Antifungal Targets Research Articles

Innovations in Antifungal Drug Discovery among Cell Envelope Synthesis Enzymes through Structural Insights.

Life-threatening systemic fungal infections occur in immunocompromised patients at an alarming rate. Current antifungal therapies face challenges like drug resistance and patient toxicity, emphasizing the need for new treatments. Membrane-bound enzymes account for a large proportion of current and potential antifungal targets, especially ones that contribute to cell wall and cell membrane biosynthesis. Moreover, structural biology has led to a better understanding of the mechanisms by which these enzymes synthesize their products, as well as the mechanism of action for some antifungals. This review summarizes the structures of several current and potential membrane-bound antifungal targets involved in cell wall and cell membrane biosynthesis and their interactions with known inhibitors or drugs. The proposed mechanisms of action for some molecules, gleaned from detailed inhibitor-protein studeis, are also described, which aids in further rational drug design. Furthermore, some potential membrane-bound antifungal targets with known inhibitors that lack solved structures are discussed, as these might be good enzymes for future structure interrogation.

Allosteric inhibition of tRNA synthetase Gln4 by N-pyrimidinyl-β-thiophenylacrylamides exerts highly selective antifungal activity

Candida species are among the most prevalent causes of systemic fungal infections, which account for ∼1.5 million annual fatalities. Here, we build on a compound screen that identified the molecule N-pyrimidinyl-β-thiophenylacrylamide (NP-BTA), which strongly inhibits Candida albicans growth. NP-BTA was hypothesized to target C.albicans glutaminyl-tRNA synthetase, Gln4. Here, we confirmed through invitro amino-acylation assays NP-BTA is a potent inhibitor of Gln4, and we defined how NP-BTA arrests Gln4's transferase activity using co-crystallography. This analysis also uncovered Met496 as a critical residue for the compound's species-selective target engagement and potency. Structure-activity relationship (SAR) studies demonstrated the NP-BTA scaffold is subject to oxidative and non-oxidative metabolism, making it unsuitable for systemic administration. In a mouse dermatomycosis model, however, topical application of the compound provided significant therapeutic benefit. This work expands the repertoire of antifungal protein synthesis target mechanisms and provides a path to develop Gln4 inhibitors.

The C-terminal protein interaction domain of the chromatin reader Yaf9 is critical for pathogenesis of Candida albicans.

Fungal infections cause a large health burden but are treated by only a handful of antifungal drug classes. Chromatin factors have emerged as possible targets for new antifungals. These targets include the reader proteins, which interact with posttranslationally modified histones to influence DNA transcription and repair. The YEATS domain is one such reader recognizing both crotonylated and acetylated histones. Here, we performed a detailed structure/function analysis of the Candida albicans YEATS domain reader Yaf9, a subunit of the NuA4 histone acetyltransferase and the SWR1 chromatin remodeling complex. We have previously demonstrated that the homozygous deletion mutant yaf9Δ/Δ displays growth defects and is avirulent in mice. Here we show that a YEATS domain mutant expected to inactivate Yaf9's chromatin binding does not display strong phenotypes in vitro, nor during infection of immune cells or in a mouse systemic infection model, with only a minor virulence reduction in vivo. In contrast to the YEATS domain mutation, deletion of the C-terminal domain of Yaf9, a protein-protein interaction module necessary for its interactions with SWR1 and NuA4, phenocopies the null mutant. This shows that the C-terminal domain is essential for Yaf9 roles in vitro and in vivo, including C. albicans virulence. Our study informs on the strategies for therapeutic targeting of Yaf9, showing that approaches taken for the mammalian YEATS domains by disrupting their chromatin binding might not be effective in C. albicans, and provides a foundation for studying YEATS proteins in human fungal pathogens.IMPORTANCEThe scarcity of available antifungal drugs and rising resistance demand the development of therapies with new modes of action. In this context, chromatin regulation may be a target for novel antifungal therapeutics. To realize this potential, we must better understand the roles of chromatin regulators in fungal pathogens. Toward this goal, here, we studied the YEATS domain chromatin reader Yaf9 in Candida albicans. Yaf9 uses the YEATS domain for chromatin binding and a C-terminal domain to interact with chromatin remodeling complexes. By constructing mutants in these domains and characterizing their phenotypes, our data indicate that the Yaf9 YEATS domain might not be a suitable therapeutic drug target. Instead, the Yaf9 C-terminal domain is critical for C. albicans virulence. Collectively, our study informs how a class of chromatin regulators performs their cellular and pathogenesis roles in C. albicans and reveals strategies to inhibit them.

