Pathogenic Filamentous Fungi Research Articles

Biochemical Identification of a Nuclear Coactivator Protein Required for AtrR-Dependent Gene Regulation in Aspergillus fumigatus.

Azole drugs represent the primary means of treating infections associated with the filamentous fungal pathogen Aspergillus fumigatus. A central player in azole resistance is the Zn2Cys6 zinc cluster-containing transcription factor AtrR. This factor stimulates expression of both the cyp51A gene, which encodes the azole drug target enzyme, as well as an ATP-binding cassette transporter-encoding gene called abcG1 (cdr1B). We used a fusion protein between AtrR and the tandem affinity purification (TAP) moiety to purify proteins that associated with AtrR from A. fumigatus. Protein fractions associated with AtrR-TAP were subjected to multidimensional protein identification technology mass spectrometry, and one of the proteins identified was encoded by the AFUA_6g08010 gene. We have designated this protein NcaA (for nuclear coactivator of AtrR). Loss of ncaA caused a reduction in voriconazole resistance and drug-induced abcG1 expression, although it did not impact induction of cyp51A transcription. We confirmed the association of AtrR and NcaA by coimmunoprecipitation from otherwise-wild-type cells. Expression of fusion proteins between AtrR and NcaA with green fluorescent protein allowed determination that these two proteins were localized in the A. fumigatus nucleus. Together, these data support the view that NcaA is required for nuclear gene transcription controlled by AtrR. IMPORTANCE Aspergillus fumigatus is a major filamentous fungal pathogen in humans and is susceptible to the azole antifungal class of drugs. However, loss of azole susceptibility has been detected with increasing frequency in the clinic, and infections associated with these azole-resistant isolates have been linked to treatment failure and worse outcomes. Many of these azole-resistant strains contain mutant alleles of the cyp51A gene, which encodes the azole drug target. A transcription factor essential for cyp51A gene transcription has been identified and designated AtrR. AtrR is required for azole-inducible cyp51A transcription, but we know little of the regulation of this transcription factor. Using a biochemical approach, we identified a new protein called NcaA that is involved in regulation of AtrR at certain target gene promoters. Understanding the mechanisms controlling AtrR function is an important goal in preventing or reversing azole resistance in this pathogen.

HIV-infected patients rarely develop invasive fungal diseases under good immune reconstitution after ART regardless high prevalence of pathogenic filamentous fungi carriage in nasopharynx/oropharynx.

We aimed to investigate the prevalence and risk factors of filamentous fungi (FF) carriage in human immunodeficiency virus (HIV)-infected patients in Guangdong province, along with its subsequent incidence of invasive fungal disease (IFD). Seven hundred and sixteen HIV-infected individuals from the outpatient clinic and 293 sex-matched healthy controls were recruited prospectively from May 1 to August 31, 2017. Fungi were isolated from oropharyngeal and nasopharyngeal swabs, then identified by morphological and molecular biological techniques. Logistic regression analysis was used to identify risk factors of pathogenic FF carriage. Pathogenic FF carriers were followed up through the end of 2019. Of the 716 included HIV-infected patients, 602 (84.1%) were male, the median age was 34 (27-42) years, and the median CD4+ count was 385 (254-542) cells/μl. Pathogenic FF were isolated in 119 (16.6%) cases with HIV infection and 40 (13.7%) healthy controls. Mucorales were found in 3 HIV-infected individuals and Talaromyces marneffei in 2 HIV-infected individuals, but not in healthy controls. History of cured opportunistic infections (OIs; OR, 1.97; 95% CI, 1.23-3.13, p = 0.004), and smoking (OR, 1.55; 95%CI, 1.03-2.32, p = 0.035) were independent risk factors of pathogenic FF carriage in HIV-infected individuals. A total of 119 pathogenic FF carriers with HIV infection were followed. During follow-up, 119 (100%) cases received antiretroviral therapy (ART) for at least 28 months, 107 (90%) cases had CD4+ counts>200 cells/μl, and none developed IFD. Pathogenic FF carriage is common in HIV-infected individuals but may not develop IFD in those who achieved immune reconstitution. Smoking and cured OIs history increase the risk of pathogenic FF carriage. Smoking abstinence and ART adherence are especially important for these patients.

