The sterol C-24 methyltransferase encoding gene, erg6, is essential for viability of Aspergillus species.

Triazoles, the most widely used class of antifungal drugs, inhibit the biosynthesis of ergosterol, a crucial component of the fungal plasma membrane. Inhibition of a separate ergosterol biosynthetic step, catalyzed by the sterol C-24 methyltransferase Erg6, reduces the virulence of pathogenic yeasts, but its effects on filamentous fungal pathogens like Aspergillus fumigatus remain unexplored. Here, we show that the lipid droplet-associated enzyme Erg6 is essential for the viability of A. fumigatus and other Aspergillus species, including A. lentulus, A. terreus, and A. nidulans. Downregulation of erg6 causes loss of sterol-rich membrane domains required for apical extension of hyphae, as well as altered sterol profiles consistent with the Erg6 enzyme functioning upstream of the triazole drug target, Cyp51A/Cyp51B. Unexpectedly, erg6-repressed strains display wild-type susceptibility against the ergosterol-active triazole and polyene antifungals. Finally, we show that erg6 repression results in significant reduction in mortality in a murine model of invasive aspergillosis. Taken together with recent studies, our work supports Erg6 as a potentially pan-fungal drug target.

A secondary mechanism of action for triazole antifungals in Aspergillus fumigatus mediated by hmg1.

Triazole antifungals function as ergosterol biosynthesis inhibitors and are frontline therapy for invasive fungal infections, such as invasive aspergillosis. The primary mechanism of action of triazoles is through the specific inhibition of a cytochrome P450 14-α-sterol demethylase enzyme, Cyp51A/B, resulting in depletion of cellular ergosterol. Here, we uncover a clinically relevant secondary mechanism of action for triazoles within the ergosterol biosynthesis pathway. We provide evidence that triazole-mediated inhibition of Cyp51A/B activity generates sterol intermediate perturbations that are likely decoded by the sterol sensing functions of HMG-CoA reductase and Insulin-Induced Gene orthologs as increased pathway activity. This, in turn, results in negative feedback regulation of HMG-CoA reductase, the rate-limiting step of sterol biosynthesis. We also provide evidence that HMG-CoA reductase sterol sensing domain mutations previously identified as generating resistance in clinical isolates of Aspergillus fumigatus partially disrupt this triazole-induced feedback. Therefore, our data point to a secondary mechanism of action for the triazoles: induction of HMG-CoA reductase negative feedback for downregulation of ergosterol biosynthesis pathway activity. Abrogation of this feedback through acquired mutations in the HMG-CoA reductase sterol sensing domain diminishes triazole antifungal activity against fungal pathogens and underpins HMG-CoA reductase-mediated resistance.

The Aspergillus fumigatus maiA gene contributes to cell wall homeostasis and fungal virulence.

In this study, two distinct in vitro infection models of Aspergillus fumigatus, using murine macrophages (RAW264.7) and human lung epithelial cells (A549), were employed to identify the genes important for fungal adaptation during infection. Transcriptomic analyses of co-incubated A. fumigatus uncovered 140 fungal genes up-regulated in common between both models that, when compared with a previously published in vivo transcriptomic study, allowed the identification of 13 genes consistently up-regulated in all three infection conditions. Among them, the maiA gene, responsible for a critical step in the L-phenylalanine degradation pathway, was identified. Disruption of maiA resulted in a mutant strain unable to complete the Phe degradation pathway, leading to an excessive production of pyomelanin when this amino acid served as the sole carbon source. Moreover, the disruption mutant exhibited noticeable cell wall abnormalities, with reduced levels of ß-glucans within the cell wall but did not show lack of chitin or mannans. The maiA-1 mutant strain induced reduced inflammation in primary macrophages and displayed significantly lower virulence in a neutropenic mouse model of infection. This is the first study linking the A. fumigatus maiA gene to fungal cell wall homeostasis and virulence.

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 ß-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.

The sterol C-24 methyltransferase encoding gene, erg6, is essential for viability of Aspergillus species.

