Biology subjects

Kerkaert, J. D.

Publications and source records attributed to Kerkaert, J. D..

6 recordsLinked to original sources

bioRxiv2026

High-throughput functional genomics and transcriptomics reveal molecular mechanisms regulating the synergistic fungicidal activity of copper and zinc

Fungal diseases cause devastating crop infestations and millions of deaths annually. To improve disease treatment development, we must understand the biological processes these treatments target and the fungal cellular responses they elicit. Current methods to identify genetic responses of fungi to antifungal drugs and fungicides typically involve laborious molecular genetics experiments or transcriptomics. We utilized high-throughput screens in the basidiomycete yeast Rhodotorula (Rhodosporidium) toruloides to rapidly identify genes associated with the fungal response to a copper-zinc fungicide developed by VM Agritech and compared identified genes to those transcriptionally activated in response to the fungicide in R. toruloides and the filamentous ascomycete Neurospora crassa. These 'omics technologies combined with molecular genetics and biochemical analyses showed copper and zinc synergize to inhibit fungal growth due, at least in part, to an increase in cellular zinc concentrations when copper is present. Intriguingly, although we identified genes involved in the copper-zinc fungicide response using both high-throughput screens and transcriptomics, there was little overlap in genes identified through the two methods. Our findings demonstrate the power of combining functional genomics and transcriptomics approaches in identifying a more comprehensive map of the genetic mechanisms necessary for responding to fungicides.

microbiology↗

bioRxiv2025

Transcriptional analysis of developing Aspergillus fumigatus biofilms reveals metabolic shifts required for biofilm maintenance

Aspergillus fumigatus is a filamentous fungus found in compost and soil that can cause invasive and/or chronic disease in a broad spectrum of individuals. Diagnosis and treatment of aspergillosis often occur during stages of infection when A. fumigatus has formed dense networks of hyphae within the lung. These dense hyphal networks are multicellular, encased in a layer of extracellular matrix, and have reduced susceptibility to contemporary antifungal drugs, characteristics which are defining features of a microbial biofilm. A mode of growth similar to these dense hyphal networks observed in vivo can be recapitulated in vitro using a static, submerged biofilm culture model. The mechanisms underlying filamentous fungal cell physiology at different stages of biofilm development remain to be defined. Here, we utilized an RNA sequencing approach to evaluate changes in transcript levels during A. fumigatus biofilm development. These analyses revealed an increase in transcripts associated with fermentation and a concomitant decrease in oxidative phosphorylation related transcripts. Further investigation revealed that ethanol and butanediol fermentation is important for mature biofilm biomass maintenance. Correspondingly, a gene (silG), a predicted transcription factor, was observed to also be required for mature biofilm biomass maintenance. Taken together, these data suggest temporal changes in A. fumigatus metabolism during biofilm development are required to maintain a fully mature biofilm. IMPORTANCEAspergillus fumigatus is the most common etiological agent of a collection of diseases termed aspergillosis. Invasive Pulmonary Aspergillosis (IPA), a severe form of aspergillosis, is highlighted by invasive growth of fungal hyphae into host lung tissue. Strains that are susceptible to antifungal therapies in vitro frequently fail to respond to treatment in vivo, resulting in high mortality rates even with treatment. It is now appreciated that this decreased antifungal efficacy in vivo is, in part, likely due to biofilm-like growth of the fungus. A. fumigatus biofilms have been shown to develop regions of limited oxygen availability that are hypothesized to induce cell quiescence and drug resistance. Understanding the mechanisms by which A. fumigatus induces, develops, and maintains biofilms to evade antifungal therapies is expected to illuminate biofilm-specific therapeutic targets. Here we present transcriptomics data of developing A. fumigatus biofilms and from these data define genes related to fungal fermentation and regulation of transcription important for maintenance of mature A. fumigatus biofilms.

microbiology↗

bioRxiv2025

Transcription factor reveals interconnected regulation of carbon source utilization and carbon catabolite repression in an oleaginous yeast