Synthesis, characterization, optical properties, biological activity and theoretical studies of a 4 nitrobenzylidene) amino) phenyl)imino)methyl)naphthalen-2-ol -based fluorescent Schiff base

A new Schiff base, 1-(E)-(4-((E) 4nitrobenzylidene) amino) phenyl)imino) methyl)naphthalen-2-ol (4NMN), was prepared from the reaction of p-phenylenediamine with 2-hydroxy-1-naphthaldehyde and 4-nitrobenzaldehyde and characterized with spectroscopic analysis. UV-VIS and NMR. Frontier molecular orbitals, molecular electrostatic potential, and chemical reactivity descriptors of the synthesized compound were studied using molecular modeling methods. The antibacterial and antifungal activities of the Schiff base were studied for its minimum inhibitory concentration. The compound showed a higher effect on yeast than against bacteria. Density functional theory (DFT) calculations were performed to study the mechanism of reaction for the synthesis of 4NMN, and the results were consistent with the experimental findings. 4NMN exhibited moderate antibacterial and antifungal activities and demonstrated higher inhibition potential against different resistant strains compared to the reference drug gentamycin. The absorption and fluorescence spectra of 4NMN were measured in different solvents, and the effect of relative polarity and acidity on the medium was observed. An inner filter effect was observed at high concentrations, and the compound showed considerable fluorescence enhancement with increasing medium viscosity and fluorescence quenching by the addition of traces of Cr1+ and Cu2+. Additionally, molecular docking studies were conducted to investigate the efficiency of antibacterial and antifungal targets.

MOB-mediated regulation of septation initiation network (SIN) signaling is required for echinocandin-induced hyperseptation in Aspergillus fumigatus.

Aspergillus fumigatus is a major invasive mold pathogen and the most frequent etiologic agent of invasive aspergillosis. The currently available treatments for invasive aspergillosis are limited in both number and efficacy. Our recent work has uncovered that the β-glucan synthase inhibitors, the echinocandins, are fungicidal against strains of A. fumigatus with defects in septation initiation network (SIN) kinase activity. These drugs are known to be fungistatic against strains with normal septation. Surprisingly, SIN kinase mutant strains also failed to invade lung tissue and were significantly less virulent in immunosuppressed mouse models. Inhibiting septation in filamentous fungi is therefore an exciting therapeutic prospect to both reduce virulence and improve current antifungal therapy. However, the SIN remains understudied in pathogenic fungi. To address this knowledge gap, we characterized the putative regulatory components of the A. fumigatus SIN. These included the GTPase, SpgA, it's two-component GTPase-activating protein, ByrA/BubA, and the kinase activators, SepM and MobA. Deletion of spgA, byrA, or bubA resulted in no overt septation or echinocandin susceptibility phenotypes. In contrast, our data show that deletion of sepM or mobA largely phenocopies disruption of their SIN kinase binding partners, sepL and sidB, respectively. Reduced septum formation, echinocandin hypersusceptibility, and reduced virulence were generated by loss of either gene. These findings provide strong supporting evidence that septa are essential not only for withstanding the cell wall disrupting effects of echinocandins but are also critical for the establishment of invasive disease. Therefore, pharmacological SIN inhibition may be an exciting strategy for future antifungal drug development.IMPORTANCESepta are important structural determinants of echinocandin susceptibility and tissue invasive growth for the ubiquitous fungal pathogen Aspergillus fumigatus. Components of the septation machinery therefore represent promising novel antifungal targets to improve echinocandin activity and reduce virulence. However, little is known about septation regulation in A. fumigatus. Here, we characterize the predicted regulatory components of the A. fumigatus septation initiation network. We show that the kinase activators SepM and MobA are vital for proper septation and echinocandin resistance, with MobA playing an essential role. Null mutants of mobA displayed significantly reduced virulence in a mouse model, underscoring the importance of this pathway for A. fumigatus pathogenesis.

