Journal of Fungi

Aspergillus fumigatus is a world-wide saprophyte filamentous fungus which released conidia, its infectious morphotype, in the atmosphere. These conidia are inhaled daily by humans and can colonize the respiratory tract, where they may develop into hyphae, the invasive morphotype. We previously showed that bronchial epithelial cells (BECs) restrict A. fumigatus virulence by inhibiting conidial germination and filament formation through a process requiring PI3K signaling and the conidial fucose-specific lectin FleA. In the present study, we are looking to identify host factors and cellular partners involved in the BEC antifungal response and to define the molecular interactions underpinning FleA recognition. For this, we analyzed transcriptome of BECs infected with A. fumigatus in the presence or absence of the PI3K inhibitor LY294002. Functional involvement of candidate genes was assessed by siRNA knockdown and readouts of fungal filamentation (microscopic scoring and galactomannan release). FleA-interacting host proteins were identified by biotin-FleA affinity co-precipitation coupled to Tandem mass spectrometry, and validated by surface plasmon resonance and biolayer interferometry. The spatiotemporal dynamics of FleA and candidate partners were analyzed by confocal microscopy and proximity ligation assay We demonstrated that BEC antifungal activity involves at least two complementary pathways: a PI3K/laminin-332 axis promoting conidial adhesion, and a FleA-dependent pathway engaging ITGB1 and MRC2 consistent with lectin uptake and trafficking toward LAMP1-positive compartments. These findings nominate FleA-host receptor interactions as attractive targets for anti-adhesive strategies against A. fumigatus. Author summaryFungal pathogens are an increasing threat to public health, as they are becoming more common and harder to treat due to rising drug resistance. Among them, Aspergillus fumigatus has been classified as a critical pathogen by the World Health Organization (WHO). This filamentous fungus delivers spores in the air daily, which are constantly inhaled by humans. In people with weakened immunity, these spores can cause a range of lung diseases known as aspergillosis, with severity ranging from mild to life-threatening. Lung epithelial cells are the first cells of the respiratory tract to encounter inhaled spores. In a previous study, we showed that bronchial cells can prevent spore from developing into filaments, the invasive form of A. fumigatus that is responsible for tissue damage. This protective effect depends of on the recognition of a fungal protein called FleA. In the present study, we identified host cell proteins that bind to FleA and transport it into intracellular compartments. Our findings suggest that these proteins help bronchial epithelial cells to internalize fungal spores, thereby blocking their transformation into the invasive filamentous form.

The sulfur metabolism is tightly regulated in cells. Cysteine, at physiological concentrations, plays a crucial role in protein assembly, as well as in coenzyme and metabolic intermediate synthesis. Additionally, cysteine is biotransformed into H2S, a gasotransmitter with several roles on cells, ranging from regulating mitochondrial metabolism to producing metabolic intermediates and mediating post-translational modification of proteins. While H2S has been shown to participate in the infection processes of animal pathogenic fungi, its role in phytopathogenic fungi remains unexplored. Here, we describe the conditions required to induce endogenous production of H2S in plant pathogenic fungi Ustilago maydis. Under these conditions, we observed an increased infection rate and more pronounced symptoms in maize plants. A label-free proteomic assay to examine adaptations of U. maydis under H2S-producing conditions shown an increased expression of extracellular enzymes required for virulence and mitochondrial proteins related to cellular respiration, ATP synthesis, and fatty acid degradation, along with enhanced expression of proteins involved in proteasomal degradation. Conversely, we found reduced expression of proteins associated with antioxidant responses, glycolysis, and the pentose phosphate pathway. These mitochondrial protein alterations correlated with increased mitochondrial biogenesis, ultrastructural changes, inhibition of the cytochrome respiratory pathway, and elevated activity of an alternative oxidase. Additionally, H2O2 production increased, while the enzymatic capacity for its detoxification decreased. Impaired lipid accumulation and altered intracellular distribution were also observed. Thus, in U. maydis, the modulation of cysteine metabolism regulates mitochondrial function, protein expression, lipid metabolism and infectious processes. Author SummaryUstilago maydis is a basidiomycete fungus that infects corn plants, inducing tumor formation. While in some countries this infection causes significant losses in maize crops, in Mexico, U. maydis, known as "huitlacoche," is celebrated as a culinary delicacy with important nutritional value. Additionally, it serves as an interesting model for studying infection by dimorphic phytopathogenic fungi. Animal models of fungal pathogenesis show that hydrogen sulfide (H2S), whether exogenously provided or induced by supplementation with its precursor cysteine, plays a role in infection processes in both the pathogen and the host cell. In this study, we explored the role of cysteine in the morphology, metabolism, and pathogenicity of U. maydis. Our findings indicate that cysteine treatment triggers an overproduction of H2S, alters mitochondrial morphology and nitrogen metabolism, and disrupts the oxidative balance in U. maydis. Furthermore, fungi to cysteine enhance tumor formation and anthocyanins accumulation in Zea mays plants. These findings suggest that H2S may play a key role in the infection efficiency of phytopathogenic fungi.

