Biology subjects

Puerner, C.

Publications and source records attributed to Puerner, C..

8 recordsLinked to original sources

bioRxiv2026

A strain-specific metabolic role for the UDP-glucose 4-epimerase Uge3 in Aspergillus fumigatus virulence

Expression of a fungal-specific sub-telomeric gene, hrmA, in Aspergillus fumigatus is important for a colony biofilm morphology termed H-MORPH, increased hypoxic fitness, and virulence in a murine model of invasive pulmonary aspergillosis (IPA). How expression of hrmA contributes to virulence and worse disease progression is ill-defined. Increased hrmA expression results in reduced attachment of the extracellular matrix (ECM) to the fungal cell wall resulting in decreased strain adherence. Fungal strains that are less adherent in vitro are typically less virulent as the ECM heteropolysaccharide galactosaminogalactan (GAG) aids in adhesion to host cells and confers protection from host responses. Here we report that the UDP-glucose 4-epimerase encoding gene required for GAG biosynthesis, uge3, is necessary for full virulence of the H-MORPH strain, hrmAREV (AF293::hrmAD304G). In contrast, loss of uge3 in the reference strain AF293 did not significantly impact virulence in the tested IPA murine model. Phenotypic, transcriptomic, and metabolic analyses of uge3 loss in the respective strain backgrounds revealed a key role for Uge3 in central carbon metabolism in a strain specific context that promotes disease progression. These results complement the known role of Uge3 in GAG biosynthesis and highlight strain specific metabolic differences in pathogenic A. fumigatus strains. IMPORTANCEAspergillus fumigatus forms adherent biofilms that contribute to its ability to persist and cause disease. However, significant strain diversity exists with regard to the morphology of A. fumigatus biofilms. A distinct colony morphotype associated with increased disease progression and low oxygen fitness, termed H-MORPH, was recently described. An additional defining feature of the H-MORPH biofilm morphotype is reduced in vitro adherence to surfaces. While reduced fungal strain adherence is most commonly associated with reductions in virulence, H-MORPH strains exhibit increased virulence relative to the well-studied N-MORPH reference strain AF293. Here we discover that the UDP-glucose 4-epimerase, Uge3, plays an important role in H-MORPH central carbon metabolism complementary to its role in production of the extracellular matrix polysaccharide galactosaminogalactan (GAG). In H-MORPH strains, this metabolic role for Uge3 becomes central to virulence. These data highlight A. fumigatus strain specific mechanisms of fungal carbon metabolism related to biofilm matrix production and fungal virulence.

microbiology↗

bioRxiv2025

Insights into Aspergillus fumigatus morphogenesis and pathogenesis through the putative lipid transporter ArvA

Aspergillus fumigatus poses a significant threat to human well-being, in part due to the increasing emergence of strains resistant to frontline antifungal therapy. In this study, we observe that the gene, arvA, is required for A. fumigatus morphogenesis, antifungal drug susceptibility, and cell wall homeostasis. Intriguingly, our study reveals novel morphological and growth aberrations in the absence of arvA. Loss of arvA results in hyper-swollen conidia that give rise to stunted, polarity-deficient hyphae in numerous environmental conditions, indicating a pivotal role for arvA in A. fumigatus morphogenesis. Surprisingly, despite these severe in vitro morphological and cell wall defects, arvA was not required for morbidity and mortality in immunologically distinct murine models of invasive pulmonary aspergillosis (IPA). However, growth in natural calf lung surfactant was able to normalize{Delta} arvA growth with the wild-type strain suggesting lung surfactant may partially complement the severe in vitro morphological defects of arvA loss in vivo. Taken together our observations reveal arvA as a mediator of A. fumigatus antifungal drug susceptibility and highlight the complex and ill-defined pulmonary nutrient environments role in mediating A. fumigatus pathogenesis and disease progression. IMPORTANCEAspergillus fumigatus is a challenging fungal pathogen in the clinic in part due to increasing azole drug resistance. In this study, we observe that loss of the A. fumigatus gene arvA results in increased azole susceptibility and significant in vitro morphological changes highlighted by hyper-swollen conidia that yield stunted and polarity deficient hyphae. Importantly, despite these severe in vitro morphological and growth abnormalities,{Delta} arvA surprisingly retains full pathogenicity and virulence in two immunologically distinct murine models of invasive pulmonary aspergillosis. These results challenge our understanding of the in-host environment and how it mediates fungal morphogenesis and pathogenesis. These results, consequently, not only enhances our understanding of the role of arvA in A. fumigatus morphogenesis and drug susceptibility, but further emphasizes the importance of in vivo animal models in fully evaluating potential antifungal drug targets.

