Development Of Antifungal Drugs Research Articles

Glutathione-mediated redox regulation in Cryptococcus neoformans impacts virulence.

The fungal pathogen Cryptococcus neoformans is well adapted to its host environment. It has several defence mechanisms to evade oxidative and nitrosative agents released by phagocytic host cells during infection. Among them, melanin production is linked to both fungal virulence and defence against harmful free radicals that facilitate host innate immunity. How C. neoformans manipulates its redox environment to facilitate melanin formation and virulence is unclear. Here we show that the antioxidant glutathione is inextricably linked to redox-active processes that facilitate melanin and titan cell production, as well as survival in macrophages and virulence in a murine model of cryptococcosis. Comparative metabolomics revealed that disruption of glutathione biosynthesis leads to accumulation of reducing and acidic compounds in the extracellular environment of mutant cells. Overall, these findings highlight the importance of redox homeostasis and metabolic compensation in pathogen adaptation to the host environment and suggest new avenues for antifungal drug development.

Antifungal activity of propafenone on Candida spp. strains: interaction with antifungals and possible mechanism of action.

Introduction. The development of new antifungal drugs has become a global priority, given the increasing cases of fungal diseases together with the rising resistance to available antifungal drugs. In this scenario, drug repositioning has emerged as an alternative for such development, with advantages such as reduced research time and costs.Gap statement. Propafenone is an antiarrhythmic drug whose antifungal activity is poorly described, being a good candidate for further study.Aim. This study aims to evaluate propafenone activity against different species of Candida spp. to evaluate its combination with standard antifungals, as well as its possible action mechanism.Methodology. To this end, we carried out tests against strains of Candida albicans, Candida auris, Candida parapsilosis, Candida tropicalis, Candida glabrata and Candida krusei based on the evaluation of the MIC, minimum fungicidal concentration and tolerance level, along with checkerboard and flow cytometry tests with clinical strains and cell structure analysis by scanning electron microscopy (SEM).Results. The results showed that propafenone has a 50% MIC ranging from 32 to 256 µg ml-1, with fungicidal activity and positive interactions with itraconazole in 83.3% of the strains evaluated. The effects of the treatments observed by SEM were extensive damage to the cell structure, while flow cytometry revealed the apoptotic potential of propafenone against Candida spp.Conclusion. Taken together, these results indicate that propafenone has the potential for repositioning as an antifungal drug.

Unsaturated fatty acid perturbation combats emerging triazole antifungal resistance in the human fungal pathogen Aspergillus fumigatus.

Contemporary antifungal therapies utilized to treat filamentous fungal infections are inhibited by intrinsic and emerging drug resistance. Consequently, there is an urgent need to develop novel antifungal compounds that are effective against drug-resistant filamentous fungi. Here, we utilized an Aspergillus fumigatus cell-based high-throughput screen to identify small molecules with antifungal activity that also potentiated triazole activity. The screen identified 16 hits with promising activity against A. fumigatus. A nonspirocyclic piperidine, herein named MBX-7591, exhibited synergy with triazole antifungal drugs and activity against pan-azole-resistant A. fumigatus isolates. MBX-7591 has additional potent activity against Rhizopus species and CO2-dependent activity against Cryptococcus neoformans. Chemical, genetic, and biochemical mode of action analyses revealed that MBX-7591 increases cell membrane saturation by decreasing oleic acid content. MBX-7591 has low toxicity in vivo and shows good efficacy in decreasing fungal burden in a murine model of invasive pulmonary aspergillosis. Taken together, our results suggest MBX-7591 is a promising hit with a novel mode of action for further antifungal drug development to combat the rising incidence of triazole-resistant filamentous fungal infections.IMPORTANCEThe incidence of infections caused by fungi continues to increase with advances in medical therapies. Unfortunately, antifungal drug development has not kept pace with the incidence and importance of fungal infections, with only three major classes of antifungal drugs currently available for use in the clinic. Filamentous fungi, also called molds, are particularly recalcitrant to contemporary antifungal therapies. Here, a recently developed Aspergillus fumigatus cell reporter strain was utilized to conduct a high-throughput screen to identify small molecules with antifungal activity. An emphasis was placed on small molecules that potentiated the activity of contemporary triazole antifungals and led to the discovery of MBX-7591. MBX-7591 potentiates triazole activity against drug-resistant molds such as A. fumigatus and has activity against Mucorales fungi. MBX-7591's mode of action involves inhibiting the conversion of saturated to unsaturated fatty acids, thereby impacting fungal membrane integrity. MBX-7591 is a novel small molecule with antifungal activity poised for lead development.