F. graminearum FgSte2 and FgSte3 are G-protein coupled receptors (GPCRs) recently shown to play roles in mediating fungal hyphal chemotropism and plant pathogenesis in response to activity arising from host-released peroxidases. Here, we follow up on the previous observation that chemotropism is dependent on both FgSte2 and FgSte3 being present at the same time; testing the possibility that this effect might be due to formation of an FgSte2-FgSte3 heterodimer. Initially the recombinant cell-surface expression of the F. graminearum GPCRs was validated in S. cerevisiae by confocal immunofluorescence microscopy. Bioluminescence resonance energy transfer analyses were subsequently conducted, where the addition of horse radish peroxidase (HRP) was found to increase the transfer of energy from the inducibly-expressed FgSte3-Nano luciferase (FgSte3-NLuc) donor, to the constitutively-expressed FgSte2-yellow fluorescent protein (FgSte2-YFP) acceptor, compared to controls. A partial response was also detected when an HRP-derived ligand-containing extract was enriched from F. graminearum spores and applied to the S. cerevisiae BRET system directly. The selectivity of the interaction was demonstrated by comparison to treatment with pheromones as well as an unrelated bovine GPCR, rhodopsin, fused to YFP as acceptor, that yielded no response when co-expressed with FgSte3-NLuc. Finally, the peroxidase-stimulated heterodimerization was validated by affinity pulldown. Taken together these findings demonstrate the formation of HRP and HRP-derived ligand stimulated heterodimers between FgSte2 and FgSte3. Outcomes are discussed from the context of the roles of ligands and reactive oxygen species in GPCR dimerization.

O_LIGrey mold disease affects fruits, vegetables and ornamental plants around the world, causing considerable losses every year. Its causing agent, the necrotrophic fungus Botrytis cinerea, produces infection cushions (IC) that are compound appressorial structures dedicated to the penetration of the plant tissues. C_LIO_LIA microarray analysis was performed to identify genes up-regulated in mature IC. The expression data were supported by RT-qPCR analysis performed in vitro and in planta, proteomic analysis of the IC secretome and mutagenesis of two candidate genes. C_LIO_LI1,231 up-regulated genes and 79 up-accumulated proteins were identified. They highlight a secretion of ROS, secondary metabolites including phytotoxins, and proteins involved in virulence: proteases, plant cell wall degrading enzymes and necrosis inducers. The role in pathogenesis was confirmed for two up-regulated fasciclin genes. DHN-melanin pathway and chitin deacetylases genes are up-regulated and the conversion of chitin into chitosan was confirmed by differential staining of the IC cell wall. In addition, up-regulation of sugar transport and sugar catabolism encoding genes was found. C_LIO_LIThese results support a role for the B. cinerea IC in plant penetration and suggest other unexpected roles for this fungal organ, in camouflage, necrotrophy or nutrition of the pathogen. C_LI