microbiology↗

bioRxiv2025

A ceramide synthase is important for filamentous fungal biofilm morphology and antifungal drug resistance

The complex structure of fungal biofilms generates microenvironments that impact the fitness of cells within the biofilm community. Contributions to fitness include the development of emergent properties resulting in the tolerance or resistance to external stressors such as rapid environmental changes and in the context of an infection, antifungal drug exposure. The biofilm developed by the filamentous fungal pathogen Aspergillus fumigatus develops zones of low oxygen which contribute to a reduction in antifungal drug susceptibility, however the genes and mechanisms involved in driving this emergent property of the biofilm are ill-defined. In this study, we utilized a transcriptomic approach to probe the biofilm structure in comparison to drug susceptible planktonic cultures to identify transcriptional patterns and genes unique to the A. fumigatus biofilm. Importantly we utilized two phenotypically diverse strains that allowed us to identify biofilm specific gene co-expression networks. One of these networks was highlighted by a gene encoding a ceramide synthase, designated barA, with a striking increase in barA transcript abundance specifically in the biofilm. Null mutants for barA in two strain backgrounds display altered biofilm morphology with some strain specific differences. Importantly, barA has a role in regulating susceptibility to ergosterol targeting antifungal drugs. These data identify biofilm specific genes in A. fumigatus for further study and highlight the importance of fungal ceramide synthases in mediating antifungal drug susceptibility in infection relevant biofilms. ImportanceBiofilms are problematic structures in the context of microbial infections due to the ability to resist both host and drug mediated attempts at tissue sterilization. Consequently, it is imperative to identify mechanisms underlying the development of these structures and the emergent properties they develop. The filamentous fungal pathogen Aspergillus fumigatus forms robust structured biofilms that are resistant to contemporary antifungal drug treatments though the mechanisms are ill-defined. In this study we compared the transcriptional landscape of two A. fumigatus reference strains grown as biofilms and in planktonic culture conditions to identify biofilm specifc genes and pathways. These analyses and subsequent genetic and phenotype studies revealed that a ceramide synthase is important for the development of antifungal drug resistant biofilms. Consequently, these data support the rationale for targeting fungal lipid homeostasis for antifungal therapeutic development, particularly in the context of biofilm mediated infections.

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↗

bioRxiv2024

Dynamic cytoplasmic fluidity during morphogenesis in a human fungal pathogen

The molecular crowding of the cytoplasm impacts a range of cellular processes. Using a fluorescent microrheological probe (GEMs), we observed a striking decrease in molecular crowding during the yeast to filamentous growth transition in the human fungal pathogen Candida albicans. This decrease in crowding is due to a decrease in ribosome concentration that results in part from an inhibition of ribosome biogenesis, combined with an increase in cytoplasmic volume; leading to a dilution of the major cytoplasmic crowder. Moreover, our results suggest that inhibition of ribosome biogenesis is a trigger for C. albicans morphogenesis.

