Characterization of spatio-temporal dynamics of the constrained network of the filamentous fungus Podospora anserina using a geomatics-based approach.

In their natural environment, fungi are subjected to a wide variety of environmental stresses which they must cope with by constantly adapting the architecture of their growing network. In this work, our objective was to finely characterize the thallus development of the filamentous fungus Podospora anserina subjected to different constraints that are simple to implement in vitro and that can be considered as relevant environmental stresses, such as a nutrient-poor environment or non-optimal temperatures. At the Petri dish scale, the observations showed that the fungal thallus is differentially affected (thallus diameter, mycelium aspect) according to the stresses but these observations remain qualitative. At the hyphal scale, we showed that the extraction of the usual quantities (i.e. apex, node, length) does not allow to distinguish the different thallus under stress, these quantities being globally affected by the application of a stress in comparison with a thallus having grown under optimal conditions. Thanks to an original geomatics-based approach based on the use of automatized Geographic Information System (GIS) tools, we were able to produce maps and metrics characterizing the growth dynamics of the networks and then to highlight some very different dynamics of network densification according to the applied stresses. The fungal thallus is then considered as a map and we are no longer interested in the quantity of material (hyphae) produced but in the empty spaces between the hyphae, the intra-thallus surfaces. This study contributes to a better understanding of how filamentous fungi adapt the growth and densification of their network to potentially adverse environmental changes.

Mutations in Podospora anserina MCM1 and VelC Trigger Spontaneous Development of Barren Fruiting Bodies.

The ascomycete Podospora anserina is a heterothallic filamentous fungus found mainly on herbivore dung. It is commonly used in laboratories as a model system, and its complete life cycle lasting eight days is well mastered in vitro. The main objective of our team is to understand better the global process of fruiting body development, named perithecia, induced normally in this species by fertilization. Three allelic mutants, named pfd3, pfd9, and pfd23 (for "promoting fruiting body development") obtained by UV mutagenesis, were selected in view of their abilities to promote barren perithecium development without fertilization. By complete genome sequencing of pfd3 and pfd9, and mutant complementation, we identified point mutations in the mcm1 gene as responsible for spontaneous perithecium development. MCM1 proteins are MADS box transcription factors that control diverse developmental processes in plants, metazoans, and fungi. We also identified using the same methods a mutation in the VelC gene as responsible for spontaneous perithecium development in the vacua mutant. The VelC protein belongs to the velvet family of regulators involved in the control of development and secondary metabolite production. A key role of MCM1 and VelC in coordinating the development of P. anserina perithecia with gamete formation and fertilization is highlighted.

Prediction and experimental evidence of different growth phases of the Podospora anserina hyphal network.

Under ideal conditions, the growth of the mycelial network of a filamentous fungus is monotonous, showing an ever increasing complexity with time. The components of the network growth are very simple and based on two mechanisms: the elongation of each hypha, and their multiplication by successive branching. These two mechanisms are sufficient to produce a complex network, and could be localized only at the tips of hyphae. However, branching can be of two types, apical or lateral, depending on its location on the hyphae, therefore imposing the redistribution of the necessary material in the whole mycelium. From an evolutionary point of view, maintaining different branching processes, with additional energy needs for structure and metabolism, is intriguing. We propose in this work to discuss the advantages of each branching type using a new observable for the network growth, allowing us to compare growth configurations. For this purpose, we build on experimental observations of the Podospora anserina mycelium growth, enabling us to feed and constrain a lattice-free modeling of this network based on a binary tree. First, we report the set of statistics related to the branches of P. anserina that we have implemented into the model. Then, we build the density observable, allowing us to discuss the succession of growth phases. We predict that density over time is not monotonic, but shows a decay growth phase, clearly separated from an other one by a stationary phase. The time of appearance of this stable region appears to be driven solely by the growth rate. Finally, we show that density is an appropriate observable to differentiate growth stress.

Role of Fungal Infections in Carcinogenesis and Cancer Development: A Literature Review.

Cancer is a serious debilitating disease and one of the most common causes of death. In recent decades the high risk of various cancers enforced scientists to discover novel prevention and treatment methods to diminish the mortality of this terrifying disease. Accordingly, its prevention can be possible in near future. Based on epidemiological evidence, there is a clear link between pathogenic fungal infections and cancer development. This association is often seen in people with weakened immune systems such as the elderly and people with acquired immunodeficiency (AIDS). Carcinoma in these people is first seen chronically and then acutely. Although the different genetic and environmental risk factors are involved in carcinogenesis, one of the most important risk factors is fungal species and infections associating with cancers etiology. Now it is known that microbial infection is responsible for initiating 2.2 million new cancer cases. In this way, many recent studies have focused on investigating the role and mechanism of fungal infections in diverse cancers occurrence. This review provides a comprehensive framework of the latest clinical findings and the association of fungal infections with versatile cancers including esophageal, gastric, colorectal, lung, cervical, skin, and ovarian cancer.