Upc2-mediated mechanisms of azole resistance in Candida auris.

Candida auris is an emerging yeast pathogen of major concern because of its ability to cause hospital outbreaks of invasive candidiasis and to develop resistance to antifungal drugs. A majority of C. auris isolates are resistant to fluconazole, an azole drug used for the treatment of invasive candidiasis. Mechanisms of azole resistance are multiple, including mutations in the target gene ERG11 and activation of the transcription factors Tac1b and Mrr1, which control the drug transporters Cdr1 and Mdr1, respectively. We investigated the role of the transcription factor Upc2, which is known to regulate the ergosterol biosynthesis pathway and azole resistance in other Candida spp. Genetic deletion and hyperactivation of Upc2 by epitope tagging in C. auris resulted in drastic increases and decreases in susceptibility to azoles, respectively. This effect was conserved in strains with genetic hyperactivation of Tac1b or Mrr1. Reverse transcription PCR analyses showed that Upc2 regulates ERG11 expression and also activates the Mrr1/Mdr1 pathway. We showed that upregulation of MDR1 by Upc2 could occur independently from Mrr1. The impact of UPC2 deletion on MDR1 expression and azole susceptibility in a hyperactive Mrr1 background was stronger than that of MRR1 deletion in a hyperactive Upc2 background. While Upc2 hyperactivation resulted in a significant increase in the expression of TAC1b, CDR1 expression remained unchanged. Taken together, our results showed that Upc2 is crucial for azole resistance in C. auris, via regulation of the ergosterol biosynthesis pathway and activation of the Mrr1/Mdr1 pathway. Notably, Upc2 is a very potent and direct activator of Mdr1.IMPORTANCECandida auris is a yeast of major medical importance causing nosocomial outbreaks of invasive candidiasis. Its ability to develop resistance to antifungal drugs, in particular to azoles (e.g., fluconazole), is concerning. Understanding the mechanisms of azole resistance in C. auris is important and may help in identifying novel antifungal targets. This study shows the key role of the transcription factor Upc2 in azole resistance of C. auris and shows that this effect is mediated via different pathways, including the regulation of ergosterol biosynthesis and also the direct upregulation of the drug transporter Mdr1.

Casein kinase 2 complex: a central regulator of multiple pathobiological signaling pathways in Cryptococcus neoformans.

The casein kinase 2 (CK2) complex has garnered extensive attention over the past decades as a potential therapeutic target for diverse human diseases, including cancer, diabetes, and obesity, due to its pivotal roles in eukaryotic growth, differentiation, and metabolic homeostasis. While CK2 is also considered a promising antifungal target, its role in fungal pathogens remains unexplored. In this study, we investigated the functions and regulatory mechanisms of the CK2 complex in Cryptococcus neoformans, a major cause of fungal meningitis. The cryptococcal CK2 complex consists of a single catalytic subunit, Cka1, and two regulatory subunits, Ckb1 and Ckb2. Our findings show that Cka1 plays a primary role as a protein kinase, while Ckb1 and Ckb2 have major and minor regulatory functions, respectively, in growth, cell cycle control, morphogenesis, stress response, antifungal drug resistance, and virulence factor production. Interestingly, triple mutants lacking all three subunits (cka1Δ ckb1Δ ckb2Δ) exhibited more severe phenotypic defects than the cka1Δ mutant alone, suggesting that Ckb1/2 may have Cka1-independent functions. In a murine model of systemic cryptococcosis, cka1Δ and cka1Δ ckb1Δ ckb2Δ mutants showed severely reduced virulence. Transcriptomic, proteomic, and phosphoproteomic analyses further revealed that the CK2 complex controls a wide array of effector proteins involved in transcriptional regulation, cell cycle control, nutrient metabolisms, and stress responses. Most notably, CK2 disruption led to dysregulation of key signaling cascades central to C. neoformans pathogenicity, including the Hog1, Mpk1 MAPKs, cAMP/PKA, and calcium/calcineurin signaling pathways. In summary, our study provides novel insights into the multifaceted roles of the fungal CK2 complex and presents a compelling case for targeting it in the development of new antifungal drugs.IMPORTANCEThe casein kinase 2 (CK2) complex, crucial for eukaryotic growth, differentiation, and metabolic regulation, presents a promising therapeutic target for various human diseases, including cancer, diabetes, and obesity. Its potential as an antifungal target is further highlighted in this study, which explores CK2's functions in C. neoformans, a key fungal meningitis pathogen. The CK2 complex in C. neoformans, comprising the Cka1 catalytic subunit and Ckb1/2 regulatory subunits, is integral to processes like growth, cell cycle, morphogenesis, stress response, drug resistance, and virulence. Our findings of CK2's role in regulating critical signaling pathways, including Hog1, Mpk1 MAPKs, cAMP/PKA, and calcium/calcineurin, underscore its importance in C. neoformans pathogenicity. This study provides valuable insights into the fungal CK2 complex, reinforcing its potential as a target for novel antifungal drug development and pointing out a promising direction for creating new antifungal agents.