Antifungal pipeline: Is there light at the end of the tunnel?

The misuse and overuse of classic antifungals have accelerated the development of resistance mechanisms, diminishing the efficacy of established therapeutic pathways and necessitating a shift towards alternative targets. Despite this pressing need for new treatments, the antifungal drug pipeline has been largely stagnant for the past three decades, primarily due to the high risks and costs associated with antifungal drug development, compounded by uncertain market returns. Extensive research durations, special patient populations and rigorous regulatory demands pose significant barriers to bringing novel antifungal agents to market. In response, the “push-pull” incentive model has emerged as a vital strategy to invigorate the pipeline and encourage innovation. This editorial critically examines the current clinical landscape and spotlights emerging antifungal agents, such as Fosmanogepix, Ibrexafungerp, and Olorofim, while also unraveling the multifaceted challenges faced in new antifungal drug development. The generation of novel antifungals offers a beacon of hope in the battle against antimicrobial resistance, but it is premature to declare them as definitive solutions. Their future role hinges on thorough clinical validation, cost-effectiveness assessments, and continuous post-marketing surveillance. Only through strategic implementation and integration with market strategies we can transform the landscape of antifungal development, addressing both the resistance crisis and the treatment challenges.

Unveiling the Anti-Candida albicans activity of bioactive fractions of mining soil-dwelling Streptomyces sp. DS104

Due to an increase in the incidence of Candida albicans infection and its growing resistance to currently used antifungal drugs, further development of more potent antimicrobials is needed. This study sought to assess the antifungal properties of Streptomyces sp. DS104, isolated from a mining soil, against C. albicans and identify the bioactive substances that are produced by this strain. Ethyl acetate supernatant's crude extract showed a high inhibition against C. albicans, with a minimal inhibitory concentration (MIC) value as low as 4 µg/mL. Two bioactive fractions (F5 and F6) have been isolated using silica gel column chromatography, demonstrating zones of inhibition against C. albicans measuring 28.33 ± 01.52 to 29.00 ± 02.64 mm. The GC-MS analysis was carried out on the purified fractions and allowed the identification of 21 compounds predominantly consisting of carboxylic acids and pyrrolopyrazine derivatives. In Fraction F5, the major compounds detected are pentadecanoic and hexadecanoic acids, constituting the highest area percentages of 41.99% and 33.77%, respectively. As for fraction F6, the predominant compounds include pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl) and pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(methylphenyl), accounting for area percentages of 20.18% and 12.16%, respectively. Various other bioactive compounds, such as isocoumarins, and phenolic compounds, had previously been identified as components with antifungal activity, were also detected. It's worth highlighting that three compounds specifically, 2-hydrazino-8-hydroxy-4-phenylquinoline, hydroxyphenylacetic acid, and 3-carboxaldehyde-1H-indole have been reported for the first time to be detected within the Streptomyces genus. These results suggested that Streptomyces sp. DS104 is a promising strain and thus appears to be a potential candidate for the development of antifungal drugs.

MIG1, TUP1 and NRG1 mediated yeast to hyphal morphogenesis inhibition in Candida albicans by ganciclovir.