Prediction and experimental evidence of the optimisation of the angular branching process in the thallus growth of Podospora anserina.

Based upon apical growth and hyphal branching, the two main processes that drive the growth pattern of a fungal network, we propose here a two-dimensions simulation based on a binary-tree modelling allowing us to extract the main characteristics of a generic thallus growth. In particular, we showed that, in a homogeneous environment, the fungal growth can be optimized for exploration and exploitation of its surroundings with a specific angular distribution of apical branching. Two complementary methods of extracting angle values have been used to confront the result of the simulation with experimental data obtained from the thallus growth of the saprophytic filamentous fungus Podospora anserina. Finally, we propose here a validated model that, while being computationally low-cost, is powerful enough to test quickly multiple conditions and constraints. It will allow in future works to deepen the characterization of the growth dynamic of fungal network, in addition to laboratory experiments, that could be sometimes expensive, tedious or of limited scope.

Involvement of VIVID in white light-responsive pigmentation, sexual development and sterigmatocystin biosynthesis in the filamentous fungus Podospora anserina.

Light serves as a source of information and regulates diverse physiological processes in living organisms. Fungi perceive and respond to light through a complex photosensory system. Fungi have evolved the desensitization mechanism to adapt to the changing light signal in a natural environment. White light exerts multiple essential impacts on the model filamentous fungus Podospora anserina. However, the light sensing and response in this species has not been investigated. In this study, we demonstrated that the loss of function of the light desensitization protein VIVID (VVD) in P. anserina triggered exacerbated light responses and therefore led to drastic morphological and physiological changes. The white light-sensitive mutant Δvvd showed growth reduction, spermatia overproduction, enhanced hyphae pigmentation and reduced oxidative stress tolerance. We observed the decreased expression level of sterigmatocystin gene cluster by transcriptome analysis and finally detected the reduced production of sterigmatocystin in Δvvd in response to white light. Our data indicate that VVD acts as a repressor of white collar complex. This study exhibits a vital role of VVD in governing white light-responsive gene expression and secondary metabolite production and contributes to a better understanding of the photoreceptor VVD in P. anserina.

Antimicrobial activity of Bacillus sp. isolated strains of wild honey.

BACKGROUND: Multi-drug resistant bacteria hazards to the health of humans could be an agent in the destruction of human generation. Natural products of Bacillus species are the main source to access progressive antibiotics that can be a good candidate for the discovery of novel antibiotics. Wild honey as a valuable food has been used in medicine with antimicrobial effects. OBJECTIVE: Bacillus strains isolated from wild honey were evaluated for the potential antimicrobial activity against human and plant bacterial and fungal pathogens. METHODS: Three bacterial isolates were identified as strain Khuz-1 (98.27% similarity with Bacillus safensis subsp. Safensis strain FO-36bT), strain Khuz-2 (99.18% similarity with Bacillus rugosus strain SPB7T), and strain Khuz-3 (99.78% similarity with Bacillus velezensis strain CR-502 T) by 16S rRNA gene sequences. The strains were characterized by their ability to inhibit the growth of human and phytopathogenic fungi. RESULTS: The results indicated that B. rugosus strain Khuz-2 inhibited the growth of phytopathogenic and human fungal more effective than other ones. It seems that the strain Khuz-2 has a suitable antimicrobial and antifungal potential as a good candidate for further pharmaceutical research. CONCLUSION: Based on the results of GC-MS, Pyrrolo [1,2-a] pyrazine-1,4-dion, hexahydro-3-(2-methylpropyle) (PPDHM) was the major compound for all strains which have a various pharmacological effect. Isolation and identification of beneficial bacteria from natural sources can play an important role in future pharmaceutical and industrial applications.

Functional Characterization of the GATA-Type Transcription Factor PaNsdD in the Filamentous Fungus Podospora anserina and Its Interplay with the Sterigmatocystin Pathway.