Cells must sense and respond to nutrients to survive. To efficiently grow in mixed carbon environments, microbes repress genes necessary to utilize carbon sources that require substantial resources to catabolize when a simpler carbon source, such as glucose, is present. This process is known as carbon catabolite repression. Canonically, in fungi, nutrient sensing transcriptional networks are composed of carbon source-specific transcription factors that activate carbon source utilization genes and carbon catabolite repression regulators, which broadly repress all nonpreferred carbon source utilization genes when a preferred carbohydrate is present. In contrast to this model, we identified a transcription factor (Cbr1) in the basidomycete yeast Rhodotorula (Rhodosporidium) toruloides that specifically inhibits glucose-mediated repression of glucose-glucose disaccharide utilization, presenting a mechanism of tailored carbon catabolite repression regulation that combats a negative feedback loop formed when glucose is released during disaccharide utilization. Cbr1 is also required for cellobiose, carboxylic acid, and fucose utilization. Using transcriptomic and molecular analyses, we demonstrated that catabolism of these carbon sources is not metabolically linked, but genes necessary for their utilization are coactivated by Cbr1 in response to each of the carbon sources. This coactivation suggests R. toruloides may encounter these carbon sources together, potentially during complex interactions among microbes in nature. Coregulation of nutrient-specific gene activation and carbon catabolite repression by a transcription factor establishes a previously uncharacterized mechanism for building nutrient sensing transcriptional networks in fungi. Characterizing diverse nutrient sensing regulatory mechanisms is critical for understanding resource acquisition during fungal pathogenesis, where carbon catabolite repression is important for virulence and drug tolerance, and metabolically engineering fungi for green biotechnology.

genetics↗

bioRxiv2021

An Alanine Aminotransferase is Required for Polysaccharide Regulation and Resistance of Aspergillus fumigatus Biofilms to Echinocandin Treatment

Alanine metabolism has been suggested as an adaptation strategy to oxygen limitation in organisms ranging from plants to mammals. Within the pulmonary infection microenvironment A. fumigatus forms biofilms with steep oxygen gradients defined by regions of oxygen limitation. A significant increase in alanine levels was observed in A. fumigatus cultured under oxygen limiting conditions. An alanine aminotransferase, AlaA, was observed to function in alanine catabolism and is required for several aspects of A. fumigatus biofilm physiology. Loss of alaA, or its catalytic activity, results in decreased adherence of biofilms through a defect in the maturation of the extracellular matrix polysaccharide galactosaminogalactan (GAG). Additionally, exposure of cell wall polysaccharides is also impacted by loss of alaA and loss of AlaA catalytic activity confers increased biofilm susceptibility to echinocandin treatment which is correlated with enhanced fungicidal activity. The increase in echinocandin susceptibility is specific to biofilms and chemical inhibition of alaA by the alanine aminotransferase inhibitor {beta}-chloro-L-alanine is sufficient to sensitize A. fumigatus biofilms to echinocandin treatment. Finally, loss of alaA increases susceptibility of A. fumigatus to in vivo echinocandin treatment in a murine model of invasive pulmonary aspergillosis. Our results provide insight into the interplay of metabolism, biofilm formation, and antifungal drug resistance in A. fumigatus and describes a mechanism of increasing susceptibility of A. fumigatus biofilms to the echinocandin class of antifungal drugs. eLife DigestAspergillus fumigatus is a ubiquitous filamentous fungus that causes an array of diseases depending on the immune status of an individual, collectively termed aspergillosis. Antifungal therapy for invasive pulmonary aspergillosis (IPA) or chronic pulmonary aspergillosis (CPA) is limited and too often ineffective. This is in part due to A. fumigatus biofilm formation within the infection environment and the resulting emergent properties, particularly increased antifungal resistance. Thus, insights into biofilm formation and mechanisms driving increased antifungal drug resistance are critical for improving existing therapeutic strategies and development of novel antifungals. In this work, we describe an unexpected observation where alanine metabolism, via the alanine aminotransferase AlaA, is required for several aspects of A. fumigatus biofilm physiology including resistance of A. fumigatus biofilms to the echinocandin class of antifungal drugs. Importantly, we observed that chemical inhibition of alanine aminotransferases is sufficient to increase echinocandin susceptibility and that loss of alaA increases susceptibility to echinocandin treatment in a murine model of IPA.

microbiology↗

bioRxiv2020

Strain specific persistence in the murine lung of Aspergillus fumigatus conidia causes an Allergic Broncho-Pulmonary Aspergillosis-like disease phenotype