BackgroundThe COVID-19 pandemic has exposed patients to severe secondary fungal infections, exacerbating clinical outcomes and devastating impact. This study conducts a systematic review with meta-analysis of secondary fungal infections (SFIs) associated with COVID-19 considering various significant parameters, such as the frequency of SFIs across the globe, species shift, gender-specific infection rates, the significance of medical history, efficacy of steroid and antifungal, treatment outcomes (mortality rate), and fungal- polymicrobial mortality analysis. MethodsA literature search was conducted on COVID-19-related fungal infection studies (2020-2024) from SCOPUS and PUBMED databases, excluding preprints. The systematic data extraction captured the PMCID, country, patient demographics (age and gender), clinical outcomes, associated pathogens, medical history, and treatment details. FindingsThe global meta-analysis of COVID-19-associated SFIs yielded 10,700 cases across 58 countries, exhibiting a significant male predominance (65.6% vs. 34.3% female). Aspergillus spp., Candida spp., and Mucorales spp. emerged as the primary fungal pathogens. The predominant six countries marking 80 % of global cases include India (46.8 %), Italy (10 %), Iran (9.4 %), France (5.1 %), Spain (4.3 %), and Egypt (4.1 %). Complication rates revealed CAM as the most prevalent (59.2%), with a 28% mortality rate. CAC (21.6%) and CAPA (19.1%) had substantially higher mortality rates, at 54% and 58%, respectively. Specific populations were highly affected, including individuals with diabetes were prone to CAM, those undergone catheterization were at increased risk of CAC, and individuals with respiratory diseases or without prior medical history were susceptible to CAPA. In both CAC and CAPA, the species shift towards the non-albicans spp. and non- fumigatus spp., associated with higher mortality. In addition, polymicrobial infection with fungal pathogens (Aspergillus spp., Candida spp., Mucorales spp.) and Multi-bacteria ( K. pneumoniae, P. aeruginosa, E. coli, S. aureus) also increased the mortality rate. Effective treatments were identified, including combining caspofungin with corticosteroids for CAC, voriconazole with dexamethasone for CAPA, and AmBisome monotherapy for CAM. InterpretationIn SFIs populations, CAM prevailed in high-density areas with relatively lower mortality rates, whereas CAC and CAPA exhibited higher mortality rates. Notably, polymicrobial infections significantly increased mortality across all SFIs. Underlying medical conditions primarily influenced the type of fungal pathogen, but treatment outcomes varied. Azole drugs and Amphotericin-B were ineffective against Candidiasis, except for caspofungins limited susceptibility. Voriconazole and AmBisome demonstrated efficacy against Aspergillosis and Mucormycosis, respectively. Additionally, steroid administration proved life-saving in CAPA and CAC cases, yet remained ineffective in CAM. FundingNone Research in contextO_ST_ABSEvidence before this studyC_ST_ABSFive years after the COVID-19 pandemic, a plethora of research has investigated SFIs associated with COVID-19, with a major focus on pathogenicity, immunomodulation, and the impact of steroids and tocilizumab. However, a critical knowledge gap persists that addresses meta-analysis on the frequency of SFIs by country, gender-specific infection rates, the significance of medical history, species shift within the fungal kingdom, its virulence expression, polymicrobial infection dynamics, synergistic effects of steroids and antifungals in this context remains understudied. To address these gaps, the meta-analysis comprehensively examines these critical aspects, shedding light on various aspects of SFIs. Added value of this studyThe systematic meta-analysis of COVID-19-associated SFIs revealed the emergence of non-candida spp. and non-fumigatus spp. Polymicrobial infection has been linked to alarming outcomes resulting in a 100% mortality rate.Similarly, the co- infection of C. albicans with non-albicans spp. A. fumigatus with non-fumigatus spp. increased the mortality rate to 100%. For other species-related, effective therapies such as the combination of caspofungin and corticosteroids against Candidiasis (CAC), voriconazole and dexamethasone (CAPA), and AmBisome monotherapy (CAM) to combat SFIs. Implications of all the available evidenceThe geographic distribution of fungal pathogens varies globally, with differing mortality rates. Deciphering their genomic characteristics will unveil insights into behaviour, transmission, and virulence, enabling targeted diagnostics, treatments, and prevention strategies. MethodologyO_ST_ABSData collectionC_ST_ABSThe peer-reviewed published case studies, multicentric studies, retrospective studies, single-center studies and cohort studies represented with individuals case files were collected using search keywords "COVID-19 and Aspergillus", COVID-19 and Candida", COVID-19 and Mucorales", "COVID Associated Pulmonary Aspergillosis", "COVID Associated Mucormycosis", COVID Associated Candidiasis", in SCOPUS and PUBMED databases. Based on the search results, the articles from Aug 2020 to May 2024 were filtered excluding the preprint articles. A total of 1981 articles that included duplicates, articles unrelated to the study as well without abstracts were eliminated. A systematic review yielded 663 eligible publications, which were subjected to independent individual case meta- analysis. The distribution included 154 studies on CAC, 240 on CAPA, and 269 on CAM (Figure 1). From each article, the details of PMCID, country, age, gender, treatment outcome (live/dead), pathogens, medical history, and usage of steroids, antibacterial & antifungal were systematically collected. For the global surveillance of COVID-19-associated SFIs, the information from review, cohort, and retrospective studies was included. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=136 SRC="FIGDIR/small/24316125v1_fig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@1d32a1aorg.highwire.dtl.DTLVardef@7130aborg.highwire.dtl.DTLVardef@132653forg.highwire.dtl.DTLVardef@1953f00_HPS_FORMAT_FIGEXP M_FIG C_FIG Meta-analysis and its statisticsA systemic global survey was conducted for the 58 reported countries with COVID- 19-associated SFIs in accordance with PRISMA guidelines. The meta-analysis surveillance was conducted considering various significant parameters, such as frequency of SFIs across the globe, species shift, gender-specific infection rates, the significance of medical history, efficacy of steroid and antifungal, treatment outcomes (mortality rate), and fungal- polymicrobial mortality analysis. For the statistical analysis, Jamovi v2.6.2 tool and other tools were used as given below. The proportional meta-analysis was studied using a random effects model to quantify the distribution of SFIs attributed to Mucorales spp., Candida spp., and Aspergillus spp., across the countries. The analysis was considered with a 95% confidence interval (Cl). The analysis also evaluated the overall effect size and sample heterogeneity as indicated by the I{superscript 2} statistic. To investigate the frequency distribution of individual species within SFI, a ClinicoPath table one was employed. This statistical approach enabled the examination of the frequency range of specific species in respective SFIs. To estimate the mortality rates, a two-outcome proportion test was conducted, providing proportion values accompanied by 95 % CI. This study calculated the overall respective SFIs ( CAM , CAPA, and CAM) as well as for specific species. For instance, the mortality rate of A. fumigatus in CAPA was determined providing insight into species-specific outcomes, which enabled a detailed understanding of mortality rates across the various SFIs and their respective causative pathogens. A survival analysis was conducted to explore the interplay between gender, age, and species-specific conditions. The Long-rank, Gehan, and Tarone-Ware tests assessed differences in survival patterns. The analysis also estimated median age at risk for each species-specific condition, by gender, using cumulative hazard functions and 95% CI. A binomial logistic regression model was employed to assess the risk of treatment outcomes with species-specific pathogens. The model calculated the probability of successful treatment outcome, accompanied by standard errors (SE) and Z-scores. These metrics enabled the evaluation of the likelihood of treatment success or failure in correlation with specific pathogens. To provide the optimal treatment strategy, a Crosstable for dependent outcome analysis was performed to predict mortality rates for the treatment outcome. This final step enabled the identification of the most effective treatment approach by examining the intersection of treatment options and mortality rates, providing actionable insights for clinicians to make data-driven decisions for the management of SFIs.