Ergosterol is a critical component of fungal plasma membranes. Although many currently available antifungal compounds target the ergosterol biosynthesis pathway for antifungal effect, current knowledge regarding ergosterol synthesis remains incomplete for filamentous fungal pathogens like Aspergillus fumigatus. Here, we show for the first time that the lipid droplet-associated sterol C-24 methyltransferase, Erg6, is essential for A. fumigatus viability. We further show that this essentiality extends to additional Aspergillus species, including A. lentulus, A. terreus, and A. nidulans. Neither the overexpression of a putative erg6 paralog, smt1, nor the exogenous addition of ergosterol could rescue erg6 deficiency. Importantly, Erg6 downregulation results in a dramatic decrease in ergosterol and accumulation in lanosterol and is further characterized by diminished sterol-rich plasma membrane domains (SRDs) at hyphal tips. Unexpectedly, erg6 repressed strains demonstrate wild-type susceptibility against the ergosterol-active triazole and polyene antifungals. Finally, repressing erg6 expression reduced fungal burden accumulation in a murine model of invasive aspergillosis. Taken together, our studies suggest that Erg6, which shows little homology to mammalian proteins, is potentially an attractive antifungal drug target for therapy of Aspergillus infections.

hapE and hmg1 Mutations Are Drivers of cyp51A-Independent Pan-Triazole Resistance in an Aspergillus fumigatus Clinical Isolate.

Aspergillus fumigatus is a ubiquitous environmental mold that can cause severe disease in immunocompromised patients and chronic disease in individuals with underlying lung conditions. Triazoles are the most widely used class of antifungal drugs to treat A. fumigatus infections, but their use in the clinic is threatened by the emergence of triazole-resistant isolates worldwide, reinforcing the need for a better understanding of resistance mechanisms. The predominant mechanisms of A. fumigatus triazole resistance involve mutations affecting the promoter region or coding sequence of the target enzyme of the triazoles, Cyp51A. However, triazole-resistant isolates without cyp51A-associated mutations are frequently identified. In this study, we investigate a pan-triazole-resistant clinical isolate, DI15-105, that simultaneously carries the mutations hapEP88L and hmg1F262del, with no mutations in cyp51A. Using a Cas9-mediated gene-editing system, hapEP88L and hmg1F262del mutations were reverted in DI15-105. Here, we show that the combination of these mutations accounts for pan-triazole resistance in DI15-105. To our knowledge, DI15-105 is the first clinical isolate reported to simultaneously carry mutations in hapE and hmg1 and only the second with the hapEP88L mutation. IMPORTANCE Triazole resistance is an important cause of treatment failure and high mortality rates for A. fumigatus human infections. Although Cyp51A-associated mutations are frequently identified as the cause of A. fumigatus triazole resistance, they do not explain the resistance phenotypes for several isolates. In this study, we demonstrate that hapE and hmg1 mutations additively contribute to pan-triazole resistance in an A. fumigatus clinical isolate lacking cyp51-associated mutations. Our results exemplify the importance of and the need for a better understanding of cyp51A-independent triazole resistance mechanisms.

Analysis of the cyp51 genes contribution to azole resistance in Aspergillus section Nigri with the CRISPR-Cas9 technique.

Cyp51 contribution to azole resistance has been broadly studied and characterized in Aspergillus fumigatus, whereas it remains poorly investigated in other clinically relevant species of the genus, such as those of section Nigri In this work, we aimed to analyze the impact of cyp51 genes (cyp51A and cyp51B) on the voriconazole (VRC) response and resistance of Aspergillus niger and Aspergillus tubingensis We generated CRISPR-Cas9 cyp51A and cyp51B knock-out mutants from strains with different genetic backgrounds and diverse patterns of azole susceptibility. Single gene deletions of cyp51 genes resulted in 2 to 16-fold decrease of the VRC Minimum Inhibitory Concentration (MIC) values, which were below the VRC Epidemiological Cutoff Value (ECV) established by the Clinical and Laboratory Standards Institute (CLSI) irrespective of their parental strains susceptibilities. Gene expression studies in the tested species confirmed that cyp51A participates more actively than cyp51B in the transcriptional response of azole stress. However, ergosterol quantification revealed that both enzymes comparably impact the total ergosterol content within the cell, as basal and VRC-induced changes to ergosterol content was similar in all cases. These data contribute to our understanding on Aspergillus azole resistance, especially in non-fumigatus species.