Alternative Oxidase – Aid or obstacle to combat the rise of fungal pathogens?

Fungal pathogens present a growing threat to both humans and global health security alike. Increasing evidence of antifungal resistance in fungal populations that infect both humans and plant species has increased reliance on combination therapies and shown the need for new antifungal therapeutic targets to be investigated. Here, we review the roles of mitochondria and fungal respiration in pathogenesis and discuss the role of the Alternative Oxidase enzyme (Aox) in both human fungal pathogens and phytopathogens. Increasing evidence exists for Aox within mechanisms that underpin fungal virulence. Aox also plays important roles in adaptability that may prove useful within dual targeted fungal-specific therapeutic approaches. As improved fungal specific mitochondrial and Aox inhibitors are under development we may see this as an emerging target for future approaches to tackling the growing challenge of fungal infection.

Synthesis and antifungal evaluation of new azole derivatives containing 1,2,3-triazole.

Invasive fungal infections caused by C. albicans are becoming increasingly serious and there is an urgent need for exploring new antifungal drugs. In the present work, a series of new azole derivatives containing a 1,2,3-triazole moiety have been prepared, and in vitro antifungal activity have been evaluated. The results revealed that most compounds showed excellent antifungal activity against C. albicans SC5314 and drug-resistant SC5314-FR. In particular, compounds 4h, 4j, 4l, 4s and 4w exhibited better antifungal activity than FLC. The preliminary mechanism study indicated that 4s could damage the integrity of the cell structure, increase the permeability of the cell membrane, and cause the leakage of cell contents of C. albicans. The molecular docking study indicated that 4s showed an obvious binding site with the target CYP51 (PDB ID: 5TL8). Therefore, 4s could be considered as a new antifungal agent targeting CYP51 for further study.

Pseudolaric acid B exerts an antifungal effect and targets SIRT1 to ameliorate inflammation by regulating Nrf2/NF-κB pathways in fungal keratitis.

Fungal keratitis (FK) is a vision-threatening infection. We aimed to explore the antifungal and anti-inflammatory effects of pseudolaric acid B (PAB) on FK and the underlying mechanisms involved. Network pharmacology utilized to acquire the potential target genes, and silent information regulator 1 (SIRT1) was consistently downregulated in Gene Expression Omnibus dataset and clinical samples. Molecular docking analysis showed that PAB and SIRT1 had good binding activity. No toxicity was observed in vivo and in vitro with a PAB concentration below 0.3μM. PAB exerted its antifungal activity by destroying the integrity of hyphae, and alleviated the severity of FK in rats by decreasing clinical scores, fungal burden and inhibiting inflammatory cell infiltration. PAB increased SIRT1 to regulate the crosstalk between nuclear factor erythroid 2-related factor 2 (Nrf2) and nuclear factor kappa-B (NF-κB), decreasing the levels of inflammatory cytokines tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6; and pattern recognition receptors, C-type lectin domain containing 7A (Dectin-1), lectin-like oxidized low-density lipoprotein receptor 1 (LOX-1), toll like receptor (TLR)-2, and TLR4 both in vivo and in vitro. However, this anti-inflammatory effect of PAB was abolished by the SIRT1 inhibitor EX527. This study provides new evidence that PAB has antifungal and anti-inflammatory effects in FK and may provide a novel therapeutic strategy for the treatment of FK.