The study of the paracoccin lectin (PCN) has provided knowledge about its role in the biology of Paracoccidioides brasiliensis and in the pathogenesis of paracoccidioidomycosis (PCM). In this context, PCN has proved to be a promising immunomodulatory agent for the exploration of vaccine target molecules and/or for diagnostic or therapeutic purposes. Previous investigations allowed establishing PCN as a factor of fungal virulence. However, the effect PCN exerts on the yeasts resistance to antifungal pharmacological agents used to treat human PCM are not known. Therefore, this work characterizes the role of PCN functional duality on virulence and susceptibility of P. brasiliensis to antifungal drugs. We show that the PCN overexpression increases the virulence of P. brasiliensis yeasts in an alternative model of infection, induces high susceptibility in vitro and in vivo of P. brasiliensis yeasts to antifungal therapy, and impact reducing relative mRNA expression of genes encoding proteins related to cell wall degradation. Conversely, PCN silencing minimized the yeasts virulence in Galleria mellonella, correlates with the lowest susceptibility to treatment with antifungal agent in vivo and impact differently from the PCN overexpression on the relative expression of markers related to P. brasiliensis yeasts cell wall remodelling. Our study demonstrates the impact of endogenous PCN on the P. brasiliensis yeasts virulence vs. susceptibility to antifungal drugs, the fungal biology, and the relationship of the yeasts-host cells.

Candidiasis is a fungal infection (mycosis) caused by an opportunistic yeast with only 8-10 pathogenic species. In spite of the multiple classes of antifungal drugs available to combat these infections, the treatment is hampered by drug toxicity, tolerability, emergence of drug resistant isolates and many more. Combination therapy has been suggested as a possible approach to improve these treatment outcomes. This study is, therefore, aimed at the analysis of the sensitivity of Candida isolates obtained from patients towards different classes of drugs affecting multiple pathways in the pathogen life cycle. Four different individual drugs with different mechanisms of action and six different combinations of these drugs at three different proportions (1:1, 1:3, and 3:1) were used for the study. All the drug sensitivity assays were performed by the agar well diffusion method. The results were statistically analyzed using Prism software. Results have shown synergistic effects of drug combinations on the sensitivity of the isolates without much variation in the stoichiometric ratios, thereby providing an alternative to monotherapy to combat the emergence of drug resistant isolates and drying existing drug pipelines.

Fungi of the order Pucciniales are obligate plant biotrophs causing rust diseases. They exhibit a complex life cycle with the production of up to five spore types, infection of two unrelated hosts and an overwintering stage. Transcription factors (TFs) are key regulators of gene expression in eukaryote cells. In order to better understand genetic programs expressed during major transitions of the rust life cycle, we surveyed the complement of TFs in fungal genomes with an emphasis on Pucciniales. We found that despite their large gene numbers, rust genomes have a reduced repertoire of TFs compared to other fungi. The proportions of C2H2 and Zinc cluster - two of the most represented TF families in fungi-indicate differences in their evolutionary relationships in Pucciniales and other fungal taxa. The cold shock protein (CSP) family showed a striking expansion in Pucciniomycotina with specific duplications in the order Pucciniales. The survey of expression profiles collected by transcriptomics along the life cycle of the poplar rust fungus revealed TF genes related to major biological transitions, e.g. response to environmental cues and host infection. Particularly, poplar rust CSPs were strongly expressed in basidia produced after the overwintering stage suggesting a possible role in dormancy exit. Expression during transition from dormant telia to basidia confirmed the specific expression of the three poplar rust CSP genes. Their heterologous expression in yeast improved cell growth after cold stress exposure, strengthening their implication in dormancy exit. This study addresses for the first time TF involved in developmental transition in the rust life cycle opening perspectives to further explore molecular regulation in the biology of the Pucciniales.

The pathogenic yeast Candida krusei is more distantly related to Candida albicans than clinically relevant CTG-clade Candida species. Its cell wall, a dynamic organelle that is the first point of interaction between pathogen and host, is relatively understudied, and its wall proteome remains unidentified to date. Here, we present an integrated study of the cell wall in C. krusei. Our comparative genomic studies and experimental data indicate that the general structure of the cell wall in C. krusei is similar to Saccharomyces cerevisiae and C. albicans and is comprised of {beta}-1,3-glucan, {beta}-1,6-glucan, chitin, and mannoproteins. However, some pronounced differences with C. albicans walls were observed, for instance, higher mannan and protein levels and altered protein mannosylation patterns. Further, despite absence of proteins with high sequence similarity to Candida adhesins, protein structure modeling identified eleven proteins related to flocculins/adhesins in S. cerevisiae or C. albicans. To obtain a proteomic comparison of biofilm and planktonic cells, C. krusei cells were grown to exponential phase and in static 24-h cultures. Interestingly, the 24-h static cultures of C. krusei yielded formation of floating biofilm (flor) rather than adherence to polystyrene at the bottom. The proteomic analysis of both conditions identified a total of 32 cell wall proteins. In line with a possible role in flor formation, increased abundance of flocculins, in particular Flo110, was observed in the floating biofilm compared to exponential cells. This study is the first to provide a detailed description of the cell wall in C. krusei including its cell wall proteome, and paves the way for further investigations on the importance of flor formation and flocculins in the pathogenesis of C. krusei. AUTHOR SUMMARYThe yeast Candida krusei is among the five most prevalent causal agents of candidiasis but its mechanisms underlying pathogenicity have been scarcely studied. This is also true for its cell wall structure, an essential organelle that governs primary host-pathogen interactions and host immune responses. Solid knowledge about cell wall synthesis and dynamics is crucial for the development of novel antifungal strategies against this pathogenic yeast. Here, through a combination of comparative genomics, protein structure modeling, and biochemical and proteomic analysis of purified walls, we present a detailed study of the cell wall composition in C. krusei and identify important architectural differences compared to C. albicans cell walls. Cell walls of C. krusei contain higher mannan and protein levels with altered mannan branching patterns, governed by expansions and reductions in gene families encoding mannosyltransferases. We also show that, in contrast to other Candida species, static cultures produce floating biofilms. Comparative wall proteomic studies of these biofilms show increased abundance of flocculins and hydrolytic enzymes, protein classes implicated in biofilm formation and primary host-pathogen interactions leading to tissue colonization. In conclusion, our study uncovers important keys towards a better molecular understanding of the virulence mechanisms of the important pathogen C. krusei.