Genomic and Molecular Identification of Genes Contributing to the Caspofungin Paradoxical Effect in Aspergillus fumigatus.

Aspergillus fumigatus is a deadly opportunistic fungal pathogen responsible for ~100,000 annual deaths. Azoles are the first line antifungal agent used against A. fumigatus, but azole resistance has rapidly evolved making treatment challenging. Caspofungin is an important second-line therapy against invasive pulmonary aspergillosis, a severe A. fumigatus infection. Caspofungin functions by inhibiting ß-1,3-glucan synthesis, a primary and essential component of the fungal cell wall. A phenomenon termed the caspofungin paradoxical effect (CPE) has been observed in several fungal species where at higher concentrations of caspofungin, chitin replaces ß-1,3-glucan, morphology returns to normal, and growth rate increases. CPE appears to occur in vivo, and it is therefore clinically important to better understand the genetic contributors to CPE. We applied genomewide association (GWA) analysis and molecular genetics to identify and validate candidate genes involved in CPE. We quantified CPE across 67 clinical isolates and conducted three independent GWA analyses to identify genetic variants associated with CPE. We identified 48 single nucleotide polymorphisms (SNPs) associated with CPE. We used a CRISPR/Cas9 approach to generate gene deletion mutants for seven genes harboring candidate SNPs. Two null mutants, ΔAfu3g13230 and ΔAfu4g07080 (dscP), resulted in reduced basal growth rate and a loss of CPE. We further characterized the dscP phosphatase-null mutant and observed a significant reduction in conidia production and extremely high sensitivity to caspofungin at both low and high concentrations. Collectively, our work reveals the contribution of Afu3g13230 and dscP in CPE and sheds new light on the complex genetic interactions governing this phenotype. IMPORTANCE This is one of the first studies to apply genomewide association (GWA) analysis to identify genes involved in an Aspergillus fumigatus phenotype. A. fumigatus is an opportunistic fungal pathogen that causes hundreds of thousands of infections and ~100,000 deaths each year, and antifungal resistance has rapidly evolved in this species. A phenomenon called the caspofungin paradoxical effect (CPE) occurs in some isolates, where high concentrations of the drug lead to increased growth rate. There is clinical relevance in understanding the genetic basis of this phenotype, since caspofungin concentrations could lead to unintended adverse clinical outcomes in certain cases. Using GWA analysis, we identified several interesting candidate polymorphisms and genes and then generated gene deletion mutants to determine whether these genes were important for CPE. Two of these mutant strains (ΔAfu3g13230 and ΔAfu4g07080/ΔdscP) displayed a loss of the CPE. This study sheds light on the genes involved in clinically important phenotype CPE.

Comparative Genomics Reveals a Single Nucleotide Deletion in pksP That Results in White-Spore Phenotype in Natural Variants of Aspergillus fumigatus.