Candida albicans is a polymorphic human fungal pathogen and the prime etiological agent responsible for candidiasis. The main two aspects of C. albicans virulence that have been suggested are yeast-to-hyphal (Y-H) morphological transitions and biofilm development. Anti-fungal agents targeting these virulence attributes enhances the antifungal drug development process. Repositioning with other non-fungal drugs offered a one of the new strategies and a potential alternative option to counter the urgent need for antifungal drug development. In the current study, an antiviral drug ganciclovir was screened as an antifungal agent against ATCC 90028, 10231 and clinical isolate (C1). Ganciclovir at 0.5mg/ml concentration reduced 50% hyphal development on a silicon-based urinary catheter and was visualized using scanning electron microscopy. Ganciclovir reduced ergosterol biosynthesis in both strains and C1 isolate of C. albicans in a concentration-dependent manner. Additionally, a gene expression profile study showed that ganciclovir treatment resulted in upregulation of hyphal-specific repressors MIG1, TUP1, and NRG1 in C. albicans. Additionally, an in vivo study on the Bombyx mori silkworm model further evidenced the virulence inhibitory ability of ganciclovir (0.5mg/ml) against C. albicans. This is the first report that explore the novel anti-morphogenic activities of ganciclovir against the pathogenic C. albicans strains, along with clinical isolates. Further, ganciclovir may be considered for therapeutic purpose after combinations with standard antifungal agents.

Antifungal activity of β-lapachone against a fluconazole-resistant Candida auris strain.

Candida spp. can be found in the human microbiome. However, immunocompromised patients are likely to develop invasive Candida infections, with mortality rates higher than 50%. The discovery of C. auris, a species that rapidly acquire antifungal resistance, increased the concern about Candida infections. The limited number of antifungal agents and the high incidence of resistance to them make imperative the development of new antifungal drugs. β-lapachone is a biological active naphthoquinone that displays antifungal activity against C. albicans and C. glabrata. The aim of this study was to evaluate if this substance affects C. auris growth and elucidate its mechanism of action. A fluconazole-resistant C. auris isolate was used in this study. The antifungal activity of β-lapachone was determined through microbroth dilution assays, and its mechanism of action was evaluated using fluorescent probes. Interaction with fluconazole and amphotericin B was assessed by disk diffusion assay and checkerboard. β-lapachone inhibited planktonic C. auris cell growth by 92.7%, biofilm formation by 84.9%, and decrease the metabolism of preformed biofilms by 87.1% at 100µg/ml. At 100µg/ml, reductions of 30% and 59% of Calcofluor White and Nile red fluorescences were observed, indicating that β-lapachone affects cell wall chitin and neutral lipids content, respectively. Also, the ratio 590nm/529nm of JC-1 decreased 52%, showing that the compound affects mitochondria. No synergism was observed between β-lapachone and fluconazole or amphotericin B. Data show that β-lapachone may be a promising candidate to be used as monotherapy to treat C. auris resistant infections.

Histone deacetylase Sir2 promotes the systemic Candida albicans infection by facilitating its immune escape via remodeling the cell wall and maintaining the metabolic activity.

Candida albicans (C. albicans) is the most common opportunistic fungal pathogen and can cause various superficial infections and even life-threatening systemic infections. To successfully propagate infection, this organism relies on the ability to express virulence-associated factors and escape host immunity. In this study, we demonstrated that the histone deacetylase Sir2 helps C. albicans adhere to host cells and escape host immunity by mediating cell wall remodeling; as a result, C. albicans successfully colonized and invaded the host in vivo. In addition, we found that Sir2 contributes to carbon utilization under hypoxic conditions, suggesting that Sir2 is important for C. albicans survival and the establishment of infection in hypoxic environments. In summary, we investigated the role of Sir2 in regulating C. albicans pathogenicity in detail; these findings provide a potential target for the development of antifungal drugs.

Effect of Cinnamaldehyde on Systemic Candida albicans Infection in Mice.