In vitro antioxidant properties and antimicrobial activity of the ethanolic extract of Senecio nutans Sch. Beep. (Asteraceae)

Context: Senecio nutans Sch. Beep. is used by the Andean population of Ayacucho-Peru as traditional medicine for various health problems. Aims: To evaluate the antioxidant and antimicrobial activity of the ethanolic extract of leaves of S. nutans against the enteropathogen Escherichia coli and the filamentous fungus Aspergillus niger. Methods: The antioxidant capacity of the ethanolic extract was evaluated by ABTS, DPPH, and FRAP assays; the antimicrobial activity was evaluated by the agar diffusion method, and determining the minimum inhibitory concentration (MIC), minimum bactericidal (MBC), and fungicidal concentration (MFC). Results: Polyphenols, terpenes, and tannins were identified; the total polyphenolic content was 67.91 ± 0.29 mg GAE/g. The ethanolic extract at 20 mg/mL showed moderate antibacterial activity (79.14 ± 0.02% inhibition, MIC and MBC >400 μg/mL on E. coli), and antifungal (88.90 ± 0.17% inhibition, MIC and MCF >400 μg/mL on A. niger); radical scavenging capacity (ABTS 10.31 ± 0.09 mM TE/g; DPPH 8.28 ± 0.07 IC50 µg/mL) and iron reducing power (FRAP 17.72 ± 0.11 mM TE/g). S. nutans turned out to be a potential antimicrobial and antioxidant species associated with the presence of its bioactive components, for which further investigation is warranted. Conclusions: The ethanolic extract of leaves S. nutans showed moderate activity against pathogenic bacteria (E. coli) and filamentous fungus (A. niger), as well as antioxidant activity in three in vitro methods.

Antimicrobial Properties, Functional Characterisation and Application of Fructobacillus fructosus and Lactiplantibacillus plantarum Isolated from Artisanal Honey.

Honey is a valuable reservoir of lactic acid bacteria (LAB) and, particularly, of fructophilic LAB (FLAB), a relatively novel subgroup of LAB whose functional potential for human and food application has yet to be explored. In this study, FLAB and LAB strains have been isolated from honeys of different floral origins and selected for their broad antimicrobial activity against typical foodborne pathogenic bacteria and spoilage filamentous fungi. The best candidates, two strains belonging to the species Lactiplantibacillus plantarum and Fructobacillus fructosus, were submitted to partial characterisation of their cell free supernatants (CFS) in order to identify the secreted metabolites with antimicrobial activity. Besides, these strains were examined to assess some major functional features, including in vitro tolerance to the oro-gastrointestinal conditions, potential cytotoxicity against HT-29 cells, adhesion to human enterocyte-like cells and capability to stimulate macrophages. Moreover, when the tested strains were applied on table grapes artificially contaminated with pathogenic bacteria or filamentous fungi, they showed a good ability to antagonise the growth of undesired microbes, as well as to survive on the fruit surface at a concentration that is recommended to develop a probiotic effect. In conclusion, both LAB and FLAB honey-isolated strains characterised in this work exhibit functional properties that validate their potential use as biocontrol agents and for the design of novel functional foods. We reported antimicrobial activity, cytotoxic evaluation, probiotic properties and direct food application of a F. fructosus strain, improving the knowledge of this species, in particular, and on FLAB, more generally.

Genome editing using a versatile vector-based CRISPR/Cas9 system in Fusarium species

Fusarium species include important filamentous fungal pathogens that can infect plants, animals, and humans. Meanwhile, some nonpathogenic Fusarium species are promising biocontrol agents against plant pathogens. Here, we developed a genome editing technology using a vector-based CRISPR/Cas9 system for Fusarium oxysporum f. sp. lycopersici (Fol). This optimized CRISPR/Cas9 system, harboring an endogenous U6 small nuclear RNA promoter for the expression of single-guide RNA and an endogenous H2B nuclear localization signal for the localization of Cas9, enabled efficient targeted gene knock-out, including in the accessory chromosomal regions in Fol. We further demonstrated single crossover-mediated targeted base editing and endogenous gene tagging. This system was also applicable for genome editing in F. oxysporum f. sp. spinaciae and F. commune without any modifications, suggesting that this CRISPR/Cas9 vector has a potential application for a broad range of researches on other Fusarium species.