Aspergillus fumigatus is a potentially deadly opportunistic human pathogen. A. fumigatus has evolved a variety of mechanisms to evade detection by the immune system. For example, the conidium surface is covered in a layer of 1,8-dihydroxynaphthalene (DHN) melanin which masks the antigen macrophages use for recognition. DHN melanin also protects conidia from ultraviolet radiation and gives A. fumigatus conidia their characteristic green-grayish color. Here, we conducted genomic analysis of two closely related white-spore natural variants of A. fumigatus in comparison to two closely related green-spore isolates to identify a genetic basis of the white-spore phenotype. Illumina whole-genome resequencing data of the four isolates was used to identify variants that were shared in the white-spore isolates and different from both the green-spore isolates and the Af293 reference genome (which is also a green-spore isolate). We identified 4,279 single nucleotide variants and 1,785 insertion/deletions fitting this pattern. Among these, we identified 64 variants predicted to be high impact, loss-of-function mutations. One of these variants is a single nucleotide deletion that results in a frameshift in pksP (Afu2g17600), the core biosynthetic gene in the DHN melanin encoding gene cluster. The frameshift mutation in the white-spore isolates leads to a truncated protein in which a phosphopantetheine attachment site (PP-binding domain) is interrupted and an additional PP-binding domain and a thioesterase domain are omitted. Growth rate analysis of white-spore and green-spore isolates at 37°C and 48°C revealed that white-spore isolates are thermosensitive. Growth rate of A. fumigatus Af293 and a pksP null mutant in the Af293 background suggests pksP is not directly involved in the thermosensitivity phenotype. Further, our study identified a mutation in a gene (Afu4g04740) associated with thermal sensitivity in yeasts which could also be responsible for the thermosensitivity of the white-spore mutants. Overall, we used comparative genomics to identify the mutation and protein alterations responsible for the white-spore phenotype of environmental isolates of A. fumigatus.

Loss of Septation Initiation Network (SIN) kinases blocks tissue invasion and unlocks echinocandin cidal activity against Aspergillus fumigatus.

Although considered effective treatment for many yeast fungi, the therapeutic efficacy of the echinocandin class of antifungals for invasive aspergillosis (IA) is limited. Recent studies suggest intense kinase- and phosphatase-mediated echinocandin adaptation in A. fumigatus. To identify A. fumigatus protein kinases required for survival under echinocandin stress, we employed CRISPR/Cas9-mediated gene targeting to generate a protein kinase disruption mutant library in a wild type genetic background. Cell wall and echinocandin stress screening of the 118 disruption mutants comprising the library identified only five protein kinase disruption mutants displaying greater than 4-fold decreased echinocandin minimum effective concentrations (MEC) compared to the parental strain. Two of these mutated genes, the previously uncharacterized A. fumigatus sepL and sidB genes, were predicted to encode protein kinases functioning as core components of the Septation Initiation Network (SIN), a tripartite kinase cascade that is necessary for septation in fungi. As the A. fumigatus SIN is completely uncharacterized, we sought to explore these network components as effectors of echinocandin stress survival. Our data show that mutation of any single SIN kinase gene caused complete loss of hyphal septation and increased susceptibility to cell wall stress, as well as widespread hyphal damage and loss of viability in response to echinocandin stress. Strikingly, mutation of each SIN kinase gene also resulted in a profound loss of virulence characterized by lack of tissue invasive growth. Through the deletion of multiple novel regulators of hyphal septation, we show that the non-invasive growth phenotype is not SIN-kinase dependent, but likely due to hyphal septation deficiency. Finally, we also find that echinocandin therapy is highly effective at eliminating residual tissue burden in mice infected with an aseptate strain of A. fumigatus. Together, our findings suggest that inhibitors of septation could enhance echinocandin-mediated killing while simultaneously limiting the invasive potential of A. fumigatus hyphae.

Overexpression of the Aspergillus fumigatus Small GTPase, RsrA, Promotes Polarity Establishment during Germination.

Cell polarization comprises highly controlled processes and occurs in most eukaryotic organisms. In yeast, the processes of budding, mating and filamentation require coordinated mechanisms leading to polarized growth. Filamentous fungi, such as Aspergillus fumigatus, are an extreme example of cell polarization, essential for both vegetative and pathogenic growth. A major regulator of polarized growth in yeast is the small GTPase Rsr1, which is essential for bud-site selection. Here, we show that deletion of the putative A. fumigatus ortholog, rsrA, causes only a modest reduction of growth rate and delay in germ tube emergence. In contrast, overexpression of rsrA results in a morphogenesis defect, characterized by a significant delay in polarity establishment followed by the establishment of multiple growth axes. This aberrant phenotype is reversed when rsrA expression levels are decreased, suggesting that correct regulation of RsrA activity is crucial for accurate patterning of polarity establishment. Despite this finding, deletion or overexpression of rsrA resulted in no changes of A. fumigatus virulence attributes in a mouse model of invasive aspergillosis. Additional mutational analyses revealed that RsrA cooperates genetically with the small GTPase, RasA, to support A. fumigatus viability.

