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.

Aspergillus fumigatus Acetate Utilization Impacts Virulence Traits and Pathogenicity.

Aspergillus fumigatus is a major opportunistic fungal pathogen of immunocompromised and immunocompetent hosts. To successfully establish an infection, A. fumigatus needs to use host carbon sources, such as acetate, present in the body fluids and peripheral tissues. However, utilization of acetate as a carbon source by fungi in the context of infection has not been investigated. This work shows that acetate is metabolized via different pathways in A. fumigatus and that acetate utilization is under the regulatory control of a transcription factor (TF), FacB. A. fumigatus acetate utilization is subject to carbon catabolite repression (CCR), although this is only partially dependent on the TF and main regulator of CCR CreA. The available extracellular carbon source, in this case glucose and acetate, significantly affected A. fumigatus virulence traits such as secondary metabolite secretion and cell wall composition, with the latter having consequences for resistance to oxidative stress, antifungal drugs, and human neutrophil-mediated killing. Furthermore, deletion of facB significantly impaired the in vivo virulence of A. fumigatus in both insect and mammalian models of invasive aspergillosis. This is the first report on acetate utilization in A. fumigatus, and this work further highlights the importance of available host-specific carbon sources in shaping fungal virulence traits and subsequent disease outcome, and a potential target for the development of antifungal strategies. IMPORTANCE Aspergillus fumigatus is an opportunistic fungal pathogen in humans. During infection, A. fumigatus is predicted to use host carbon sources, such as acetate, present in body fluids and peripheral tissues, to sustain growth and promote colonization and invasion. This work shows that A. fumigatus metabolizes acetate via different pathways, a process that is dependent on the transcription factor FacB. Furthermore, the type and concentration of the extracellular available carbon source were determined to shape A. fumigatus virulence determinants such as secondary metabolite secretion and cell wall composition. Subsequently, interactions with immune cells are altered in a carbon source-specific manner. FacB is required for A. fumigatus in vivo virulence in both insect and mammalian models of invasive aspergillosis. This is the first report that characterizes acetate utilization in A. fumigatus and highlights the importance of available host-specific carbon sources in shaping virulence traits and potentially subsequent disease outcome.

Novel Biological Functions of the NsdC Transcription Factor in Aspergillus fumigatus.

The fungal zinc finger transcription factor NsdC is named after, and is best known for, its essential role in sexual reproduction (never in sexual development). In previous studies with Aspergillus nidulans, it was also shown to have roles in promotion of vegetative growth and suppression of asexual conidiation. In this study, the function of the nsdC homologue in the opportunistic human pathogen A. fumigatus was investigated. NsdC was again found to be essential for sexual development, with deletion of the nsdC gene in both MAT1-1 and MAT1-2 mating partners of a cross leading to complete loss of fertility. However, a functional copy of nsdC in one mating partner was sufficient to allow sexual reproduction. Deletion of nsdC also led to decreased vegetative growth and allowed conidiation in liquid cultures, again consistent with previous findings. However, NsdC in A. fumigatus was shown to have additional biological functions including response to calcium stress, correct organization of cell wall structure, and response to the cell wall stressors. Furthermore, virulence and host immune recognition were affected. Gene expression studies involving chromatin immunoprecipitation (ChIP) of RNA polymerase II (PolII) coupled to next-generation sequencing (Seq) revealed that deletion of nsdC resulted in changes in expression of over 620 genes under basal growth conditions. This demonstrated that this transcription factor mediates the activity of a wide variety of signaling and metabolic pathways and indicates that despite the naming of the gene, the promotion of sexual reproduction is just one among multiple roles of NsdC.IMPORTANCEAspergillus fumigatus is an opportunistic human fungal pathogen and the main causal agent of invasive aspergillosis, a life-threatening infection especially in immunocompromised patients. A. fumigatus can undergo both asexual and sexual reproductive cycles, and the regulation of both cycles involves several genes and pathways. Here, we have characterized one of these genetic determinants, the NsdC transcription factor, which was initially identified in a screen of transcription factor null mutants showing sensitivity when exposed to high concentrations of calcium. In addition to its known essential roles in sexual reproduction and control of growth rate and asexual reproduction, we have shown in the present study that A. fumigatus NsdC transcription factor has additional previously unrecognized biological functions including calcium tolerance, cell wall stress response, and correct cell wall organization and functions in virulence and host immune recognition. Our results indicate that NsdC can play novel additional biological functions not directly related to its role played during sexual and asexual processes.

Genome-wide transcriptome analysis of Aspergillus fumigatus exposed to osmotic stress reveals regulators of osmotic and cell wall stresses that are SakAHOG1 and MpkC dependent.

Invasive aspergillosis is predominantly caused by Aspergillus fumigatus, and adaptations to stresses experienced within the human host are a prerequisite for the survival and virulence strategies of the pathogen. The central signal transduction pathway operating during hyperosmotic stress is the high osmolarity glycerol mitogen-activated protein kinase cascade. A. fumigatus MpkC and SakA, orthologues of the Saccharomyces cerevisiae Hog1p, constitute the primary regulator of the hyperosmotic stress response. We compared A. fumigatus wild-type transcriptional response to osmotic stress with the ΔmpkC, ΔsakA, and ΔmpkC ΔsakA strains. Our results strongly indicate that MpkC and SakA have independent and collaborative functions during the transcriptional response to transient osmotic stress. We have identified and characterized null mutants for four A. fumigatus basic leucine zipper proteins transcription factors. The atfA and atfB have comparable expression levels with the wild-type in ΔmpkC but are repressed in ΔsakA and ΔmpkC ΔsakA post-osmotic stress. The atfC and atfD have reduced expression levels in all mutants post-osmotic stress. The atfA-D null mutants displayed several phenotypes related to osmotic, oxidative, and cell wall stresses. The ΔatfA and ΔatfB were shown to be avirulent and to have attenuated virulence, respectively, in both Galleria mellonella and a neutropenic murine model of invasive pulmonary aspergillosis.