S4.1d In vitro activity of eight antifungals against 3343 filamentous pathogenic fungi collected during 18 years at the French National Reference Center for invasive mycosis and antifungals

S4.1 Treatment of rare mold infections in 2021: the role of new and old antifungals, September 22, 2022, 10:30 AM - 12:00 PM Background & ObjectivesThe NRCMA oversees the surveillance of invasive fungal diseases in France. As part of our expertise, we perform antifungal susceptibility testing based on European Committee on Antimicrobial Susceptibility Testing (EUCAST) methodology on all isolates. The antifungal profiles help us monitor the emergence of resistant isolates, determine the susceptibility pattern of wild-type strains to new antifungals, and in specific cases, determine the relationship between clinical failure and selection/emergence of a less susceptible isolate.We review the Minimum inhibitory concentrations (MIC) distribution of eight antifungals on clinical isolates of filamentous fungi identified at the NRCMA from 2003 to 2021.MethodsSpecies identification was performed by a combination of morphological features and multilocus sequencing. Only strains that produced enough conidia or spores were tested. In vitro susceptibility testing was performed according to the EUCAST procedure. Eight antifungal agents were used: triazoles [itraconazole, voriconazole, posaconazole, isavuconazole (since 2015)], echinocandins (caspofungin, micafungin), amphothericin B, and terbinafine. The concentrations inhibiting 50% (MIC50) and 90% (MIC90) of the isolates were determined for species with at least 5 and 10 isolates, respectively. For Aspergillus fumigatus isolates exhibiting high azole MICs, we sequenced the cyp51A gene for mutation screening.ResultsMICs were obtained for 3343 pathogenic strains. We identified Aspergillus spp. including cryptic species (32%), Fusarium spp. (21%), Mucorales (18%), phaeohyphomycetes (10%), i.e., Alternaria spp., Curvularia spp., Fonsecaea, Exophiala, Phaeoacremonium, rare hyphomycetes (9%), i.e., Rasamsonia, Paecilomyces, Trichoderma, Scopulariopsis. Scedosporium spp. (7%) and emergent pathogens such as Nannizziopsis obscura (1%), dimorphic fungi (1%), or to a less extent basidiomycetous molds. Examples of MIC results are presented in Table 1.For the nine genera of the order Mucorales, amphotericin B (AmB) exhibited low MICs except for Cunninghamella species and Saksenaea vasiformis. Posaconazole had variable activity depending on the species (high MIC values were observed for Mucor species) while voriconazole and echinocandins had none.In section Fumigati, amphotericin B MIC values were high due to the intrinsic resistance of cryptic species Aspergillus lentulus and A. udagawae. Other cryptic species were identified such as A. hiratsukae isolates which exhibited low MICs to all antifungals.Members of Aspergillus section Nidulantes such as A. nidulans and A. quadrilineatus showed variable MICs to caspofungin and amphotericin B.Among melanized fungi, Alternaria infectoria and A. alternata species group displayed low MICs of all azoles with the exception of voriconazole.Four Exophiala species were analyzed and had similar azole MICs, E. spinifera being the species with the lowest values.Multi-drug resistant profiles were observed especially in species belonging to Microascales such as Lomentospora prolificans, Microascus cirrosus, and Scopulariopsis brevicaulis. Members of Fusarium species complexes had predictably high MIC values to all antifungals including isavuconazole. Only Fusarium dimerum species complex had low MICs to amphotericin B.Further distribution analysis is ongoing. These large datasets provide a baseline for monitoring the emergence of antifungal resistance in our country and supports the fact that MIC determination should be performed for rare/emergent species and also in case of infections due to cryptic species, which would benefit patient management.

Biofilm Formation and Structure in the Filamentous Fungus Fusarium graminearum, a Plant Pathogen.