Mechanisms of triazole resistance in Aspergillus fumigatus.

The ubiquitous fungal pathogen Aspergillus fumigatus is the primary cause of opportunistic mould infections in humans. Aspergilli disseminate via asexual conidia passively travelling through air currents to germinate within a broad range of environs, wherever suitable nutrients are found. Though the average human inhales hundreds of conidia daily, A. fumigatus invasive infections primarily affect the immunocompromised. At-risk individuals can develop often fatal invasive disease for which therapeutic options are limited. Regrettably, the global insurgence of isolates resistant to the triazoles, the frontline antifungal class used in medicine and agriculture to control A. fumigatus, is complicating the treatment of patients. Triazole antifungal resistance in A. fumigatus has become recognized as a global, yet poorly comprehended, problem. Due to a multitude of factors, the magnitude of resistant infections and their contribution to treatment outcomes are likely underestimated. Current studies suggest that human drug-resistant infections can be either environmentally acquired or de novo host selected during patient therapy. While much concerning development of resistance is yet unknown, recent investigations have revealed assorted underlying mechanisms enabling triazole resistance within individual clinical and environmental isolates. This review will provide an overview of triazole resistance as it is currently understood, as well as highlight some of the prominent biological mechanisms associated with clinical and environmental resistance to triazoles in A. fumigatus.

Characterization of the Efflux Capability and Substrate Specificity of Aspergillus fumigatus PDR5-like ABC Transporters Expressed in Saccharomyces cerevisiae.

This research analyzed six Aspergillus fumigatus genes encoding putative efflux proteins for their roles as transporters. TheA. fumigatus genes abcA, abcC, abcF, abcG, abcH, and abcI were cloned into plasmids and overexpressed in a Saccharomyces cerevisiae strain in which the highly active endogenous ABC transporter gene PDR5 was deleted. The activity of each transporter was measured by efflux of rhodamine 6G and accumulation of alanine ß-naphthylamide. The transporters AbcA, AbcC, and AbcF had the strongest efflux activities of these compounds. All of the strains with plasmid-expressed transporters had more efflux activity than did the PDR5-deleted background strain. We performed broth microdilution drug susceptibility testing and agar spot assays using an array of compounds and antifungal drugs to determine the transporter specificity and drug susceptibility of the strains. The transporters AbcC and AbcF showed the broadest range of substrate specificity, while AbcG and AbcH had the narrowest range of substrates. Strains expressing the AbcA, AbcC, AbcF, or AbcI transporter were more resistant to fluconazole than was the PDR5-deleted background strain. Strains expressing AbcC and AbcF were additionally more resistant to clotrimazole, itraconazole, ketoconazole, and posaconazole than was the background strain. Finally, we analyzed the expression levels of the genes by reverse transcription-quantitative PCR (RT-qPCR) in triazole-susceptible and -resistant A. fumigatus clinical isolates. All of these transporters are expressed at a measurable level, and transporter expression varied significantly between strains, demonstrating the high degree of phenotypic variation, plasticity, and divergence of which this species is capable.IMPORTANCE One mechanism behind drug resistance is altered export out of the cell. This work is a multifaceted analysis of membrane efflux transporters in the human fungal pathogen A. fumigatus Bioinformatics evidence infers that there is a relatively large number of genes in A. fumigatus that encode ABC efflux transporters. However, very few of these transporters have been directly characterized and analyzed for their potential role in drug resistance.Our objective was to determine if these undercharacterized proteins function as efflux transporters and then to better define whether their efflux substrates include antifungal drugs used to treat fungal infections. We chose six A. fumigatus potential plasma membrane ABC transporter genes for analysis and found that all six genes produced functional transporter proteins. We used two fungal systems to look for correlations between transporter function and drug resistance. These transporters have the potential to produce drug-resistant phenotypes in A. fumigatus Continued characterization of these and other transporters may assist in the development of efflux inhibitor drugs.