To investigate the therapeutic efficacy of cinnamaldehyde (CA) on systemic Candida albicans infection in mice and to provide supportive data for the development of novel antifungal drugs. Ninety BALB/c mice were randomly divided into 3 groups according to a random number table: CA treatment group, fluconazole (positive control) group, and Tween saline (negative control) group, with 30 mice in each group. Initially, all groups of mice received consecutive intraperitoneal injections of cyclophosphamide at 200 mg/kg for 2 days, followed by intraperitoneal injection of 0.25 mL C. albicans fungal suspension (concentration of 1.0 × 107 CFU/mL) on the 4th day, to establish an immunosuppressed systemic Candida albicans infection animal model. Subsequently, the mice were orally administered CA, fluconazole and Tween saline, at 240, 240 mg/kg and 0.25 mL/kg respectively for 14 days. After a 48-h discontinuation of treatment, the liver, small intestine, and kidney tissues of mice were collected for fungal direct microscopic examination, culture, and histopathological examination. Additionally, renal tissues from each group of mice were collected for (1,3)- β -D-glucan detection. The survival status of mice in all groups was monitored for 14 days of drug administration. The CA group exhibited a fungal clearance rate of C. albicans above 86.7% (26/30), significantly higher than the fluconazole group (60.0%, 18/30, P<0.01) and the Tween saline group (30.0%, 9/30, P<0.01). Furthermore, histopathological examination in the CA group revealed the disappearance of inflammatory cells and near-normal restoration of tissue structure. The (1,3)-β-D-glucan detection value in the CA group (860.55 ± 126.73 pg/mL) was significantly lower than that in the fluconazole group (1985.13 ± 203.56 pg/mL, P<0.01) and the Tween saline group (5910.20 ± 320.56 pg/mL, P<0.01). The mouse survival rate reached 90.0% (27/30), higher than the fluconazole group (60.0%, 18/30) and the Tween saline group (30.0%, 9/30), with a significant difference between the two groups (both P<0.01). CA treatment exhibited significant therapeutic efficacy in mice with systemic C. albicans infection. Therefore, CA holds potential as a novel antifungal agent for targeted treatment of C. albicans infection.

FRET Assays for the Identification of C. albicans HSP90-Sba1 and Human HSP90α-p23 Binding Inhibitors.

Heat shock protein 90 (HSP90) is a critical target for anticancer and anti-fungal-infection therapies due to its central role as a molecular chaperone involved in protein folding and activation. In this study, we developed in vitro Förster Resonance Energy Transfer (FRET) assays to characterize the binding of C. albicans HSP90 to its co-chaperone Sba1, as well as that of the homologous human HSP90α to p23. The assay for human HSP90α binding to p23 enables selectivity assessment for compounds aimed to inhibit the binding of C. albicans HSP90 to Sba1 without affecting the physiological activity of human HSP90α. The combination of the two assays is important for antifungal drug development, while the assay for human HSP90α can potentially be used on its own for anticancer drug discovery. Since ATP binding of HSP90 is a prerequisite for HSP90-Sba1/p23 binding, ATP-competitive inhibitors can be identified with the assays. The specificity of binding of fusion protein constructs-HSP90-mNeonGreen (donor) and Sba1-mScarlet-I (acceptor)-to each other in our assay was confirmed via competitive inhibition by both non-labeled Sba1 and known ATP-competitive inhibitors. We utilized the developed assays to characterize the stability of both HSP90-Sba1 and HSP90α-p23 affinity complexes quantitatively. Kd values were determined and assessed for their precision and accuracy using the 95.5% confidence level. For HSP90-Sba1, the precision confidence interval (PCI) was found to be 70-120 (100 ± 20) nM while the accuracy confidence interval (ACI) was 100-130 nM. For HSP90α-p23, PCI was 180-260 (220 ± 40) nM and ACI was 200-270 nM. The developed assays were used to screen a nucleoside-mimetics library of 320 compounds for inhibitory activity against both C. albicans HSP90-Sba1 and human HSP90α-p23 binding. No novel active compounds were identified. Overall, the developed assays exhibited low data variability and robust signal separation, achieving Z factors > 0.5.

Fungal Plasma Membrane H+-ATPase: Structure, Mechanism, and Drug Discovery.

The fungal plasma membrane H+-ATPase (Pma1) pumps protons out of the cell to maintain the transmembrane electrochemical gradient and membrane potential. As an essential P-type ATPase uniquely found in fungi and plants, Pma1 is an attractive antifungal drug target. Two recent Cryo-EM studies on Pma1 have revealed its hexameric architecture, autoinhibitory and activation mechanisms, and proton transport mechanism. These structures provide new perspectives for the development of antifungal drugs targeting Pma1. In this article, we review the history of Pma1 structure determination, the latest structural insights into Pma1, and drug discoveries targeting Pma1.