Sterol regulatory element-binding proteins (SREBPs) are a family of transcription factors known to regulate sterol biosynthesis and homeostasis in fungi. For this reason they have a role in several biological processes, including virulence, fungicide tolerance, hypoxia adaptation, lipid and carbohydrate metabolisms, and iron homeostasis. While the biological function of SREBPs in yeast and filamentous fungal species pathogenic to humans and plants is known, their role in fungal biocontrol agents (BCAs) is still elusive. This study aimed to investigate the biological and regulatory function of SREBPs in the BCA Clonostachys rosea, with a focus on their role in fungicide tolerance, hypoxia adaptation and antagonisms. The C. rosea genome contains two genes (sre1 and sre2) coding for SREBPs and one gene each coding for Insulin induced gene (INSIG) and SREBP cleavage-activating protein (SCAP), required for SREBP-mediated ergosterol biosynthesis in fungi. Deletion of sre1 resulted in mutants with pleiotropic effects, including the reduced ability to grow on media supplemented with proline (active ingredient prothioconazole) and cantus (active ingredient boscalid) fungicides, hypoxia mimicking agent CoCl2, cell wall stressor SDS, and increased growth rate on medium supplemented with caffeine, compared with C. rosea wild type (WT). In addition, the antagonistic ability against the fungal hosts Botrytis cinerea and Rhizoctonia solani was affected when sre1 was deleted. However, no significant difference between sre2 deletion strains and C. rosea WT was found for any of the tested phenotypes. To investigate the regulatory role of SRE1, the transcriptome of C. rosea WT and a sre1 deletion strain was analyzed. The transcriptome analysis identified differentially expressed genes in the sre1 deletion strain associated with carbohydrate and lipid metabolism, respiration, iron homeostasis, and xenobiotic tolerance. Moreover, genes coding for polyketide synthases and chitinases with a proven antimicrobial role were downregulated in the mutant, corroborating the reduced antagonism phenotypes. In summary, this work sheds light on the regulation role of transcription factor SRE1 while also exploring its effect on regulating the antagonistic activity and fungicide resistance of C. rosea, giving us helpful knowledge to design applications of this organism in IPM strategies.

Candida albicans Prn1 is a protein that shares similarities with mammalian Pirin but with an unknown function in the yeast. Orthologues of Prn1 have been identified in other pathogenic fungi but not in Saccharomyces cerevisiae, suggesting a relationship with pathogenesis. Prn1 increase in abundance after H2O2 treatment has been shown previously, thus, in the present work, C. albicans prn1{Delta} mutant and the corresponding wild-type strain SN250 have been treated with H2O2 and their response was studied by quantitative differential proteomics. These assays indicated a lower increase of proteins with oxidoreductase activity after treatment in the prn1{Delta} strain compared to the wild type, as well as an increase in proteasome-activating proteins and a decrease in translation-involved proteins. Accordingly, Prn1 absence, under H2O2 treatment, led to a lower survival rate and a higher percentage of apoptosis, together with higher reactive oxygen species levels and higher proteasome activity. Besides, remarkable differences in the abundance of some transcription factors were observed between the two strains. Mnl1, involved in Prn1 expression, Bas1, Tiff33, and orf19.1150 presented an inverse pattern of expression under H2O2 treatment respect to Nrg1, a Mnl1 antagonist. Interestingly, orf19.4850, a protein orthologue to S. cerevisiae Cub1, has shown to be involved in the response to H2O2 presenting a conserved proteasome function. Under basal conditions, the proteomics results indicate a possible involvement of Prn1 in mitochondrial oxidative stress detoxication. Our experiments confirm Prn1 as a relevant actor in the oxidative response. ImportanceCandida albicans is a human opportunistic pathogen included in the WHO fungal priority pathogens list. The increase in resistant strains necessitates the discovery of new targets for antifungal therapies. Our research sheds light on the important role of the previously uncharacterized C. albicans protein Prn1 during the oxidative stress response. Study of the proteome remodelling under oxidative stress unveils the role of Prn1 in the decreased reactive oxygen species levels and the consequences, such as death by apoptosis and necrosis or cell growth delay. A proteomics approach allowed the identification of several proteins potentially involved in Prn1 activity, such as oxidoreductases and transcription factors. The lack of Prn1 orthologues in Saccharomyces cerevisiae but the presence in other Candida and Aspergillus species implicates this protein in pathogenesis and suggests that it may serve as a candidate for new drug targets.