Genome-Wide Association for Itraconazole Sensitivity in Non-resistant Clinical Isolates of Aspergillus fumigatus.

Aspergillus fumigatus is a potentially lethal opportunistic pathogen that infects over ~200,000 people and causes ~100,000 deaths per year globally. Treating A. fumigatus infections is particularly challenging because of the recent emergence of azole-resistance. The majority of studies focusing on the molecular mechanisms underlying azole resistance have examined azole-resistant isolates. However, isolates that are susceptible to azoles also display variation in their sensitivity, presenting a unique opportunity to identify genes contributing to azole sensitivity. Here, we used genome-wide association (GWA) analysis to identify loci involved in azole sensitivity by analyzing the association between 68,853 SNPs and itraconazole (ITCZ) minimum inhibitory concentration (MIC) in 76 clinical isolates of A. fumigatus from Japan. Population structure analysis suggests the presence of four distinct populations, with ITCZ MICs distributed relatively evenly across populations. We independently conducted GWA when treating ITCZ MIC as a quantitative trait and a binary trait, and identified two SNPs with strong associations in both analyses. These SNPs fell within the coding regions of Afu2g02220 and Afu2g02140. We functionally validated Afu2g02220 by knocking it out using a CRISPR/Cas9 approach, because orthologs of this gene are involved in sterol modification and ITCZ targets the ergosterol biosynthesis pathway. Knockout strains displayed no difference in growth compared to the parent strain in minimal media, yet a minor but consistent inhibition of growth in the presence of 0.15 µg/ml ITCZ. Our results suggest that GWA paired with efficient gene deletion is a powerful and unbiased strategy for identifying the genetic basis of complex traits in A. fumigatus.

Differential requirements of protein geranylgeranylation for the virulence of human pathogenic fungi.

Protein prenylation is a crucial post-translational modification largely mediated by two heterodimeric enzyme complexes, farnesyltransferase and geranylgeranyltransferase type-I (GGTase-I), each composed of a shared α-subunit and a unique ß-subunit. GGTase-I enzymes are validated drug targets that contribute to virulence in Cryptococcus neoformans and to the yeast-to-hyphal transition in Candida albicans. Therefore, we sought to investigate the importance of the α-subunit, RamB, and the ß-subunit, Cdc43, of the A. fumigatus GGTase-I complex to hyphal growth and virulence. Deletion of cdc43 resulted in impaired hyphal morphogenesis and thermo-sensitivity, which was exacerbated during growth in rich media. The Δcdc43 mutant also displayed hypersensitivity to cell wall stress agents and to cell wall synthesis inhibitors, suggesting alterations of cell wall biosynthesis or stress signaling. In support of this, analyses of cell wall content revealed decreased amounts of ß-glucan in the Δcdc43 strain. Despite strong in vitro phenotypes, the Δcdc43 mutant was fully virulent in two models of murine invasive aspergillosis, similar to the control strain. We further found that a strain expressing the α-subunit gene, ramB, from a tetracycline-inducible promoter was inviable under non-inducing in vitro growth conditions and was virtually avirulent in both mouse models. Lastly, virulence studies using C. albicans strains with tetracycline-repressible RAM2 or CDC43 expression revealed reduced pathogenicity associated with downregulation of either gene in a murine model of disseminated infection. Together, these findings indicate a differential requirement for protein geranylgeranylation for fungal virulence, and further inform the selection of specific prenyltransferases as promising antifungal drug targets for each pathogen.

Mutations in hmg1, Challenging the Paradigm of Clinical Triazole Resistance in Aspergillus fumigatus.