Synergistic effects of putative Ca2+-binding sites of calmodulin in fungal development, temperature stress and virulence of Aspergillus fumigatus

ABSTRACT In pathogenic fungi, calcium-calmodulin-dependent serine-threonine-specific phosphatase calcineurin is involved in morphogenesis and virulence. Therefore, calcineurin and its tightly related protein complexes are attractive antifungal drug targets. However, there is limited knowledge available on the relationship between in vivo Ca2+-binding sites of calmodulin (CaM) and its functions in regulating stress responses, morphogenesis, and pathogenesis. In the current study, we demonstrated that calmodulin is required for hyphal growth, conidiation, and virulence in the human fungal pathogen, Aspergillus fumigatus. Site-directed mutations of calmodulin revealed that a single Ca2+-binding site mutation had no significant effect on A. fumigatus hyphal development, but multiple Ca2+-binding site mutations exhibited synergistic effects, especially when cultured at 42 °C, indicating that calmodulin function in response to temperature stress depends on its Ca2+-binding sites. Western blotting implied that mutations in Ca2+-binding sites caused highly degraded calmodulin fragments, suggesting that the loss of Ca2+-binding sites results in reduced protein stability. Moreover, normal intracellular calcium homeostasis and the nuclear translocation of the transcriptional factor CrzA are dependent on Ca2+-binding sites of AfCaM, demonstrating that Ca2+-binding sites of calmodulin are required for calcium signalling and its major transcription factor CrzA. Importantly, in situ mutations for four Ca2+-binding sites of calmodulin resulted in an almost complete loss of virulence in the Galleria mellonella wax moth model. This study shed more light on the functional characterization of putative calcium-binding sites of calmodulin in the morphogenesis and virulence of A. fumigatus, which enhances our understanding of calmodulin biological functions in cells of opportunistic fungal pathogens.

Reprogramming in Candida albicans Gene Expression Network under Butanol Stress Abrogates Hyphal Development.

Candida albicans is the causative agent of invasive fungal infections. Its hyphae-forming ability is regarded as one of the important virulence factors. To unravel the impact of butanol on Candida albicans, it was placed in O+ve complete human serum with butanol (1% v/v). The Candida transcriptome under butanol stress was then identified by mRNA sequencing. Studies including electron microscopy demonstrated the inhibition of hyphae formation in Candida under the influence of butanol, without any significant alteration in growth rate. The numbers of genes upregulated in the butanol in comparison to the serum alone were 1061 (20 min), 804 (45 min), and 537 (120 min). Candida cells exhibited the downregulation of six hypha-specific transcription factors and the induction of four repressor/regulator genes. Many of the hypha-specific genes exhibited repression in the medium with butanol. The genes related to adhesion also exhibited repression, whereas, among the heat-shock genes, three showed inductions in the presence of butanol. The fungal-specific genes exhibited induction as well as repression in the butanol-treated Candida cells. Furthermore, ten upregulated genes formed the core stress gene set in the presence of butanol. In the gene ontology analysis, enrichment of the processes related to non-coding RNA, ribosome biosynthesis, and metabolism was observed in the induced gene set. On the other side, a few GO biological process terms, including biofilm formation and filamentous growth, were enriched in the repressed gene set. Taken together, under butanol stress, Candida albicans is unable to extend hyphae and shows growth by budding. Many of the genes with perturbed expression may have fitness or virulence attributes and may provide prospective sites of antifungal targets against C. albicans.