ABSTRACTBiofilms are protective structures for pathogens of plants and animals, in which cells are shielded from host defense responses and antimicrobial treatments. Although biofilms are well studied in bacterial pathogens, their development and structure in filamentous fungi, as well as their role in pathogenicity, are poorly understood. We show that the economically important plant pathogen Fusarium graminearum, a filamentous fungus, forms biofilms in vitro, which adhere to polystyrene, a hydrophobic surface. The biofilms have complex hyphal structures surrounded by a polymeric matrix that consists primarily of polysaccharides and extracellular nucleic acids, and lack lipids. Pellicles are formed in liquid cultures, floating biofilm masses that are common in bacterial biofilms, and noted but undescribed in filamentous fungal biofilms. Commonly, F. graminearum grows as hyphal colonies; however, on media which lack electron acceptors, an altered morphology is formed with predominantly short, bulbous hyphae embedded in the matrix. Supplementation of the biofilm-inducing medium with an electron acceptor restores the filamentous hyphal morphology, demonstrating that the formation of bulbous hyphae is due, at least in part, to oxidative stress. Plant hosts infected with pathogens generally respond by producing reactive oxygen species, commonly produced as a defense response. Thus, the formation of biofilms strongly suggests a role in protecting cells from host responses during the course of plant disease.IMPORTANCE Fusarium graminearum is a filamentous fungal pathogen that causes Fusarium head blight (FHB) in cereal crops, leading to devastating crop losses. We have demonstrated the ability of this pathogen to form biofilms. Biofilms are likely to be important in the disease cycle of F. graminearum and other plant pathogens, protecting cells from plant defenses and environmental conditions. Towards this end, we have characterized the formation of biofilms in F. graminearum in vitro, which, together with ongoing characterization of their association with host plants, provides a basis for understanding the functionality of biofilms in the pathogen disease cycle.

Identification and antifungal susceptibility profile of filamentous fungi isolated from cases of healthcare associated infections

Although, bacteria have been considered as most common cause of healthcare associated infections (HCAI), however recent years have witnessed increased isolation of fungal pathogens. species, Mucorales, spp., spp. and spp. are predominant fungal pathogens isolated from cases of HCAI. As compared to bacterial nosocomial infections, fungal infections are generally difficult to diagnose and treat. Nosocomial mycoses are associated with high mortality and morbidity. The present study was conducted in the tertiary care academic hospital with an aim to identify filamentous fungal pathogen isolated from HCAI and study its antifungal susceptibility profile. Filamentous fungi isolated from cases of HCAI from intensive care unit (MICU) were included. They were identified by standard mycological techniques and antifungal susceptibility profile was studied. Out of 50 fungal pathogens, spp. were isolated from 21 (42%) cases, whereas 29 (58%) isolates were filamentous fungi. spp. (51.7%) were predominant among filamentous fungal pathogen. (27.6%) was the predominant isolate. Use of broad-spectrum antibiotics followed by neutropenia and presence of indwelling medical devices were common risk factors associated with HCAI due to filamentous fungi. Amphotericin B resistance was observed in 6.9% of isolates whereas 33.3% were resistant to fluconazole. Invasive fungal infection (IFI) though less common is associated with increased morbidity and mortality in patients admitted to ICU. Neutropenia and use of broad spectrum antibiotics are important risk factors for IFI. is the most common filamentous fungus cause of IFI. More emphasis should be given on rapid diagnosis, prompt treatment and strict compliance with infection prevention and control practices.

Isolation and Characterization of Dermatophytes from Students Bathing Places

Background: Dermatophyte comprises of a vast range of filamentous pathogenic fungi that infect keratinous tissues of skin (the stratum corneum layer), hair, and nail in humans and animals via their keratinase enzymes which are capable of causing superficial infections. Aim: This study aimed to isolate and characterized dermatophytes isolated from students bathing places. Methodology: Four sampling sites were chosen from both male and female students bathing places; the floor, walls, sinks and door handles. Three hundred samples were collected and cultured on sterile plates containing Potato dextrose agar using spread plate method. The plates were incubated at 350C for 72 hours. Results: Out of three hundred samples collected from the various sampling sites, a total of four hundred and ten fungi were isolated, 286 (70%) were dermatophytes belonging to three genera (Trichophyton, Microsporum and Epidermorphyton) while124 (30%) were non dermatophytes. The female bathing places yielded 167(58%) and 48(39%) dermatophytes and Candida species respectively higher than male counterparts which yielded 119 (42%) dermatophytes without any species of Candida. There was no significant difference between the isolates from female and male bathrooms P ≥ 0.05. Conclusion: Hence the students bathing places were found to be rich in pathogenic and potentially pathogenic fungi and need thorough disinfectants to avoid dermatophytes infections’ like Tinea pedis, Tinea corporis Tinea capitis and Tinea cruris.