In the following research, a novel antifungal culture method was discussed with the aim of developing bacterial culture media in which fungal growth is inhibited and bacterial growth is stimulated. This technique is based on the use of lactic acid as an antifungal agent, due to its capacity to neutralise the electrochemical gradient of fungal cell membranes, thus inhibiting their development. Based on this principle, the Minimum Inhibitory Concentration (MIC) of lactic acid in the culture media was investigated by changing its concentrations. After the preparation of the solutions, microbiological seeding was carried out in the different experimental groups. Data were collected using a quadrat sampling technique for subsequent statistical analysis using the T-test coefficient. As a result, the minimum inhibitory concentration of lactic acid was found to be 20%, where fungal growth was inhibited, and bacterial growth was stimulated. RESUMENEn la presente investigacion, se discutio acerca de un novedoso metodo de cultivo antifungico con la finalidad de desarrollar medios de cultivo bacterianos; en los que el crecimiento de hongos se viera inhibido y el bacteriano estimulado. Esta tecnica se basa en el uso del acido lactico como agente antifungico, debido a la capacidad de este acido para neutralizar el gradiente electroquimico de las membranas celulares de los hongos, inhibiendo asi su desarrollo. A partir de esta premisa, se investigo la Concentracion Inhibitoria Minima (CIM) del acido lactico en los medios de cultivo mediante un cambio en sus concentraciones. Posterior a la preparacion de las soluciones, se procedio a la siembra microbiologica en los diferentes grupos experimentales. Los datos fueron recolectados mediante una tecnica de muestreo por cuadrantes para su posterior analisis estadistico mediante el coeficiente de la Prueba T. Como resultado, se encontro que la concentracion inhibitoria minima del acido lactico fue del 20%, donde el desarrollo fungico se vio inhibido y el bacteriano estimulado. Published ElectronicallyO_LIBackground and Aims Lactic acid has been shown to have a negative effect on the chemical activity of fungal cell membranes and is therefore proposed to be used as an antifungal agent in growth media. C_LIO_LIMethods In the current study, an 88% lactic acid solution was used to produce agar solutions for culture media to assess the extent to which lactic acid can inhibit fungal growth. Subsequently, a quadrant sampling technique and a statistical study were used to measure the extent to which lactic acid can inhibit the growth of fungi. C_LIO_LIKey Results An inversely proportional correlation was found where lactic acid inhibited fungal growth. Specifically, the minimum inhibitory concentration of lactic acid was 20% and the results were supported by mathematical correlation and a statistical T-test, where 95% reliability was assumed. C_LIO_LIConclusions These results are consistent with the limited research into the molecular pathways of lactic acid in fungal organisms, which establishes, as in this research, that lactic acid negatively affects fungi and their development in culture media. C_LI

Witches broom disease (WBD) is a major constraint for cacao production in the Americas. The severe socioeconomic impact of WBD encouraged the evaluation of different control strategies, including the use of strobilurin fungicides. These molecules inhibit mitochondrial respiration, thus impairing ATP generation and leading to oxidative stress. These chemicals, however, have proven ineffective against the WBD pathogen Moniliophthora perniciosa. Here, we demonstrate that M. perniciosa tolerates high concentrations of strobilurins under in vitro conditions and highlight a set of molecular alterations that correlate with strobilurin tolerance in this fungus. Short-term exposure of M. perniciosa to the commercial strobilurin azoxystrobin led to the up-regulation of genes encoding enzymes of the glyoxylate cycle, gluconeogenesis, and fatty acid and amino acid catabolism, indicating that the fungal metabolism is remodeled to compensate for reduced ATP production. Furthermore, cell division, ribosome biogenesis, and sterol metabolism were repressed, which agrees with the impaired mycelial growth on azoxystrobin. Genes associated with cellular detoxification and response to oxidative stress (e.g., cytochrome P450s, membrane transporters and glutathione s-transferases) were strongly induced by the drug and represent potential strategies used by the pathogen to mitigate the toxic effects of the fungicide. Remarkably, exposure of M. perniciosa to azoxystrobin resulted in the spontaneous generation of a mutant with increased resistance to strobilurin. Comparative genomics and transcriptomics revealed alterations that may explain the resistance phenotype, including a large deletion in a putative transcriptional regulator and significant changes in the mutant transcriptome. Overall, this work provides important advances towards a comprehensive understanding of the molecular basis of strobilurin resistance in a tropical fungal pathogen. This is a fundamental step to efficiently employ these fungicides in agriculture and to prevent the emergence of strobilurin resistance.

BackgroundCytochalasans are a large family of fungal metabolites which inhibit actin polymerization and ultimately lead to a broad range of biological effects in different assays. Investigations into the biosynthesis of cytochalasans has revealed that the cytochrome P450 monooxygenase (P450s) tailoring enzymes possess a somewhat relaxed substrate-specificity and may accept structurally-related intermediates for oxidation, partly explaining the variety of structural variations observed in this family of molecules. In this study, we investigate a broad range of P450 enzymes via combinatorial biosynthesis to better understand their substrate scope and potential applications as biocatalysts. ResultsGenome mining enabled us to identify cryptic cytochalasan biosynthetic gene clusters (BGCs) in six different species of fungi, each with at least two P450 enzymes encoded. Comparative genomics identified a cryptic thioredoxin-like enzyme encoded in cytochalasan BGCs that co-occurs with the gene encoding a Baeyer-Villiger monooxygenase. Heterologous expression of seven P450s in Magnaporthe grisea mutant strains, lacking P450s required for pyrichalasin H biosynthesis, enabled functional characterization of three P450s, two of which were previously cryptic. The experimental results, combined with phylogenetic analysis of the P450 sequences, reveal subtle information regarding the structures of the associated cytochalasans and begins to explain why some P450s are inactive on the substrates available to them. ConclusionsThe P450 enzymes involved in cytochalasan biosynthesis are known to be site-selective in their native host but also possess intrinsic promiscuity due to being able to modify structurally-related analogues. By investigating a diverse set of P450s from characterized and cryptic BGCs, we were able to identify that the stereochemistry of functional groups around the cytochalasan backbone is more restrictive than the size of the macrocycle when introducing the P450 enzyme to non-native substrates.