Reviewing the mechanism of action and results of clinical studies on the antifungal drug ibrexafungerp

Introduction. Vulvovaginal candidiasis is an extremely common pathology of the female genital organs, leading to a long-term recurrent course and multiple complications. Although currently it is widely known about developing antibiotic resistance of bacterial pathogens, it is necessary to remember about similar phenomenon observed in other groups of infectious agents. In this regard, fungal infection also requires development of new therapeutic techniques and medicinal antifungal drugs, such as ibrexafungerp. Aim: to analyze available publications revealing the mechanism of action, efficacy, antifungal spectrum and results of clinical trials for a new oral antifungal drug ibrexafungerp. Materials and Methods. A search for publications in the electronic databases PubMed, eLibrary and ClinicalTrials.gov, published over the last 25 years was conducted using the following keywords in Russian and English: “candidiasis”, “vulvovaginal candidiasis”, “antifungal drugs”, “ibrexafungerp”, “clinical trials”, “mechanism of action”. Articles were assessed according to PRISMA guidelines. The titles and abstracts of identified publications were independently reviewed to retrieve relevant full text studies. After the selection procedure, 46 articles were included in the review. Results. This review provides information on the creation of the drug ibrexafungerp, its mechanism of action, the activity against a relatively wide range of pathogens, as well as the results from 13 ongoing and completed clinical trials in patients with fungal infection. Conclusion. The analysis of ibrexafungerp-related clinical studies showed its good oral bioavailability, high antifungal efficacy, so that its one-day dosage may further eliminate a need for unnecessarily long hospitalization and complex dosing schedules, thereby increasing adherence to therapy and odds for treatment success.

Codon usage bias of secretory protein in Fusarium oxysporum f. sp. cubense tropical race 4.

Banana Fusarium oxysporum f. sp. cubense tropical race 4 (Foc-TR4) is a highly destructive pathogen that infects nearly all major banana cultivars and has a tendency to spread further. Secreted proteins play a crucial role in the process of Fusarium wilt infection in bananas. In this study, we analyzed the codon usage bias (CUB) of the Foc-TR4 classical secretory protein genome for the first time and observed a strong bias toward codons ending with C. We found that 572 out of the 14,543 amino acid sequences in the Foc-TR4 genome exhibited characteristics of classical secretory proteins. The CUB was largely influenced by selection optimization pressure, as indicated by the ENC value and neutral plot analysis. Among the identified codons, such as UCC and CCC, 11 were found to be optimal for Foc-TR4 gene expression. Codons with higher GC content and a C base in the third position showed greater selectivity. The CUB in the secretory proteins encoded by Foc-TR4 provides insights into their evolutionary patterns, contributing to the development and screening of novel and effective antifungal drugs.

Mechanisms of antifungal resistance and developments in alternative strategies to combat Candida albicans infection.

Candida albicans is a commensal fungus that infects the humans and becomes an opportunistic pathogen particularly in immuno-compromised patients. Among the Candida genus, yeast C. albicans is the most frequently incriminated species and is responsible for nearly 50-90% of human candidiasis, with vulvovaginal candidiasis alone, affecting about 75% of the women worldwide. One of the significant virulence traits in C. albicans is its tendency to alternate between the yeast and hyphae morphotypes, accounting for the development of multi-drug resistance in them. Thus, a thorough comprehension of the decision points and genes controlling this transition is necessary, to understand the pathogenicity of this, naturally occurring, pernicious fungus. Additionally, the formation of C. albicans biofilm is yet another pathogenesis trait and a paramount cause of invasive candidiasis. Since 1980 and in 90s, wide spread use of immune-suppressing therapies and over prescription of fluconazole, a drug used to treat chronic fungal infections, triggered the emergence of novel anti-fungal drug development. Thus, this review thoroughly elucidates the diseases associated with C. albicans infection as well as the anti-fungal resistance mechanism associated with them and identifies the emerging therapeutic agents, along with a rigorous discussion regarding the future strategies that can possibly be adopted for the cure of this deleterious pathogen.