Differential Roles of Three α-Crystallin Domain-Containing sHsps of Beauveria bassiana in Asexual Development, Multiple Stress Tolerance and Virulence.

Small heat shock proteins (sHsps) containing conserved α-crystallin domain play important roles in many cellular processes, but little is known about the functions of sHsps in filamentous entomopathogens. Here, three sHsps of Hsp20, Hsp30a, and Hsp30b were characterized in Beauveria bassiana, a filamentous fungal insect pathogen that serves as the main source of wide-spectrum fungal insecticides. The results demonstrated that these three genes are interrelated at the transcriptional level under normal and heat-shocked conditions. Meanwhile, all the deletion mutants showed significant but differential changes in cell wall integrity, antioxidant activity, hyphal tolerance to carbendazim fungicide, conidial tolerance to 45 °C wet heat and virulence. However, only Δhsp30b showed growth defects on rich and minimal media at 25 °C and Δhsp30a displayed the reduction in conidiophores and conidia. Moreover, the single deletion of hsp30a and hsp30b caused the decreases in hyphal growth at 34 °C and conidial tolerance to UV-B irradiation. Our findings provide a global insight into vital roles of hsp20, hsp30a, and hsp30b in asexual development, environmental adaptation, and fungal virulence of B. bassiana.

The Endoplasmic Reticulum Cargo Receptor FgErv14 Regulates DON Production, Growth and Virulence in Fusarium graminearum.

Fusarium graminearum is a plant filamentous pathogenic fungi and the predominant causal agent of Fusarium head blight (FHB) in cereals worldwide. The regulators of the secretory pathway contribute significantly to fungal mycotoxin synthesis, development, and virulence. However, their roles in these processes in F. graminearum remain poorly understood. Here, we identified and functionally characterized the endoplasmic reticulum (ER) cargo receptor FgErv14 in F. graminearum. Firstly, it was observed that FgErv14 is mainly localized in the ER. Then, we constructed the FgErv14 deletion mutant (ΔFgerv14) and found that the absence of the FgErv14 caused a serious reduction in vegetative growth, significant defects in asexual and sexual reproduction, and severely impaired virulence. Furthermore, we found that the ΔFgerv14 mutant exhibited a reduced expression of TRI genes and defective toxisome generation, both of which are critical for deoxynivalenol (DON) biosynthesis. Importantly, we found the green fluorescent protein (GFP)-tagged FgRud3 was dispersed in the cytoplasm, whereas GFP-FgSnc1-PEM was partially trapped in the late Golgi in ΔFgerv14 mutant. These results demonstrate that FgErv14 mediates anterograde ER-to-Golgi transport as well as late secretory Golgi-to-Plasma membrane transport and is necessary for DON biosynthesis, asexual and sexual reproduction, vegetative growth, and pathogenicity in F. graminearum.

Regulatory Roles of Histone Modifications in Filamentous Fungal Pathogens.

Filamentous fungal pathogens have evolved diverse strategies to infect a variety of hosts including plants and insects. The dynamic infection process requires rapid and fine-tuning regulation of fungal gene expression programs in response to the changing host environment and defenses. Therefore, transcriptional reprogramming of fungal pathogens is critical for fungal development and pathogenicity. Histone post-translational modification, one of the main mechanisms of epigenetic regulation, has been shown to play an important role in the regulation of gene expressions, and is involved in, e.g., fungal development, infection-related morphogenesis, environmental stress responses, biosynthesis of secondary metabolites, and pathogenicity. This review highlights recent findings and insights into regulatory mechanisms of histone methylation and acetylation in fungal development and pathogenicity, as well as their roles in modulating pathogenic fungi–host interactions.

Fungal Infections as an Uprising Threat to Human Health: Chemosensitization of Fungal Pathogens With AFP From Aspergillus giganteus.