The RSC and the homologous chromatin remodeling complexes are known to regulate cell cycle progression in various organisms, including Saccharomyces cerevisiae, Drosophila, and Homo sapiens. In this work, we characterized the role of two novel CTG clade-specific proteins (Nri1 and Nri2) of the RSC complex in the regulation of cell cycle progression in a critical priority fungal pathogen, Candida albicans. We observed that Nri1, alone or along with Nri2, regulates cell cycle progression at multiple stages. The nri1{Delta}/{Delta} and nri1{Delta}/{Delta} nri2{Delta}/{Delta} mutants exhibited transient cell cycle arrest, defective spindle morphology, and cytokinesis. Transcriptomic analysis supported these mutant phenotypes and indicated a broad role of Nri proteins in the cell cycle. From our results, we conclude that Nri proteins are crucial for C. albicans proliferation and fitness. ImportanceThe composition of the essential RSC chromatin remodeling complex exhibits species-specific divergence, harboring unique subunits with distinct functions. In this study, we report that two fungal CTG clade-specific proteins of the C. albicans RSC complex, namely Nri1 and Nri2 can promote C. albicans fitness through regulating its cell cycle progression at multiple stages. Fitness defect along with stressor sensitivity and differential expression of the genes regulating pathogenesis in the nri mutants indicate potentiality of the Nri proteins as anti-Candida drug targets.

Aspergillus fumigatus is a human fungal pathogen that causes a disease named aspergillosis. Echinocandins, such as the fungistatic drug caspofungin (CAS) are used as second-line therapy. Some A. fumigatus clinical isolates can survive and grow in higher CAS concentrations, a phenomenon known as "caspofungin paradoxical effect" (CPE). Here we investigate if CPE is due to a subpopulation of conidia produced by a CAS tolerant strain, indicative of a persistence phenotype or is caused by all the conidia which would be consistent with a tolerance phenotype. We evaluated 67 A. fumigatus clinical isolates for CPE growth and used a novel CPE Index (CPEI) classified them as CPE+ (CPEI [≥] 0.40) or CPE- (CPEI [≤] 0.20). Conidia produced by three CPE+ clinical isolates, CEA17 (CPEI=0.52), Af293 (CPEI=0.64), CM7555 (CPEI=0.58) all showed the ability to grow in high levels of CAS while all conidia produced by the CPE- isolate IFM61407 (CPEI=0.12) strain showed no evidence of tolerance. Given the importance of calcium/calcineurin/transcription factor CrzA pathway in CPE regulation, we also evaluated {Delta}crzAAf293 (CPE-) and {Delta}crzACEA17 (CPE+) conidia tolerance to CAS. All {Delta}crzACEA17 conidia showed CPE+ while 100 % of {Delta}crzAAf293 spores are CPE-. As all spores derived from an individual strain are phenotypically indistinct with respect to CPE it is likely that CPE is a genetically encoded adaptive trait that should be considered an antifungal tolerant phenotype. As the CPEI shows that the strength of the CPE is not uniform between strains we propose that the mechanisms that govern this phenomenon are multi-factorial.

ObjectivesCaspofungin is an echinocandin antifungal agent that inhibits synthesis of glucan required for the fungal cell wall. Resistance is mediated by mutation of Fks1 glucan synthase, among which S645P is the most common resistance-associated polymorphism. Rapamycin is a macrolide that inhibits the mechanistic target of rapamycin (mTOR) protein kinase activity. This study investigated the interaction between rapamycin and caspofungin in inhibiting the growth of wild type Candida albicans and Fks1 S645P mutant clinical isolate and wild type Candida lusitaniae and genetically engineered isogenic strain with Fks1 S645P mutation at equivalent position. MethodsInteractions between caspofungin and rapamycin were evaluated using the microdilution checkerboard method in liquid medium. The results were analysed using the fractional inhibitory concentration (FIC) index and the response surface (RS) analysis according to the Bliss model. ResultsSynergy between rapamycin and caspofungin was shown for C. albicans and C. lusitaniae strains by RS analysis of the checkerboard tests. Synergy was observed in strains sensitive and resistant to caspofungin. Weak subinhibitory concentrations of rapamycin were sufficient to restore caspofungin susceptibility. ConclusionsWe report here for the first time synergy between caspofungin and rapamycin in Candida species. Synergy was shown for strains susceptible and resistant to caspofungin. This study highlights the role of the TOR pathway in sensing antifungal-mediated cell wall stress and in modulating the cellular response to echinocandins in Candida yeasts.