The model ascomycete Podospora anserina, distinguished by its strict sexual development, is a prolific but yet unexploited reservoir of natural products. The GATA-type transcription factor NsdD has been characterized by the role in balancing asexual and sexual reproduction and governing secondary metabolism in filamentous fungi. In the present study, we functionally investigated the NsdD ortholog PaNsdD in P. anserina. Compared to the wild-type strain, vegetative growth, ageing processes, sexual reproduction, stress tolerance, and interspecific confrontations in the mutant were drastically impaired, owing to the loss of function of PaNsdD. In addition, the production of 3-acetyl-4-methylpyrrole, a new metabolite identified in P. anserina in this study, was significantly inhibited in the ΔPaNsdD mutant. We also demonstrated the interplay of PaNsdD with the sterigmatocystin biosynthetic gene pathway, especially as the deletion of PaNsdD triggered the enhanced red-pink pigment biosynthesis that occurs only in the presence of the core polyketide synthase-encoding gene PaStcA of the sterigmatocystin pathway. Taken together, these results contribute to a better understanding of the global regulation mediated by PaNsdD in P. anserina, especially with regard to its unexpected involvement in the fungal ageing process and its interplay with the sterigmatocystin pathway. IMPORTANCE Fungal transcription factors play an essential role in coordinating multiple physiological processes. However, little is known about the functional characterization of transcription factors in the filamentous fungus Podospora anserina. In this study, a GATA-type regulator PaNsdD was investigated in P. anserina. The results showed that PaNsdD was a key factor that can control the fungal ageing process, vegetative growth, pigmentation, stress response, and interspecific confrontations and positively regulate the production of 3-acetyl-4-methylpyrrole. Meanwhile, a molecular interaction was implied between PaNsdD and the sterigmatocystin pathway. Overall, loss of function of PaNsdD seems to be highly disadvantageous for P. anserina, which relies on pure sexual reproduction in a limited life span. Therefore, PaNsdD is clearly indispensable for the survival and propagation of P. anserina in its complex ecological niches.

Complementary Strategies to Unlock Biosynthesis Gene Clusters Encoding Secondary Metabolites in the Filamentous Fungus Podospora anserina.

The coprophilous ascomycete Podospora anserina is known to have a high potential to synthesize a wide array of secondary metabolites (SMs). However, to date, the characterization of SMs in this species, as in other filamentous fungal species, is far less than expected by the functional prediction through genome mining, likely due to the inactivity of most SMs biosynthesis gene clusters (BGCs) under standard conditions. In this work, our main objective was to compare the global strategies usually used to deregulate SM gene clusters in P. anserina, including the variation of culture conditions and the modification of the chromatin state either by genetic manipulation or by chemical treatment, and to show the complementarity of the approaches between them. In this way, we showed that the metabolomics-driven comparative analysis unveils the unexpected diversity of metabolic changes in P. anserina and that the integrated strategies have a mutual complementary effect on the expression of the fungal metabolome. Then, our results demonstrate that metabolite production is significantly influenced by varied cultivation states and epigenetic modifications. We believe that the strategy described in this study will facilitate the discovery of fungal metabolites of interest and will improve the ability to prioritize the production of specific fungal SMs with an optimized treatment.

Variation in chemical composition and antimalarial activities of two samples of Terminalia albida collected from separate sites in Guinea.

BACKGROUND: The disparity of harvesting locations can influence the chemical composition of a plant species, which could affect its quality and bioactivity. Terminalia albida is widely used in traditional Guinean medicine whose activity against malaria has been validated in vitro and in murine models. The present work investigated the antimalarial properties and chemical composition of two samples of T. albida collected from different locations in Guinea. METHOD: T. albida samples were collected in different locations in Guinea, in Dubréka prefecture (West maritime Guinea) and in Kankan prefecture (eastern Guinea). The identity of the samples was confirmed by molecular analysis. In vitro antiplasmodial activity of the two extracts was determined against the chloroquine resistant strain PfK1. In vivo, extracts (100 mg/kg) were tested in two experimental murine models, respectively infected with P. chabaudi chabaudi and P. berghei ANKA. The chemical composition of the two samples was assessed by ultra-high-performance liquid chromatography coupled to high resolution mass spectrometry. RESULTS: In vitro, the Dubréka sample (TaD) was more active with an IC50 of 1.5 µg/mL versus 8.5 µg/mL for the extract from Kankan (TaK). In vivo, the antiparasitic effect of TaD was substantial with 56% of parasite inhibition at Day 10 post-infection in P. chabaudi infection and 61% at Day 8 in P. berghei model, compared to 14 and 19% inhibition respectively for the treatment with TaK. In addition, treatment with TaD further improved the survival of P. berghei infected-mice by 50% at Day 20, while the mortality rate of mice treated with Tak was similar to the untreated group. The LC/MS analysis of the two extracts identified 38 compounds, 15 of which were common to both samples while 9 and 14 other compounds were unique to TaD and TaK respectively. CONCLUSION: This study highlights the variability in the chemical composition of the species T. albida when collected in different geographical locations. These chemical disparities were associated with variable antimalarial effects. From a public health perspective, these results underline the importance of defining chemical fingerprints related to botanical species identification and to biological activity, for the plants most commonly used in traditional medicine.