Discovery and Development of 4-Hydroxyphenylpyruvate Dioxygenase as a Novel Crop Fungicide Target.

Plant pathogenic fungi pose a significant threat to crop yields and quality, and the emergence of fungicide resistance has further exacerbated the problem in agriculture. Therefore, there is an urgent need for efficient and environmentally friendly fungicides. In this study, we investigated the antifungal activity of (+)-Usnic acid and its inhibitory effect on crop pathogenic fungal 4-hydroxyphenylpyruvate dioxygenases (HPPDs) and determined the structure of Zymoseptoria tritici HPPD (ZtHPPD)-(+)-Usnic acid complex. Thus, the antifungal target of (+)-Usnic acid and its inhibitory basis toward HPPD were uncovered. Additionally, we discovered a potential lead fungicide possessing a novel scaffold that displayed remarkable antifungal activities. Furthermore, our molecular docking analysis revealed the unique binding mode of this compound with ZtHPPD, explaining its high inhibitory effect. We concluded that HPPD represents a promising target for the control of phytopathogenic fungi, and the new compound serves as a novel starting point for the development of fungicides and dual-purpose pesticides.

Discovery of Highly Efficient Novel Antifungal Lead Compounds Targeting Succinate Dehydrogenase: Pyrazole-4-carboxamide Derivatives with an N-Phenyl Substituted Amide Fragment.

Developing environmentally friendly fungicides is crucial to tackle the issue of rising pesticide resistance. In this study, a series of novel pyrazole-4-carboxamide derivatives containing N-phenyl substituted amide fragments were designed and synthesized. The structures of target compounds were confirmed by 1H NMR, 13C NMR, and HRMS, and the crystal structure of the most active compound N-(1-(4-(4-(tert-butyl)benzamido)phenyl)propan-2-yl)-3-(difluoromethyl)-N-methoxy-1-methyl-1H-pyrazole-4-carboxamide (U22) was further determined by X-ray single-crystal diffraction. The bioassay results indicated that the 26 target compounds possessed good in vitro antifungal activity against Sclerotinia sclerotiorum with EC50 values for compounds U12, U13, U15, U16, U18, U22, and U23 being 4.17 ± 0.46, 8.04 ± 0.71, 7.01 ± 0.71, 12.77 ± 1.00, 8.11 ± 0.70, 0.94 ± 0.11, and 9.48 ± 0.83 μg·mL-1, respectively, which were the similar to controls bixafen (6.70 ± 0.47 μg·mL-1), fluxapyroxad (0.71 ± 0.14 μg·mL-1), and pydiflumetofen (0.06 ± 0.01 μg·mL-1). Furthermore, in vivo antifungal activity results against S. sclerotiorum indicated that compounds U12 (80.6%) and U22 (89.9%) possessed excellent preventative efficacy at 200 μg·mL-1, which was the same as the control pydiflumetofen (82.4%). Scanning electron microscopy and transmission electron microscopy studies found that the compound U22 could destroy the hyphal morphology and damage mitochondria, cell membranes, and vacuoles. The results of molecular docking of compound U22 and pydiflumetofen with succinate dehydrogenase (SDH) indicated they interact well with the active site of SDH. This study validated our approach and design strategy to produce compounds with an enhanced biological activity as compared to the parent structure.

Is the C-Terminal Domain an Effective and Selective Target for the Design of Hsp90 Inhibitors against Candida Yeast?

Improving the armamentarium to treat invasive candidiasis has become necessary to overcome drug resistance and the lack of alternative therapy. In the pathogenic fungus Candida albicans, the 90-kDa Heat-Shock Protein (Hsp90) has been described as a major regulator of virulence and resistance, offering a promising target. Some human Hsp90 inhibitors have shown activity against Candida spp. in vitro, but host toxicity has limited their use as antifungal drugs. The conservation of Hsp90 across all species leads to selectivity issues. To assess the potential of Hsp90 as a druggable antifungal target, the activity of nine structurally unrelated Hsp90 inhibitors with different binding domains was evaluated against a panel of Candida clinical isolates. The Hsp90 sequences from human and yeast species were aligned. Despite the degree of similarity between human and yeast N-terminal domain residues, the in vitro activities measured for the inhibitors interacting with this domain were not reproducible against all Candida species. Moreover, the inhibitors binding to the C-terminal domain (CTD) did not show any antifungal activity, with the exception of one of them. Given the greater sequence divergence in this domain, the identification of selective CTD inhibitors of fungal Hsp90 could be a promising strategy for the development of innovative antifungal drugs.