Aspergillus fumigatus is a filamentous fungus which can cause multiple diseases in humans. Allergic Broncho-pulmonary Aspergillosis (ABPA) is a disease diagnosed primarily in Cystic Fibrosis patients caused by a severe allergic response often to long-term A. fumigatus colonization in the lungs. Mice develop an allergic response to repeated inhalation of A. fumigatus spores; however, no strains have been identified that can survive long-term in the mouse lung and cause ABPA-like disease. We characterized A. fumigatus strain W72310 by whole genome sequencing and in vitro and in vivo viability assays in comparison to a common reference strain, CEA10. W72310 was resistant to leukocyte-mediated killing and persisted in the mouse lung longer than CEA10, a phenotype that correlated with greater resistance to oxidative stressors, hydrogen peroxide and menadione, in vitro. In animals both sensitized and challenged with W72310, conidia, but not hyphae, were viable in the lungs for up to 21 days in association with eosinophilic airway inflammation, airway leakage, serum IgE, and mucus production. W72310-sensitized mice that were recall-challenged with conidia had increased inflammation, Th1 and Th2 cytokines, and airway leakage compared to controls. Collectively, our studies demonstrate that a unique strain of A. fumigatus resistant to leukocyte killing can persist in the mouse lung in conidial form and elicit features of ABPA-like disease. IMPORTANCEAllergic Broncho-pulmonary Aspergillosis (ABPA) patients often present with long-term colonization of Aspergillus fumigatus. Current understanding of ABPA pathogenesis has been complicated by a lack of long-term in vivo fungal persistence models. We have identified a clinical isolate of A. fumigatus, W72310, which persists in the murine lung and causes an ABPA-like disease phenotype. Surprisingly, while viable, W72310 showed little to no growth beyond the conidial stage in the lung. This indicates that it is possible that A. fumigatus can cause allergic disease in the lung without any significant hyphal growth. The identification of this strain of A. fumigatus can not only be used to better understand disease pathogenesis of ABPA and potential anti-fungal treatments, but also to identify features of fungal strains that drive long-term fungal persistence in the lung. Consequently, these observations are a step toward helping resolve the long-standing question when to utilize antifungal therapies in patients with ABPA and fungal allergic type diseases.

microbiology↗

bioRxiv2019

A Ssd1 homolog impacts trehalose and chitin biosynthesis and contributes to virulence in Aspergillus fumigatus

Regulation of fungal cell wall biosynthesis is critical to maintain cell wall integrity in the face of dynamic fungal infection microenvironments. In this study, we observe that a yeast ssd1 homolog, ssdA, in the filamentous fungus Aspergillus fumigatus is involved in trehalose and cell wall homeostasis. An ssdA null mutant strain exhibited an increase in trehalose levels and a reduction in colony growth rate. Over-expression of ssdA in contrast perturbed trehalose biosynthesis and reduced conidia germination rates. The ssdA null mutant strain was more resistant to cell wall perturbing agents while over-expression of ssdA promoted increased sensitivity. Over-expression of ssdA significantly increased chitin levels and both loss and over-expression of ssdA altered sub-cellular localization of the class V chitin synthase CsmA. Strikingly, over-expression of ssdA abolished adherence to abiotic surfaces and severely attenuated the virulence of A. fumigatus in a murine model of invasive pulmonary aspergillosis. In contrast, despite the severe in vitro fitness defects observed upon loss of ssdA, neither surface adherence or murine survival was impacted. In conclusion, A. fumigatus SsdA plays a critical role in cell wall homeostasis that alters fungal-host interactions.\n\nImportanceLife threatening infections caused by the filamentous fungus Aspergillus fumigatus are increasing along with a rise in fungal strains resistant to contemporary antifungal therapies. The fungal cell wall and the associated carbohydrates required for its synthesis and maintenance are attractive drug targets given that many genes encoding proteins involved in cell wall biosynthesis and integrity are absent in humans. Importantly, genes and associated cell wall biosynthesis and homeostasis regulatory pathways remain to be fully defined in A. fumigatus. In this study, we identify SsdA, a model yeast Ssd1p homolog, as an important component of trehalose and fungal cell wall biosynthesis in A. fumigatus that consequently impacts fungal virulence in animal models of infection.

microbiology↗