Botrydial, botcinic acid and their derivatives are major phytotoxic metabolites produced by the necrotrophic fungal pathogen Botrytis cinerea. These phytotoxins are able to induce programmed cell death in the host and thereby promote plant susceptibility to B. cinerea. We observed that a{Delta} bot2{Delta}boa6 double mutant strain, which synthesizes neither botrydial nor botcinic acid, was almost avirulent on tomato leaves when the disease assay was performed using synthetic minimal Gamborg B5 medium. However, virulence of this mutant was restored when the inoculation medium was supplemented with yeast extract. Further virulence assays which compared the double mutant with other multiple mutants using both inoculation media, revealed a prominent contribution of botrydial and botcinic acid to the full virulence of B. cinerea. Therefore, we performed an RNA-sequencing experiment to identify B. cinerea genes that contribute to the phenotypic switch from an "incompatible" to a "compatible" interaction between tomato and the{Delta} bot2{Delta}boa6 double mutant. Four genes encoding cell death-inducing effector proteins were upregulated in B. cinerea by the addition of yeast extract, and their transcript profiles grouped within a co-expression module that was positively correlated with the compatible interaction. Functional analyses of these effector genes were performed by overexpressing them individually in the{Delta} bot2{Delta}boa6 background, followed by disease assays with the Gamborg B5 medium without yeast extract. ImportanceThe grey mould fungus Botrytis cinerea is a model for necrotrophic plant pathogens due to its wide host range, economic impact, well-assembled genome, and versatile mechanisms for inducing host cell death during colonization. Botrydial and botcinic acid have previously been characterized as major phytotoxins produced by B. cinerea. However, studies from different groups reported variable results regarding the contributions of these phytotoxins to fungal virulence. Here we demonstrate that botrydial and botcinic acid make a prominent contribution to the full virulence of B. cinerea, by performing infection assays with mutants that are defective in phytotoxin production and/or multiple cell death-inducing proteins using different inoculation media. This work highlights the pivotal roles of these phytotoxins as compared to other virulence factors, as well as the significant impact of inoculation conditions on compatible and incompatible interactions between the fungus and its hosts.

Candida auris is frequently associated with biofilm-related invasive infections. The resistant profile of these biofilms necessitates innovative therapeutic options, where quorum sensing may be a potential target. Farnesol and tyrosol are two fungal quorum-sensing molecules with antifungal effects at supraphysiological concentrations. To date there has been no high-throughput comparative molecular analysis regarding the background of farnesol- or tyrosol-related effects against C. auris biofilms. Here, we performed genome-wide transcript profiling with C. auris biofilms following 75 M farnesol or 15 mM tyrosol exposure using transcriptome sequencing (RNA-Seq). The analysis highlighted that the number of up-regulated genes (a minimum 1.5-fold increase) was 686 and 138 for tyrosol and farnesol, respectively, while 662 and 199 genes were down-regulated (a minimum 1.5-fold decrease) for tyrosol and farnesol, respectively. The overlap between tyrosol- and farnesol-responsive genes was considerable (101 and 116 overlapping up-regulated and down-regulated genes, respectively). Genes involved in biofilm events, glycolysis, ergosterol biosynthesis, fatty acid oxidation, iron metabolism, and autophagy were primarily affected in treated cells. Farnesol caused an 89.9%, 73.8%, and 32.6% reduction in the calcium, magnesium, and iron content, respectively, whereas tyrosol resulted an 82.6%, 76.6%, and 81.2% decrease in the calcium, magnesium, and iron content compared to the control, respectively. Moreover, the complexation of farnesol, but not tyrosol, with ergosterol is impeded in the presence of exogenous ergosterol, resulting in a minimum inhibitory concentration increase in the quorum-sensing molecules. This study revealed several farnesol- and tyrosol-specific responses, which will contribute to the development of alternative therapies against C. auris biofilms. ImportanceCandida auris is a multidrug-resistant fungal pathogen, which is frequently associated with biofilm related infections. Candida-derived quorum-sensing molecules (farnesol and tyrosol) play a pivotal role in the regulation of fungal morphogenesis and biofilm development. Furthermore, they may have remarkable anti-biofilm effects, especially at supraphysiological concentrations. Innovative therapeutic approaches interfering with quorum-sensing may be a promising future strategy against C. auris biofilms; however, limited data are currently available concerning farnesol-induced and tyrosol-related molecular effects in C. auris. Here, we detected several genes involved in biofilm events, glycolysis, ergosterol biosynthesis, fatty acid oxidation, iron metabolism, and autophagy, which were primarily influenced following farnesol or tyrosol exposure. Moreover, calcium, magnesium, and iron homeostasis were also significantly affected. These results reveal molecular events that provide definitive explanations for the observed anti-biofilm effect; furthermore, they support the development of novel therapeutic approaches against C. auris biofilms.

PurposeA new category of systemic co-infections that emerged with the COVID-19 pandemic is known as COVID-19-associated (CA) fungal infections, which include pulmonary aspergillosis (CAPA), candidiasis (CAC), and mucormycosis (CAM). We aimed to study the association between patient characteristics of hospitalized COVID-19 patients, COVID-19 comorbidities, and COVID-19 therapies with secondary non-superficial fungal infections. MethodsWe performed descriptive and regression analyses of data from 4,999 hospitalized COVID-19 patients from the University of Kentucky Healthcare (UKHC) system. ResultsThe patients with secondary systemic fungal infections had a 6-fold higher risk of death than those without such infections. Generally, the risk factors for severe COVID-19 (age, obesity, cardiovascular disease, diabetes, and lack of COVID-19 vaccination) were strong predictors of a secondary fungal infection. However, several characteristics had much higher risks, suggesting that a causative link may be at play: ICU admission, mechanical ventilation, length of hospital stay, and steroid use. ConclusionsIn sum, this study found that the known risk factors for severe COVID-19 disease, age, diabetes, cardiovascular disease, obesity, ventilation, and high steroid doses were all predictors of a secondary fungal infection. Steroid therapy may need to be modified to account for a risk or a presence of a fungal infection in vulnerable patients.