Biology subjects

Busch, R. J.

Publications and source records attributed to Busch, R. J..

3 recordsLinked to original sources

bioRxiv2026

Inhibition of Calcineurin Results in Increased Susceptibility to Voriconazole in the White-Nose Syndrome Fungus Pseudogymnoascus destructans.

Pseudogymnoascus destructans is a psychrophilic ascomycete fungus that causes White-Nose Syndrome in North American hibernating bats. P. destructans invades the tissue present in bats' noses and wing membranes, causing the characteristic white nose and cupping erosion in the wings. P. destructans exhibits characteristics of a hemibiotrophic fungal pathogen, in which P. destructans can invade the host cells without inducing damage. During this stage, P. destructans hyphae assume a morphology similar to fungal plant pathogens during the biotrophic stage of infection. However, the mechanism by which P. destructans regulates its morphology and virulence is not fully understood. The phosphatase calcineurin is a key regulator of fungal stress response and virulence and could play a role in P. destructans virulence. Calcineurin can be inhibited using FK506 or Cyclosporin A (CSA). FK506 interacts with FKBP12, while CSA interacts with Cyclosporin A, and the protein-drug complex then inhibits calcineurin activity. Here, we show that P. destructans is intrinsically resistant to one calcineurin inhibitor (FK506), possibly because P. destructans FKBP12 has a lysine instead of the conserved arginine at position 89, which has been shown to reduce the affinity of the FKBP12-FK506 complex for calcineurin. Using cyclosporin A, we found that calcineurin is important for fungal growth. Additionally, calcineurin inhibition increases voriconazole inhibition. In contrast, calcineurin inhibition was antagonistic to the anti-cell wall drug micafungin. Taken together, calcineurin's role in fungal growth and response to voriconazole is conserved in P. destructans but may play a unique role in P. destructans response to micafungin.

microbiology↗

bioRxiv2025

Deletion of core septin gene aspB in Aspergillus fumigatus results in fungicidal activity of caspofungin

Septins are a family of GTP-binding proteins found in many eukaryotic lineages. Although highly conserved throughout many eukaryotes, their functions vary across species. In Aspergillus fumigatus, the etiological agent of invasive aspergillosis, septins participate in a variety of processes, including conidiation, septation, and responses to cell wall stress. Previous studies determined that the {Delta}aspB strain had a greater sensitivity to anti-cell wall drugs, especially the echinocandins, yet mechanisms behind this augmented sensitivity are unknown. We performed cell viability staining of the deletion strains after caspofungin exposure and found that the {Delta}aspA, {Delta}aspB, and {Delta}aspC strains had significantly lower cell viability. Concomitant with the reduced viability, deletion strains are more susceptible to caspofungin on solid media. These results indicate that the septin cytoskeleton is important for A. fumigatus survival in the presence of caspofungin. Due to the potential of improved therapeutic outcome, we followed up using a neutropenic murine model of invasive aspergillosis. Animals infected with the {Delta}aspB strain and treated with caspofungin showed improved survival compared to the animals infected with akuBKU80 wild-type or complemented strains. Additionally, histological analysis showed reduced fungal burden and inflammation in the {Delta}aspB infected, caspofungin-treated group. Affinity purification coupled with quantitative proteomics identified proteins involved in the septin-dependent response to caspofungin, including four candidate interactors involved in cell wall stress response. Deletion of these candidate genes resulted in increased susceptibility to caspofungin and moderately reduced viability post-drug exposure. Taken together, these data suggest that septin AspB contributes to the fungistatic response to caspofungin. Author SummaryInvasive aspergillosis is a pulmonary disease caused by the fungus Aspergillus fumigatus that primarily occurs in immunocompromised patients. Invasive aspergillosis has a high mortality rate, ranging from 50-90%. Therapy options are limited due to few available drugs with fungicidal activity and growing global drug resistance. Treatment typically starts with triazoles, which target the fungal cell membrane. If unsuccessful, an echinocandin, which targets the cell wall, is given as a salvage therapy in the U.S. Echinocandins, including caspofungin, are fungistatic against A. fumigatus, slowing growth of the fungus rather than killing it. Due to this, echinocandins have a high therapeutic failure rate. Previous work suggests that deletion of the cytoskeletal septin genes increases sensitivity to caspofungin. Here we describe our finding that the septin genes aspA, aspB, and aspC are involved in the fungal response to caspofungin. Additionally, the deletion of aspB results in fungicidal activity of this otherwise fungistatic drug. These findings show promise for novel therapy options that block the septin-mediated response to caspofungin.

microbiology↗

bioRxiv2024

Strategies for Genetic Manipulation of the Halotolerant Black Yeast H. werneckii: Ectopic DNA integration and marker-free CRISPR/Cas9 Trasformation

Hortaea werneckii is a halotolerant black yeast commonly found in hypersaline environments. This yeast is also the causative agent of tinea nigra, a superficial mycosis of the palm of the hand and soles of the feet of humans. In addition to their remarkable halotolerance, this black yeast exhibits an unconventional cell division cycle, alternating between fission and budding cell division. Cell density and the salt concentration in their environment regulate which cell division cycle H. werneckii uses. Although H. werneckii have been extensively studied due to their unique physiology and cell biology, deciphering the underlying mechanisms behind these remarkable phenotypes has been limited due to the lack of genetic tools available. Here, we report a new ectopic integration protocol for H. werneckii using PEG-CaCl2 mediated protoplast transformation. This approach relies on a drug (hygromycin B) resistance gene to select for successful integration of the genetic construct. The same construct was used to express cytosolic green fluorescent protein. Finally, we developed a marker-free CRISPR/Cas9 protocol for targeted gene deletion using the melanin synthesis pathway as a visual reporter of successful transformation. These transformation strategies will allow testing hypotheses related to H. werneckii cell biology and physiology. ImportanceHortaea werneckii is a remarkable yeast capable of growing in high salt concentration, and its cell division cycle alternates between fission-like and budding. For these unique attributes, H. werneckii has gathered interest in a research program studying extremophile fungi and cell division. Most of our understanding of H. werneckii biology comes from genomic analyses, usage of drugs to target a particular pathway or heterologous expression of its gene in S. cerevisiae. Nonetheless, H. werneckii has remained genetically intractable. Here, we report on two strategies to transform H. werneckii: ectopic integration of a plasmid and gene deletion using CRISPR/Cas9. These approaches will be fundamental to expanding the experimental techniques available to study H. werneckii, including live cell imaging of cellular processes and reverse genetic approaches.

microbiology↗