Aspergillus fumigatus is the predominant pathogen of invasive aspergillosis, a disease state credited with over 200,000 life-threatening infections each year. The triazole class of antifungals are clinically essential to the treatment of invasive aspergillosis, both as frontline and as salvage therapy. Unfortunately, resistance to the triazoles among A. fumigatus isolates is now increasingly reported worldwide, and a large proportion of this resistance remains unexplained. In this work, we characterize the contributions of previously identified mechanisms of triazole resistance, including mutations in the sterol-demethylase-encoding gene cyp51A, overexpression of sterol-demethylase genes, and overexpression of the efflux pump-encoding gene abcC, among a large collection of highly triazole-resistant clinical A. fumigatus isolates. Upon revealing that these mechanisms alone cannot substantiate the majority of triazole resistance exhibited by this collection, we subsequently describe the identification and characterization of a novel genetic determinant of triazole resistance. Mutations in the 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase-encoding gene, hmg1, were identified in a majority of triazole-resistant clinical isolates in our collection. Introduction of three different hmg1 mutations, predicted to encode residue alterations in the conserved sterol sensing domain of Hmg1, resulted in significantly increased resistance to the triazole class of agents. Additionally, correction of a hmg1 mutation in a pan-triazole-resistant clinical isolate of A. fumigatus with a novel Cas9-ribonucleoprotein-mediated system was shown to restore clinical susceptibility to all triazole agents. Mutations in hmg1 were also shown to lead to the accumulation of ergosterol precursors, such as eburicol, by sterol profiling, while not altering the expression of sterol-demethylase genes.IMPORTANCEAspergillus fumigatus is the predominant pathogen of invasive aspergillosis, a disease state credited with over 200,000 life-threatening infections annually. The triazole class of antifungals are clinically essential to the treatment of invasive aspergillosis. Unfortunately, resistance to the triazoles among A. fumigatus isolates is now increasingly reported worldwide. In this work, we challenge the current paradigm of clinical triazole resistance in A. fumigatus, by first demonstrating that previously characterized mechanisms of resistance have nominal impact on triazole susceptibility and subsequently identifying a novel mechanism of resistance with a profound impact on clinical triazole susceptibility. We demonstrate that mutations in the HMG-CoA reductase gene, hmg1, are common among resistant clinical isolates and that hmg1 mutations confer resistance to all clinically available triazole antifungals.

SH3-class Ras guanine nucleotide exchange factors are essential for Aspergillus fumigatus invasive growth.

Proper hyphal morphogenesis is essential for the establishment and progression of invasive disease caused by filamentous fungi. In the human pathogen Aspergillus fumigatus, signalling cascades driven by Ras and Ras-like proteins orchestrate a wide variety of cellular processes required for hyphal growth. For activation, these proteins require interactions with Ras-subfamily-specific guanine nucleotide exchange factors (RasGEFs). Although Ras-protein networks are essential for virulence in all pathogenic fungi, the importance of RasGEF proteins is largely unexplored. A. fumigatus encodes four putative RasGEFs that represent three separate classes of RasGEF proteins (SH3-, Ras guanyl nucleotide-releasing protein [RasGRP]-, and LTE-class), each with fungus-specific attributes. Here, we show that the SH3-class and RasGRP-class RasGEFs are required for properly timed polarity establishment during early growth and branch emergence as well as for cell wall stability. Further, we show that SH3-class RasGEF activity is essential for polarity establishment and maintenance, a phenotype that is, at least, partially independent of the major A. fumigatus Ras proteins, RasA and RasB. Finally, loss of both SH3-class RasGEFs resulted in avirulence in multiple models of invasive aspergillosis. Together, our findings suggest that RasGEF activity is essential for the integration of multiple signalling networks to drive invasive growth in A. fumigatus.

Emerging threat of triazole-resistant Aspergillus fumigatus.