cell biology↗

bioRxiv2024

Giant transposons promote strain heterogeneity in a major fungal pathogen

Fungal infections are difficult to prevent and treat in large part due to strain heterogeneity which confounds diagnostic predictability. Yet the genetic mechanisms driving strain-to-strain variation remain poorly understood. Here, we determined the extent to which Starships--giant transposons capable of mobilizing numerous fungal genes--generate genetic and phenotypic variability in the opportunistic human pathogen Aspergillus fumigatus. We analyzed 519 diverse strains, including 11 newly sequenced with long-read technology and multiple isolates of the same reference strain, to reveal 20 distinct Starships that are generating genomic heterogeneity over timescales relevant for experimental reproducibility. Starship-mobilized genes encode diverse functions, including known biofilm-related virulence factors and biosynthetic gene clusters, and many are differentially expressed during infection and antifungal exposure in a strain-specific manner. These findings support a new model of fungal evolution wherein Starships help generate variation in genome structure, gene content and expression among fungal strains. Together, our results demonstrate that Starships are a previously hidden mechanism generating genotypic and, in turn, phenotypic heterogeneity in a major human fungal pathogen. ImportanceNo "one size fits all" option exists for treating fungal infections in large part due to genetic and phenotypic variability among strains. Accounting for strain heterogeneity is thus fundamental for developing efficacious treatments and strategies for safeguarding human health. Here, we report significant progress towards achieving this goal by uncovering a previously hidden mechanism generating heterogeneity in the human fungal pathogen Aspergillus fumigatus: giant transposons called Starships that span dozens of kilobases and mobilize fungal genes as cargo. By conducting a systematic investigation of these unusual transposons in a single fungal species, we demonstrate their contributions to population-level variation at the genome, pangenome and transcriptome levels. The Starship compendium we develop will not only help predict variation introduced by these elements in laboratory experiments but will serve as a foundational resource for determining how Starships impact clinically-relevant phenotypes, such as antifungal resistance and pathogenicity.

microbiology↗

bioRxiv2023

The Cystic Fibrosis treatment, Trikafta, affects growth, viability, and cell wall of Aspergillus fumigatus biofilms

People with Cystic Fibrosis (pwCF) commonly test positive for the pathogenic fungus, Aspergillus fumigatus, which is associated with a decline in lung function. Trikafta is a recently approved therapy for pwCF that improves quantity and function of the CFTR protein, however, it is not known how Trikafta affects microbial communities in the lung. Therefore, the aim of this study was to determine whether Trikafta directly affects A. fumigatus growth and biology. While Trikafta did not impact the viability of A. fumigatus conidia, treatment of A. fumigatus biofilms with Trikafta reduced overall biofilm biomass. This finding was associated with increased membrane permeability, decreased viability, and reduced metabolic activity following long-term treatment of biofilms. Trikafta-induced membrane permeability and biomass reduction was partially blocked with the calcium channel inhibitor Verapamil and fully blocked by the mammalian CFTR inhibitor GlyH-101. Trikafta-induced biomass reduction and metabolic activity was shown to be regulated by the High Osmolarity Glycerol (HOG) pathway gene sakA. Trikafta treatment also induced resistance to the cell wall stressor calcofluor white, susceptibility to the antifungal caspofungin, and decreased inflammatory responses from murine bone marrow cells. Collectively, these results reveal that Trikafta affects infection-relevant A. fumigatus biology and host-microbial interactions. IMPORTANCEPeople with Cystic Fibrosis (pwCF) commonly test positive for the pathogenic fungus, Aspergillus fumigatus, which is associated with a decline in lung function. Trikafta is a recently approved therapy for pwCF that improves quantity and function of the CFTR protein, however, it is not known how Trikafta affects microbial communities in the CF lung. Here we report that Trikafta has anti-fungal activity against the infection relevant form of A. fumigatus, termed biofilms. When exposed to Trikafta A. fumigatus biofilms lose their viability. In addition, exposure to Trikafta enhances the susceptibility of A. fumigatus biofilms to the clinically utilized anti-fungal drug caspofungin. These data suggest Trikafta impacts the cell wall of A. fumigatus and suggests the potential for using Trikafta to augment existing anti-fungal therapy.

microbiology↗

bioRxiv2022

CF-Seq, An Accessible Web Application for Rapid Re-Analysis of Cystic Fibrosis Pathogen RNA Sequencing Studies

Researchers studying cystic fibrosis (CF) pathogens have produced numerous RNA-seq datasets which are available in the gene expression omnibus (GEO). Although these studies are publicly available, substantial computational expertise and manual effort are required to compare similar studies, visualize gene expression patterns within studies, and use published data to generate new experimental hypotheses. Furthermore, it is difficult to filter available studies by domain-relevant attributes such as strain, treatment, or media, or for a researcher to assess how a specific gene responds to various experimental conditions across studies. To reduce these barriers to data re-analysis, we have developed an R Shiny application called CF-Seq, which works with a compendium of 147 studies and 1,446 individual samples from 13 clinically relevant CF pathogens. The application allows users to filter studies by experimental factors and to view complex differential gene expression analyses at the click of a button. Here we present a series of use cases that demonstrate the application is a useful and efficient tool for new hypothesis generation. (CFSeq: http://scangeo.dartmouth.edu/CFSeq/)

bioinformatics↗