Occurrence and intensity of systemic invasive fungal infections have significantly risen in recent decades with large amount of mortality and morbidity rates at global level. Treatment therapy lies on the current antifungal interventions and are often limited due to the emergence of resistance to antifungal agents. Chemosensitization of fungal strains to the conventional antimycotic drugs are of growing concern. Current antifungal drugs often have been reported with poor activity and side effects to the host and have a few number of targets to manifest their efficacy on the pathogens. Indiscriminately, the aforementioned issues have been easily resolved by the development of new intervention strategies. One such approach is to employ combinational therapy that has exhibited a great level of inhibitions than that of a single compound. Chemosensitization of pathogenic mycoses to commercial antifungal drugs could be drastically enhanced by co-application of chemosensitizers along with the conventional drugs. Chemosensitizers could address the resistance mechanisms evolved in the pathogenic fungi and targeting the system to make the organism susceptible to commercially and clinically proven antifungal drugs. However, this strategy has not been overreached to the greater level, but it needs much attention to fight against not only with the pathogen but combat the resistance mechanisms of pathogens to drugs. Natural compounds including plant compounds and microbial proteins act as potential chemosensitizers to break the resistance in mycoses. Aspergillus giganteus, a filamentous fungus, is known to produce a cysteine rich extracellular protein called as antifungal protein (AFP). AFP has shown enhanced efficacy against several filamentous and non-filamentous fungal pathogens. On the basis of the reported studies on its targeted potential against pathogenic mycoses, AFP would be fabricated as a good chemosensitizer to augment the fungicidal efficacy of commercial antimycotic drugs. This paper reviews on breakthrough in the discovery of antifungal drugs along with the resistance patterns of mycoses to commercial drugs followed by the current intervention strategies applied to augment the fungicidal potential of drugs.

Systematic contributions of CFEM domain-containing proteins to iron acquisition are essential for interspecies interaction of the filamentous pathogenic fungus Beauveria bassiana.

Common in fungal extracellular membrane (CFEM) domain is unique in fungal proteins and some of which contribute to iron acquisition in yeast. However, their roles in iron acquisition remain largely unknown in filamentous fungi. In this study, 12 CFEM-containing proteins were bioinformatically identified in the filamentous entomopathogenic fungus Beauveria bassiana, and the roles of 11 genes were genetically characterized. Transmembrane helices were critical for their association with intracellular membranes, and their number varied among proteins. Eleven CFEM genes significantly contribute to vegetative growth under iron starvation and virulence. Notably, the virulence of most disruptants could be significantly weakened by a decrease in iron availability, in which the virulence of ΔBbcfem7 and 8 strains was partially recovered by exogenous hemin. ΔBbcfem7 and 8 mutants displayed defective competitiveness against the sister entomopathogenic fungus Beauveria brongniartii. All 11 disruptants displayed impaired growth in the antagonistic assay with the saprotrophic fungus Aspergillus niger, which could be repressed by exogenous ferric ions. These findings not only reveal the systematic contributions of CFEM proteins to acquire two forms of iron (i.e. heme and ferric ion) in the entire lifecycle of entomopathogenic fungi but also help to better understand the mechanisms of fungus-host and inter-fungus interactions.

Antimicrobial Efficacy of Edible Mushroom Extracts: Assessment of Fungal Resistance

Antimicrobial efficacy of the water or methanolic extracts of three medicinal mushrooms Taiwanofungus camphoratus, Agaricus blazei Murrill, and Ganoderma lucidum (Curtis) P. Karst were investigated against yeast and filamentous fungal pathogens as well as against commensal and pathogenic bacteria. The methanolic extract of T. camphoratus (TcM) exhibited both potent antifungal and antibacterial activity, while the water extract of T. camphoratus (TcW) showed limited antibacterial activity against Listeria monocytogenes. Neither the methanolic nor water extracts of A. blazei and G. lucidum exhibited antimicrobial activity. In the risk assessment testing monitoring the development of fungal tolerance to mushroom extracts in food matrices, two P. expansum mitogen-activated protein kinase (MAPK) mutants exhibited a tolerance to TcM. In a proof-of-concept bioassay using the natural benzoic salicylaldehyde (SA), P. expansum and A. fumigatus MAPK antioxidant mutants showed similar tolerance to SA, suggesting that natural ingredients in TcM such as benzoic derivatives could negatively affect the efficacy of TcM when antioxidant mutants are targeted. Conclusion: TcM could be developed as a food ingredient having antimicrobial potential. The antimicrobial activity of TcM operates via the intact MAPK antioxidant signaling system in microbes, however, mutants lacking genes in the MAPK system escape the toxicity triggered by TcM. Therefore, caution should be exercised in the use of TcM so as to not adversely affect food safety and quality by triggering the resistance of antioxidant mutants in contaminated food.