Functional characterization of the sterigmatocystin secondary metabolite gene cluster in the filamentous fungus Podospora anserina: involvement in oxidative stress response, sexual development, pigmentation and interspecific competitions.

Filamentous fungi are known as prolific untapped reservoirs of diverse secondary metabolites, where genes required for their synthesis are organized in clusters. The bioactive properties of these compounds are closely related to their functions in fungal biology, which are not well understood. In this study, we focused on the Podospora anserina gene cluster responsible for the biosynthesis of sterigmatocystin (ST). Deletion of the PaStcA gene encoding the polyketide synthase and overexpression (OE) of the PaAflR gene encoding the ST-specific transcription factor in P. anserina were performed. We showed that growth of PaStcAΔ was inhibited in the presence of methylglyoxal, while OE-PaAflR showed a little inhibition, indicating that ST production may enhance oxidative stress tolerance in P. anserina. We also showed that the OE-PaAflR strain displayed an overpigmented thallus mediated by the melanin pathway. Overexpression of PaAflR also led to sterility. Interspecific confrontation assays showed that ST-overexpressed strains produced a high level of peroxides and possessed a higher competitiveness against other fungi. Comparative metabolite profiling demonstrated that PaStcAΔ strain was unable to produce ST, while OE-PaAflR displayed a ST overproduction. This study contributes to a better understanding of ST in P. anserina, especially with regard to its involvement in fungal physiology.

Characterization of three multicopper oxidases in the filamentous fungus Podospora anserina: A new role of an ABR1-like protein in fungal development?

The Podospora anserina genome contains a large family of 15 multicopper oxidases (MCOs), including three genes encoding a FET3-like protein, an ABR1-like protein and an ascorbate oxidase (AO)-like protein. FET3, ABR1 and AO1 are involved in global laccase-like activity since deletion of the relevant genes led to a decrease of activity when laccase substrate (ABTS) was used as substrate. However, contrary to the P. anserina MCO proteins previously characterized, none of these three MCOs seemed to be involved in lignocellulose degradation and in resistance to phenolic compounds and oxidative stress. We showed that the bulk of ferroxidase activity was clearly due to ABR1, and only in minor part to FET3, although ABR1 does not contain all the residues typical of FET3 proteins. Moreover, we showed that ABR1, related to the Aspergillus fumigatus ABR1 protein, was clearly and specifically involved in pigmentation of ascospores. Surprisingly, phenotypes were more severe in mutants lacking both abr1 and ao1. Deletion of the ao1 gene led to an almost total loss of AO activity. No direct involvement of AO1 in fungal developmental process in P. anserina was evidenced, except in a abr1Δ background. Overall, unlike other previously characterized MCOs, we thus evidence a clear involvement of ABR1 protein in fungal development.

Inositol-phosphate signaling as mediator for growth and sexual reproduction in Podospora anserina.

The molecular pathways involved in the development of multicellular fruiting bodies in fungi are still not well known. Especially, the interplay between the mycelium, the female tissues and the zygotic tissues of the fruiting bodies is poorly documented. Here, we describe PM154, a new strain of the model ascomycetes Podospora anserina able to mate with itself and that enabled the easy recovery of new mutants affected in fruiting body development. By complete genome sequencing of spod1, one of the new mutants, we identified an inositol phosphate polykinase gene as essential, especially for fruiting body development. A factor present in the wild type and diffusible in mutant hyphae was able to induce the development of the maternal tissues of the fruiting body in spod1, but failed to promote complete development of the zygotic ones. Addition of myo-inositol in the growth medium was able to increase the number of developing fruiting bodies in the wild type, but not in spod1. Overall, the data indicated that inositol and inositol polyphosphates were involved in promoting fruiting body maturation, but also in regulating the number of fruiting bodies that developed after fertilization. The same effect of inositol was seen in two other fungi, Sordaria macrospora and Chaetomium globosum. Key role of the inositol polyphosphate pathway during fruiting body maturation appears thus conserved during the evolution of Sordariales fungi.

