In this study, we utilized a Drosophila melanogaster infection model to analyze the immunological responses to C. parapsilosis. Our results demonstrate that the Toll pathway in Drosophila controls C. parapsilosis proliferation as the Toll signaling mutant MyD88-/- flies are highly susceptible to C. parapsilosis. We also confirmed that the MyD88-/- fly is a convenient invertebrate animal model to analyze virulence properties of different species and strains from the C. parapsilosis sensu lato complex as C. orthopsilosis, C. metapsilosis proved to be less virulent than C. parapsilosis sensu stricto and the N-mannan deficient C. parapsilosis och1Δ/Δ strain showed attenuated pathogenicity in this immunodeficient Drosophila background. We also found that Persephone protease is not required for detection and activation of Toll pathway during C. parapsilosis infection. Furthermore, we observed that Drosophila ß-glucan receptor deficient flies where more sensitive to C. parapsilosis compared to wild-type flies; however, we could not find a clear dependence on the recognition of this receptor and the cell wall ß-glucan exposure-induced host response. These studies establish this D. melanogaster infection model as an efficient tool in deciphering immune responses to C. parapsilosis as well as for assessing virulence factors produced by this emerging fungal predator.

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.

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.

Symbiotic NCR Peptide Fragments Affect the Viability, Morphology and Biofilm Formation of Candida Species.

The increasing rate of fungal infections causes global problems not only in human healthcare but agriculture as well. To combat fungal pathogens limited numbers of antifungal agents are available therefore alternative drugs are needed. Antimicrobial peptides are potent candidates because of their broad activity spectrum and their diverse mode of actions. The model legume Medicago truncatula produces >700 nodule specific cysteine-rich (NCR) peptides in symbiosis and many of them have in vitro antimicrobial activities without considerable toxicity on human cells. In this work we demonstrate the anticandidal activity of the NCR335 and NCR169 peptide derivatives against five Candida species by using the micro-dilution method, measuring inhibition of biofilm formation with the XTT (2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide) assay, and assessing the morphological change of dimorphic Candida species by microscopy. We show that both the N- and C-terminal regions of NCR335 possess anticandidal activity as well as the C-terminal sequence of NCR169. The active peptides inhibit biofilm formation and the yeast-hypha transformation. Combined treatment of C. auris with peptides and fluconazole revealed synergistic interactions and reduced 2-8-fold the minimal inhibitory concentrations. Our results demonstrate that shortening NCR peptides can even enhance and broaden their anticandidal activity and therapeutic potential.

Complex and Controversial Roles of Eicosanoids in Fungal Pathogenesis.

The prevalence of fungal infections has increased in immunocompromised patients, leading to millions of deaths annually. Arachidonic acid (AA) metabolites, such as eicosanoids, play important roles in regulating innate and adaptative immune function, particularly since they can function as virulence factors enhancing fungal colonization and are produced by mammalian and lower eukaryotes, such as yeasts and other fungi (Candida albicans, Histoplasma capsulatum and Cryptococcus neoformans). C. albicans produces prostaglandins (PG), Leukotrienes (LT) and Resolvins (Rvs), whereas the first two have been well documented in Cryptococcus sp. and H. capsulatum. In this review, we cover the eicosanoids produced by the host and fungi during fungal infections. These fungal-derived PGs have immunomodulatory functions analogous to their mammalian counterparts. Prostaglandin E2 (PGE2) protects C. albicans and C. parapsilosis cells from the phagocytic and killing activity of macrophages. H. capsulatum PGs augment the fungal burden and host mortality rates in histoplasmosis. However, PGD2 potentiates the effects and production of LTB4, which is a very potent neutrophil chemoattractant that enhances host responses. Altogether, these data suggest that eicosanoids, mainly PGE2, may serve as a new potential target to combat diverse fungal infections.

Enhancing the chemical transformation of Candida parapsilosis.

Candida parapsilosis is a leading cause of invasive mycoses and the major cause of nosocomial fungaemia amongst low and very low birth weight neonates. However, the molecular and physiological characteristics of this fungus remain understudied. To advance our knowledge about the pathobiology of this pathogen, we sought to develop and validate an effective method for chemical transformation of C. parapsilosis. Chemical transformation is the primary procedure for introducing foreign DNA into Candida yeast as it requires no special equipment, although its performance efficacy drops rapidly when the size of the transforming DNA increases. To define optimal conditions for chemical transformation in C. parapsilosis, we selected a leucine auxotroph laboratory strain. We identified optimal cell density for transformation, incubation times, inclusion of specific enhancing chemicals, and size and amounts of DNA fragments that resulted in maximized transformation efficiency. We determined that the inclusion of dimethyl sulfoxide was beneficial, but dithiothreitol pretreatment reduced colony recovery. As a result, the modified protocol led to a 20-55-fold increase in transformation efficiency, depending on the size of the transforming fragment. We validated the modified methodology with prototrophic isolates and demonstrated that the new approach resulted in the recovery of significantly more transformants in 5 of 6 isolates. Additionally, we identified a medium in which transformation competent yeast cells could safely be maintained at -80°C for up to 6 weeks that reduces laboratory work and shortens the overall procedure. These modifications will significantly aid further investigations into the genetic basis for virulence in C. parapsilosis.

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.