Differential Functions of Individual Transcription Factor Binding Sites in the Tandem Repeats Found in Clinically Relevant cyp51A Promoters in Aspergillus fumigatus.

ABSTRACTAspergillus fumigatus is the major filamentous fungal pathogen in humans. The gold standard treatment of A. fumigatus is based on azole drug use, but the appearance of azole-resistant isolates is increasing at an alarming rate. The cyp51A gene encodes the enzymatic target of azole drugs, and azole-resistant alleles of cyp51A often have an unusual genetic structure containing a duplication of a 34- or 46-bp region in the promoter causing enhanced gene transcription. These tandem repeats are called TR34 and TR46 and produce duplicated binding sites for the SrbA and AtrR transcription factors. Using site-directed mutagenesis, we demonstrate that both the SrbA (sterol response element [SRE]) and AtrR binding sites (AtrR response element [ATRE]) are required for normal cyp51A gene expression. Loss of either the SRE or ATRE from the distal 34-bp repeat of the TR34 promoter (further 5′ from the transcription start site) caused loss of expression of cyp51A and decreased voriconazole resistance. Surprisingly, loss of these same binding sites from the proximal 34- or 46-bp repeat led to increased cyp51A expression and voriconazole resistance. These data indicate that these duplicated regions in the cyp51A promoter function differently. Our findings suggest that the proximal 34- or 46-bp repeat in cyp51A recruits a corepressor that requires multiple factors to act while the distal repeat is free of this repression and provides the elevated cyp51A expression caused by these promoter duplications.

Cell Wall Integrity and Its Industrial Applications in Filamentous Fungi.

Signal transduction pathways regulating cell wall integrity (CWI) in filamentous fungi have been studied taking into account findings in budding yeast, and much knowledge has been accumulated in recent years. Given that the cell wall is essential for viability in fungi, its architecture has been analyzed in relation to virulence, especially in filamentous fungal pathogens of plants and humans. Although research on CWI signaling in individual fungal species has progressed, an integrated understanding of CWI signaling in diverse fungi has not yet been achieved. For example, the variety of sensor proteins and their functional differences among different fungal species have been described, but the understanding of their general and species-specific biological functions is limited. Our long-term research interest is CWI signaling in filamentous fungi. Here, we outline CWI signaling in these fungi, from sensor proteins required for the recognition of environmental changes to the regulation of cell wall polysaccharide synthesis genes. We discuss the similarities and differences between the functions of CWI signaling factors in filamentous fungi and in budding yeast. We also describe the latest findings on industrial applications, including those derived from studies on CWI signaling: the development of antifungal agents and the development of highly productive strains of filamentous fungi with modified cell surface characteristics by controlling cell wall biogenesis.

The Caspofungin Paradoxical Effect is a Tolerant "Eagle Effect" in the Filamentous Fungal Pathogen Aspergillus fumigatus.

ABSTRACTCell responses against antifungals other than resistance have rarely been studied in filamentous fungi, while terms such as tolerance and persistence are well-described for bacteria and increasingly examined in yeast-like organisms. Aspergillus fumigatus is a filamentous fungal pathogen that causes a disease named aspergillosis, for which caspofungin (CAS), a fungistatic drug, is used as a second-line therapy. Some A. fumigatus clinical isolates can survive and grow in CAS concentrations above the minimum effective concentration (MEC), a phenomenon known as “caspofungin paradoxical effect” (CPE). Here, we evaluated the CPE in 67 A. fumigatus clinical isolates by calculating recovery rate (RR) values, where isolates with an RR of ≥0.1 were considered CPE+ while isolates with an RR of <0.1 were classified as CPE–. Conidia produced by three CPE+ clinical isolates, CEA17 (RR = 0.42), Af293 (0.59), and CM7555 (0.38), all showed the ability to grow in high levels of CAS, while all conidia produced by the CPE– isolate IFM61407 (RR = 0.00) showed no evidence of paradoxical growth. Given the importance of the calcium/calcineurin/transcription factor-CrzA pathway in CPE regulation, we also demonstrated that all ΔcrzACEA17 (CPE+) conidia exhibited CPE while 100% of ΔcrzAAf293 (CPE–) did not exhibit CPE. Because all spores derived from an individual strain were 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. Because the RR parameter showed that the strength of the CPE was not uniform between strains, we propose that the mechanisms which govern this phenomenon are multifactorial.