Bilirubin oxidase-like proteins from Podospora anserina: promising thermostable enzymes for application in transformation of plant biomass.

Plant biomass degradation by fungi is a critical step for production of biofuels, and laccases are common ligninolytic enzymes envisioned for ligninolysis. Bilirubin oxidases (BODs)-like are related to laccases, but their roles during lignocellulose degradation have not yet been fully investigated. The two BODs of the ascomycete fungus Podospora anserina were characterized by targeted gene deletions. Enzymatic assay revealed that the bod1(Δ) and bod2(Δ) mutants lost partly a thermostable laccase activity. A triple mutant inactivated for bod1, bod2 and mco, a previously investigated multicopper oxidase gene distantly related to laccases, had no thermostable laccase activity. The pattern of fruiting body production in the bod1(Δ) bod2(Δ) double mutant was changed. The bod1(Δ) and bod2(Δ) mutants were reduced in their ability to grow on ligneous and cellulosic materials. Furthermore, bod1(Δ) and bod2(Δ) mutants were defective towards resistance to phenolic substrates and H2 O2 , which may also impact lignocellulose breakdown. Double and triple mutants were more affected than single mutants, evidencing redundancy of function among BODs and mco. Overall, the data show that bod1, bod2 and mco code for non-canonical thermostable laccases that participate in the degradation of lignocellulose. Thanks to their thermal stability, these enzymes may be more promising candidate for biotechnological application than canonical laccases.

Systematic gene deletions evidences that laccases are involved in several stages of wood degradation in the filamentous fungus Podospora anserina.

Transformation of plant biomass into biofuels may supply environmentally friendly alternative biological sources of energy. Laccases are supposed to be involved in the lysis of lignin, a prerequisite step for efficient breakdown of cellulose into fermentable sugars. The role in development and plant biomass degradation of the nine canonical laccases belonging to three different subfamilies and one related multicopper oxidase of the Ascomycota fungus Podospora anserina was investigated by targeted gene deletion. The 10 genes were inactivated singly, and multiple mutants were constructed by genetic crosses. lac6(Δ), lac8(Δ) and mco(Δ) mutants were significantly reduced in their ability to grow on lignin-containing materials, but also on cellulose and plastic. Furthermore, lac8(Δ), lac7(Δ), mco(Δ) and lac6(Δ) mutants were defective towards resistance to phenolic substrates and H2 O2 , which may also impact lignocellulose breakdown. Double and multiple mutants were generally more affected than single mutants, evidencing redundancy of function among laccases. Our study provides the first genetic evidences that laccases are major actors of wood utilization in a fungus and that they have multiple roles during this process apart from participation in lignin lysis.

Systematic deletion of homeobox genes in Podospora anserina uncovers their roles in shaping the fruiting body.

Higher fungi, which comprise ascomycetes and basidiomycetes, play major roles in the biosphere. Their evolutionary success may be due to the extended dikaryotic stage of their life cycle, which is the basis for their scientific name: the Dikarya. Dikaryosis is maintained by similar structures, the clamp in basidiomycetes and the crozier in ascomycetes. Homeodomain transcription factors are required for clamp formation in all basidiomycetes studied. We identified all the homeobox genes in the filamentous ascomycete fungus Podospora anserina and constructed deletion mutants for each of these genes and for a number of gene combinations. Croziers developed normally in these mutants, including those with up to six deleted homeogenes. However, some mutants had defects in maturation of the fruiting body, an effect that could be rescued by providing wild-type maternal hyphae. Analysis of mutants deficient in multiple homeogenes revealed interactions between the genes, suggesting that they operate as a complex network. Similar to their role in animals and plants, homeodomain transcription factors in ascomycetes are involved in shaping multicellular structures.

Crucial role of a dicarboxylic motif in the catalytic center of yeast RNA polymerases.