Uncovering a Novel cyp51A Mutation and Antifungal Resistance in Aspergillus fumigatus through Culture Collection Screening.

Aspergillus fumigatus is an important concern for immunocompromised individuals, often resulting in severe infections. With the emergence of resistance to azoles, which has been the therapeutic choice for Aspergillus infections, monitoring the resistance of these microorganisms becomes important, including the search for mutations in the cyp51A gene, which is the gene responsible for the mechanism of action of azoles. We conducted a retrospective analysis covering 478 A. fumigatus isolates. This comprehensive dataset comprised 415 clinical isolates and 63 isolates from hospital environmental sources. For clinical isolates, they were evaluated in two different periods, from 1998 to 2004 and 2014 to 2021; for environmental strains, one strain was isolated in 1998, and 62 isolates were evaluated in 2015. Our primary objectives were to assess the epidemiological antifungal susceptibility profile; trace the evolution of resistance to azoles, Amphotericin B (AMB), and echinocandins; and monitor cyp51A mutations in resistant strains. We utilized the broth microdilution assay for susceptibility testing, coupled with cyp51A gene sequencing and microsatellite genotyping to evaluate genetic variability among resistant strains. Our findings reveal a progressive increase in Minimum Inhibitory Concentrations (MICs) for azoles and AMB over time. Notably, a discernible trend in cyp51A gene mutations emerged in clinical isolates starting in 2014. Moreover, our study marks a significant discovery as we detected, for the first time, an A. fumigatus isolate carrying the recently identified TR46/F495I mutation within a sample obtained from a hospital environment. The observed cyp51A mutations underscore the ongoing necessity for surveillance, particularly as MICs for various antifungal classes continue to rise. By conducting resistance surveillance within our institution's culture collection, we successfully identified a novel TR46/F495I mutation in an isolate retrieved from the hospital environment which had been preserved since 1998. Moreover, clinical isolates were found to exhibit TR34/L98H/S297T/F495I mutations. In addition, we observed an increase in MIC patterns for Amphotericin B and azoles, signaling a change in the resistance pattern, emphasizing the urgent need for the development of new antifungal drugs. Our study highlights the importance of continued monitoring and research in understanding the evolving challenges in managing A. fumigatus infections.

Proteome-Wide Analysis of Lysine 2-Hydroxyisobutyrylation in Aspergillus fumigatus

Aspergillus fumigatus is the significant causative agent in cases of invasive aspergillosis, leading to a high mortality rate in immunocompromised patients. A comprehensive understanding of its growth patterns and metabolic processes within the host is a critical prerequisite for the development of effective antifungal strategies. Lysine 2-hydroxyisobutyrylation (Khib) is a highly conserved protein posttranslational modifications (PTM) found in various organisms. In this study, we investigate the biological impact of Khib in A. fumigatus. Using a combination of antibody enrichment with the conventional LC–MS/MS method, the pattern of Khib-modification in proteins and their respective sites were analyzed in a wild type strain of A. fumigatus. Our findings revealed 3494 Khib-modified proteins with a total of 18,091 modified sites in this strain. Functional enrichment analysis indicated that these Khib-modified proteins participate in a diverse range of cellular functions, spanning various subcellular locations such as ribosome biosynthesis, protein synthesis and nucleocytoplasmic transport. Notably, when compared with other reported eukaryotes, A. fumigatus exhibited consistently higher numbers of Khib-modified proteins, suggesting the potential significance of this modification in this organism. An interesting observation is the prevalence of Khib modifications in most enzymes involved in the ergosterol synthesis pathway. The insights gathered from this study provide new avenue for studying PTM-associated mechanisms in fungal growth and offer potential implication for antifungal drug development.

Associations between Genomic Variants and Antifungal Susceptibilities in the Archived Global Candida auris Population.