Natural Salicylaldehyde for Fungal and Pre- and Post-Emergent Weed Control

A sustainable, alternative weed control strategy is developed using salicylaldehyde (SA; 2-hydroxybenzaldehyde) as an active ingredient. SA is a natural, redox-active small molecule listed as a Generally Recognized As Safe food additive by the European Food Safety Authority and the United States Food and Drug Administration. The repurposing of SA determined that SA possesses both pre- and post-emergent herbicidal, fumigant activity, where the emitted SA from the source completely prevented the germination of plant seeds and/or the growth of the germinated plants. As a proof-of-concept, we developed agricultural byproducts (tree nutshell particles) as SA delivery vehicles to the soil, thus helping the growers’ sustainable byproduct recycling program, necessary for carbon sequestration. In plate assays, SA emitted from the nutshell vehicles (0.15 to 1.6 M) completely prevented the germination of six invasive or native weed seeds (monocots, dicots). In Magenta vessel assays, SA emitted from the nutshell vehicles (0.8 to 1.6 M) not only prevented the germination (pre-emergent) of Lagurus ovatus (Bunny Tails Grass) seeds but also inhibited the growth (post-emergent) of the germinated weeds. We determined further that soil covering (soil pasteurization) could be one of the practices to effectively deliver SA to the soil, whereby 1.6 M of SA emitted from the nutshell vehicles prevented the germination of the L. ovatus seeds maintained in soil trays covered with plastic tarp at 22 °C, while 0.8 M SA allowed partial (15%) germination of the weed seeds. Of note, SA also possesses an intrinsic antifungal activity that overcomes the tolerance of the stress signaling mutants of filamentous fungal pathogens (Aspergillus fumigatus, Penicillium expansum) to the phenylpyrrole fungicide fludioxonil. Environmental degradation data available in the public database indicate that, once released to the environment, SA will be broken down in the air by sunlight or microorganisms and, thus, is not built up in aquatic organisms. Altogether, SA can serve as a safe, potent pesticide (herbicidal, fungicidal) ingredient that promotes sustainable crop production by lowering the pesticide burden in fields.

Crystal structure of ArOYE6 reveals a novel C-terminal helical extension and mechanistic insights into the distinct class III OYEs from pathogenic fungi.

Old yellow enzymes (OYEs) play a critical role in antioxidation, detoxification and ergot alkaloid biosynthesis processes in various organisms. The yeast- and bacteria-like OYEs have been structurally characterized earlier, however, filamentous fungal pathogens possess a novel OYE class, that is, class III, whose biochemical and structural intricacies remain unexplored to date. Here, we present the 1.6 Å X-ray structure of a class III member, OYE 6 from necrotrophic fungus Ascochyta rabiei (ArOYE6), in flavin mononucleotide (FMN)-bound form (PDB ID-7FEV) and provide mechanistic insights into their catalytic capability. We demonstrate that ArOYE6 exists as a monomer in solution, forms (β/α)8 barrel structure harbouring non-covalently bound FMN at cofactor binding site, and utilizes reduced nicotinamide adenine dinucleotide phosphate as its preferred reductant. The large hydrophobic cavity situated above FMN, specifically accommodates 12-oxo-phytodienoic acid and N-ethylmaleimide substrates as observed in ArOYE6-FMN-substrate ternary complex models. Site-directed mutations in the conserved catalytic (His196, His199 and Tyr201) and FMN-binding (Lys249 and Arg348) residues render the enzyme inactive. Intriguingly, the ArOYE6 structure contains a novel C-terminus (369-445 residues) made of three α-helices, which stabilizes the FMN binding pocket as its mutation/truncation lead to complete loss of FMN binding. Moreover, the loss of the extended C-terminus does not alter the monomeric nature of ArOYE6. In this study, for the first time, we provide the structural and biochemical insights for a fungi-specific class III OYE homologue and dissect the oxidoreductase mechanism. Our findings hold broad biological significance during host-fungus interactions owing to the conservation of this class among pathogenic fungi, and would have potential implications in the pharmacochemical industry.