Towards a High-Affinity Peptidomimetic Targeting Proliferating Cell Nuclear Antigen from Aspergillus fumigatus.

Invasive fungal infections (IFIs) are prevalent in immunocompromised patients. Due to alarming levels of increasing resistance in clinical settings, new drugs targeting the major fungal pathogen Aspergillus fumigatus are required. Attractive drug targets are those involved in essential processes like DNA replication, such as proliferating cell nuclear antigens (PCNAs). PCNA has been previously studied in cancer research and presents a viable target for antifungals. Human PCNA interacts with the p21 protein, outcompeting binding proteins to halt DNA replication. The affinity of p21 for hPCNA has been shown to outcompete other associating proteins, presenting an attractive scaffold for peptidomimetic design. p21 has no A. fumigatus homolog to our knowledge, yet our group has previously demonstrated that human p21 can interact with A. fumigatus PCNA (afumPCNA). This suggests that a p21-based inhibitor could be designed to outcompete the native binding partners of afumPCNA to inhibit fungal growth. Here, we present an investigation of extensive structure-activity relationships between designed p21-based peptides and afumPCNA and the first crystal structure of a p21 peptide bound to afumPCNA, demonstrating that the A. fumigatus replication model uses a PIP-box sequence as the method for binding to afumPCNA. These results inform the new optimized secondary structure design of a potential peptidomimetic inhibitor of afumPCNA.

Identification and functional characterization of the putative members of the CTDK-1 kinase complex as regulators of growth and development in Aspergillus nidulans and Aspergillus fumigatus.

Aspergillus fumigatus has been included by the World Health Organization in the priority list of fungal pathogens because (i) it causes 90% of invasive aspergillosis cases, with a high mortality rate, and (ii) infections are becoming increasingly resistant to azole antifungals. A. nidulans is an opportunistic pathogen and a saprotroph which has served during the last 80 years as a reference system for filamentous fungi. Here, we characterized the role in morphogenesis and development of the putative transcriptional cyclin/kinase complex CTDK-1 in both aspergilli. The null mutants of the corresponding genes showed delayed germination, aberrant conidiophore development, and inhibition of cleistothecia production. While in higher eukaryotes this complex is formed only by a cyclin and a kinase, the fungal complex would incorporate a fungal-specific third component, FlpB, which would enable the interaction between the kinase (Stk47) and the cyclin (FlpA) and may be used as a target for antifungals.

TrCla4 promotes actin polymerization at the hyphal tip and mycelial growth in Trichophyton rubrum.