Invasive aspergillosis is a leading cause of morbidity and mortality among immunocompromised populations and is predicted to cause more than 200 000 life-threatening infections each year. Aspergillus fumigatus is the most prevalent pathogen isolated from patients with invasive aspergillosis, accounting for more than 60% of all cases. Currently, the only antifungal agents available with consistent activity against A. fumigatus are the mould-active triazoles and amphotericin B, of which the triazoles commonly represent both front-line and salvage therapeutic options. Unfortunately, the treatment of infections caused by A. fumigatus has recently been further complicated by the global emergence of triazole resistance among both clinical and environmental isolates. Mutations in the A. fumigatus sterol-demethylase gene cyp51A, overexpression of cyp51A and overexpression of efflux pump genes are all known to contribute to resistance, yet much of the triazole resistance among A. fumigatus still remains unexplained. Also lacking is clinical experience with therapeutic options for the treatment of triazole-resistant A. fumigatus infections and mortality associated with these infections remains unacceptably high. Thus, further research is greatly needed to both better understand the emerging threat of triazole-resistant A. fumigatus and to develop novel therapeutic strategies to combat these resistant infections.

Whole-genome sequencing reveals highly specific gene targeting by in vitro assembled Cas9-ribonucleoprotein complexes in Aspergillus fumigatus.

BACKGROUND: CRISPR/Cas9-based genome editing is quickly becoming a powerful tool within the field of fungal genetics. Adaptation of CRISPR/Cas9 systems are allowing for rapid and highly efficient gene targeting within fungi. We recently reported the adaptation of a simple CRISPR/Cas9 system for gene deletion that is effective across multiple genetic backgrounds of Aspergillus fumigatus. This system employs in vitro assembly of Cas9 ribonucleoproteins (RNPs) coupled with micro-homology repair templates for gene deletion. Although highly efficient at gene targeting in wild type genetic backgrounds of A. fumigatus, the potential for our system to produce unwanted off-target mutations has not been addressed. RESULTS: Next-generation Illumina sequencing was used to identify genome mutations among transformants isolated from standard (no Cas9) and Cas9-mediated integration of a hygromycin deletion cassette. Two different concentrations of Cas9 were utilized to examine the association of Cas9 concentration with total numbers and types of genomic mutations. For each of the three test groups (zero, low, and high Cas9), three transformants were sequenced and compared to the parent strain. Bioinformatics analyses revealed the average number of total mutations to be similar among all three test groups. A. fumigatus transformation using standard, non-Cas9-mediated methods resulted in an average of 373 ± 28 mutations. In comparison, transformation with in vitro assembled Cas9-RNPs using either high (1 µg/µl) or low (0.5 µg/µl) levels of Cas9 resulted in an average of 326 ± 19 and 395 ± 69 mutations, respectively. In all cases, the vast majority of mutations identified were intergenic. No correlation between the amount of Cas9 utilized for transformation and the overall number of mutations was found. Finally, the specific type of mutation introduced during the transformation process was not Cas9-dependent, as both single-nucleotide polymorphisms and insertion/deletion events were not significantly different between the experimental groups. CONCLUSIONS: CRISPR/Cas9-based genome editing in A. fumigatus using in vitro assembled RNPs coupled with microhomology templates is a reliable method of gene targeting. This system is highly efficient and is not associated with increased off-target mutations caused by introduction of the Cas9 nuclease.

Commonly Used Oncology Drugs Decrease Antifungal Effectiveness against Candida and Aspergillus Species.

The incidence of invasive fungal infections has risen significantly in recent decades as medical interventions have become increasingly aggressive. These infections are extremely difficult to treat due to the extremely limited repertoire of systemic antifungals, the development of drug resistance, and the extent to which the patient's immune function is compromised. Even when the appropriate antifungal therapies are administered in a timely fashion, treatment failure is common, even in the absence of in vitro microbial resistance. In this study, we screened a small collection of FDA-approved oncolytic agents for compounds that impact the efficacy of the two most widely used classes of systemic antifungals against Candida albicans, Candida glabrata, and Aspergillus fumigatus We have identified several drugs that enhance fungal growth in the presence of azole antifungals and examine the potential that these drugs directly affect fungal fitness, specifically antifungal susceptibility, and may be contributing to clinical treatment failure.