Candida auris is a recently emerged human fungal pathogen that has posed a significant threat to public health. Since its first identification in 2009, this fungus has caused nosocomial infections in over 47 countries across all inhabited continents. As of May 2023, the whole-genome sequences of over 4000 strains have been reported and a diversity of mutations, including in genes known to be associated with drug resistance in other human fungal pathogens, have been described. Among them, 387 strains contained antifungal-susceptibility information for which different methods might be used depending on the drugs and/or investigators. In most reports on C. auris so far, the number of strains analyzed was very small, from one to a few dozen, and the statistical significance of the relationships between these genetic variants and their antifungal susceptibilities could not be assessed. In this study, we conducted genome-wide association studies on individual clades based on previously published C. auris isolates to investigate the statistical association between genomic variants and susceptibility differences to nine antifungal drugs belonging to four major drug categories: 5-fluorocytosine, amphotericin B, fluconazole, voriconazole, itraconazole, posaconazole, anidulafungin, caspofungin, and micafungin. Due to the small sample sizes for Clades II, V, and VI, this study only assessed Clades I, III, and IV. Our analyses revealed 15 single nucleotide polymorphisms (SNPs) in Clade I (10 in coding and 5 in noncoding regions), 24 SNPs in Clade III (11 in coding and 13 in noncoding regions), and 13 SNPs in clade IV (10 in coding and 3 in noncoding regions) as statistically significantly associated with susceptibility differences to one or more of the nine antifungal drugs. While four SNPs in genes encoding lanosterol 14-α-demethylase (ERG11) and the catalytic subunit of 1,3-beta-D-glucan synthase (FKS1) were shared between clades, including the experimentally confirmed Ser639Phe/Pro missense substitutions in FKS1 for echinocandin resistance, most of the identified SNPs were clade specific, consistent with their recent independent origins. Interestingly, the majority of the antifungal resistance-associated SNPs were novel, and in genes and intergenic regions that have never been reported before as associated with antifungal resistance. While targeted study is needed to confirm the role of each novel SNP, the diverse mechanisms of drug resistance in C. auris revealed here indicate both challenges for infection control and opportunities for the development of novel antifungal drugs against this and other human fungal pathogens.

A CUG codon-adapted anchor-away toolkit for functional analysis of genes in Candida albicans.

Molecular genetic studies thus far have identified ~27% open-reading frames as being essential for the vegetative growth of Candida albicans in rich medium out of a total 6,198 haploid set of open reading frames. However, a major limitation has been to construct rapid conditional alleles of essential C. albicans genes with near quantitative depletion of encoded proteins. Here, we have developed a toolbox for rapid and conditional depletion of genes that would aid studies of gene function of both essential and non-essential genes.

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.

Evaluation of antibacterial, antifungal and antibiofilm activities of A. baumannii-derived tannase and gallic acid against uropathogenic microorganisms

One of the most prevalent infectious diseases and a key driver of antibiotic prescriptions in pediatrics is urinary tract infection (UTI). Due to the emergence of more resistant uropathogenic bacterial and fungal strains, current treatments are no longer effective, necessitating the urgent development of novel antibacterial and antifungal drugs. In this study, the antifungal, antibacterial, and anti-biofilm capabilities of compounds, such as tannase (TN) and gallic acid (GA), which were produced from a novel natural source, Acinetobacter baumannii (AB11) bacteria, were assessed for the inactivation of uropathogenic microorganisms (UMs). Ammonium sulphate precipitation, ion exchange, high-performance liquid chromatography, and gel filtration were used to purify TN and GA that were isolated from A. baumannii. A 43.08 % pure TN with 1221.2 U/mg specific activity and 10.51 mg/mL GA was obtained. The antibacterial, antifungal and anti-biofilm activities of TN and GA were evaluated against UMs and compared to those of commercially available antibiotics including sulfamethoxazole (SXT), levofloxacin (LEV), ciprofloxacin (CIP), amikacin (Ak), and nitrofurantoin (F). The results showed that TN and GA were superior to commercial antibiotics in their ability to inactivate UMs and considerably reduced biofilms formation. Additionally, the GA emerges as the top substitute for currently available medications, demonstrating superior antibacterial and antibiofilm properties against all UMs evaluated in this study. The results of this investigation showed that A. baumannii-derived TN and GA could be utilized as an alternative medication to treat UTIs.