Dermatophytes invade and colonize host superficial tissues via hyphal growth. Although cytoskeletal reorganization and its regulation are essential for hyphal growth, the molecular mechanisms in dermatophytes and their applicability as antifungal drug targets remain poorly understood. The p21-activated kinase (PAK) is a downstream effector of the small GTPases Rac and CDC42, also known as p21, and is involved in various molecular and cellular functions, including actin polymerization and cell morphogenesis. In this study, we investigated the contribution of the PAK protein TrCla4 to morphogenesis and mycelial growth in Trichophyton rubrum, the most frequently isolated fungus in dermatophytosis (athlete's foot). The actin polymerization inhibitor, cytochalasin A inhibited actin accumulation at the hyphal tip and mycelial growth of T. rubrum, suggesting the involvement of the actin cytoskeleton in mycelial growth. In the Trcla4 knockout strain (ΔTrcla4), we observed defects in mycelial growth, hyphal branching, and the accumulation of polymerized actin at the hyphal tip. Chemical inhibitors of TrRac-dependent TrCla4 kinase activity, FRAX486 and IPA-3, also inhibited spore germination and mycelial growth. Interestingly, ΔTrcla4 showed no additional inhibition of mycelial growth when treated with these inhibitors, indicating that their inhibitory effects are primarily mediated through TrCla4. In an invertebrate dermatophyte infection model, animals infected with ΔTrcla4 had higher survival rates than those infected with the wild-type, and IPA-3 and FRAX486 treatments both significantly improved animal survival rates. These results suggest that the dermatophyte PAK promotes mycelial growth by facilitating actin polymerization at the hyphal tip, making it a potential therapeutic target for dermatophytosis. IMPORTANCE Superficial fungal infections, such as athlete's foot, affect more than 10% of the world's population and have a significant impact on quality of life. Despite the fact that treatment-resistant fungi are a concern, there are just a few antifungal drug targets accessible, as opposed to the wide range of therapeutic targets found in bacterial infections. As a result, additional alternatives are sought. In this study, we generated a PAK TrCla4 deletion strain (∆Trcla4) of Trichophyton rubrum. The ∆Trcla4 strain exhibited deficiencies in mycelial growth, hyphal morphology, and polarized actin localization at the hyphal tip. IPA-3 and FRAX486, small chemical inhibitors of mammalian PAK, were discovered to limit fungal mycelial proliferation. According to our findings, fungal PAKs are interesting therapeutic targets for the development of new antifungal medicines.

The Aspergillus fumigatus UPR is variably activated across nutrient and host environments and is critical for the establishment of corneal infection.

The Aspergillus fumigatus unfolded protein response (UPR) is a two-component relay consisting of the ER-bound IreA protein, which splices and activates the mRNA of the transcription factor HacA. Spliced hacA accumulates under conditions of acute ER stress in vitro, and UPR null mutants are hypovirulent in a murine model of invasive pulmonary infection. In this report, we demonstrate that a hacA deletion mutant (ΔhacA) is furthermore avirulent in a model of fungal keratitis, a corneal infection, and an important cause of ocular morbidity and unilateral blindness worldwide. Interestingly, we demonstrate that A. fumigatus hacA is spliced in infected lung samples, but not in the cornea, suggesting the amount of ER stress experienced by the fungus varies upon the host niche. To better understand how the UPR contributes to fungal cell biology across a spectrum of ER-stress levels, we employed transcriptomics on the wild-type and ΔhacA strains in glucose minimal media (low stress), glucose minimal media with dithiothreitol (high stress), and gelatin minimal media as a proxy for the nutrient stress encountered in the cornea (mid-level stress). These data altogether reveal a unique HacA-dependent transcriptome under each condition, suggesting that HacA activity is finely-tuned and required for proper fungal adaptation in each environment. Taken together, our results indicate that the fungal UPR could serve as an important antifungal target in the setting of both invasive pulmonary and corneal infections.

Biological evaluation, GC–MS profiling, and molecular docking studies of some essential oils against postharvest pathogens of maize

Considering the use of essential oils (EOs) as biopesticides due to their ability to effectively control postharvest pathogens, EOs-based formulations, and coatings became a priority. The present work aimed to screen essential oils from eight different plant species with anti-fungal properties along with the phytochemical analysis and molecular docking insight. Furthermore, the selected essential oil was applied as an edible coating to examine its protective effect against the attack of isolated postharvest pathogens on maize seedlings and rescue the seedling’s growth. Eight EOs were screened for their antimicrobial potential against Fusarium oxysporum and Xanthomonas campestris. Later on, the identification of the chemical constituents of the best-performing essential oil (Bergamot oil) was performed by GC–MS analysis. Different coating formulations consisting of essential oils and chitosan at 0.25% and 0.5% concentrations were applied to maize seeds to protect against postharvest pathogens. Formulation containing the bergamot essential oil showed the highest protective potential against a fungal and bacterial postharvest pathogen of maize under in-vitro conditions. The molecular docking study showed that the linalool and linalyl acetate present in essential oil comprises strong interactions with antifungal target proteins.