The catalytic center of yeast RNA polymerase II and III contains an acidic loop borne by their second largest subunit (Rpb2-(832)GYNQED(837), Rpc128-(764)GYDIED(769)) and highly conserved in all cellular and viral DNA-dependent RNA polymerases. A site-directed mutagenesis of this dicarboxylic motif reveals its strictly essential character in RNA polymerase III, with a slightly less stringent pattern in RNA polymerase II, where rpb2-E836Q and other substitutions completely prevent growth, whereas rpb2-E836A combines a dominant growth defect with severe lethal sectoring. A mild but systematic increase in RNA polymerase occupancy and a strict dependency on the transcript cleavage factor TFIIS (Dst1) also suggest a slower rate of translocation or higher probability of transcriptional stalling in this mutation. A conserved nucleotide triphosphate funnel domain binds the Rpb2-(832)GYNQED(837) loop by an Rpb2-R(1020)/Rpb2-D(837) salt-bridge. Molecular dynamic simulations reveal a second bridge (Rpb1-K(752)/Rpb2-E(836)), which may account for the critical role of the invariant Rpb2-E(836). Rpb2-E(836) and the funnel domain are not found among the RNA-dependent eukaryotic RNA polymerases and may thus represent a specific adaptation to double-stranded DNA templates.

Recombination proteins mediate meiotic spatial chromosome organization and pairing.

Meiotic chromosome pairing involves not only recognition of homology but also juxtaposition of entire chromosomes in a topologically regular way. Analysis of filamentous fungus Sordaria macrospora reveals that recombination proteins Mer3, Msh4, and Mlh1 play direct roles in all of these aspects, in advance of their known roles in recombination. Absence of Mer3 helicase results in interwoven chromosomes, thereby revealing the existence of features that specifically ensure "entanglement avoidance." Entanglements that remain at zygotene, i.e., "interlockings," require Mlh1 for resolution, likely to eliminate constraining recombinational connections. Patterns of Mer3 and Msh4 foci along aligned chromosomes show that the double-strand breaks mediating homologous alignment have spatially separated ends, one localized to each partner axis, and that pairing involves interference among developing interhomolog interactions. We propose that Mer3, Msh4, and Mlh1 execute all of these roles during pairing by modulating the state of nascent double-strand break/partner DNA contacts within axis-associated recombination complexes.

Non-canonical DNA transcription enzymes and the conservation of two-barrel RNA polymerases.

DNA transcription depends on multimeric RNA polymerases that are exceptionally conserved in all cellular organisms, with an active site region of >500 amino acids mainly harboured by their Rpb1 and Rpb2 subunits. Together with the distantly related eukaryotic RNA-dependent polymerases involved in gene silencing, they form a monophyletic family of ribonucleotide polymerases with a similarly organized active site region based on two double-Psi barrels. Recent viral and phage genome sequencing have added a surprising variety of putative nucleotide polymerases to this protein family. These proteins have highly divergent subunit composition and amino acid sequences, but always contain eight invariant amino acids forming a universally conserved catalytic site shared by all members of the two-barrel protein family. Moreover, the highly conserved 'funnel' and 'switch 2' components of the active site region are shared by all putative DNA-dependent RNA polymerases and may thus determine their capacity to transcribe double-stranded DNA templates.

Nonsense and missense mutations in FCY2 and FCY1 genes are responsible for flucytosine resistance and flucytosine-fluconazole cross-resistance in clinical isolates of Candida lusitaniae.

The aim of this work was to elucidate the molecular mechanisms of flucytosine (5FC) resistance and 5FC/fluconazole (FLC) cross-resistance in 11 genetically and epidemiologically unrelated clinical isolates of Candida lusitaniae. We first showed that the levels of transcription of the FCY2 gene encoding purine-cytosine permease (PCP) in the isolates were similar to that in the wild-type strain, 6936. Nucleotide sequencing of the FCY2 alleles revealed that 5FC and 5FC/FLC resistance could be correlated with a cytosine-to-thymine substitution at nucleotide 505 in the fcy2 genes of seven clinical isolates, resulting in a nonsense mutation and in a putative nonfunctional truncated PCP of 168 amino acids. Reintroducing a FCY2 wild-type allele at the fcy2 locus of a ura3 auxotrophic strain derived from the clinical isolate CL38 fcy2(C505T) restored levels of susceptibility to antifungals comparable to those of the wild-type strains. In the remaining four isolates, a polymorphic nucleotide was found in FCY1 where the nucleotide substitution T26C resulted in the amino acid replacement M9T in cytosine deaminase. Introducing this mutated allele into a 5FC- and 5FC/FLC-resistant fcy1Delta strain failed to restore antifungal susceptibility, while susceptibility was obtained by introducing a wild-type FCY1 allele. We thus found a correlation between the fcy1 T26C mutation and both 5FC and 5FC/FLC resistances. We demonstrated that only two genetic events occurred in 11 unrelated clinical isolates of C. lusitaniae to support 5FC and 5FC/FLC resistance: either the nonsense mutation C505T in the fcy2 gene or the missense mutation T26C in the fcy1 gene.