Nanobody-mediated proteolysis of Aspergillus fumigatus transcription factors in vitro and in vivo

Aspergillus fumigatus (Afu) is the primary human filamentous fungal pathogen and has been designated by the World Health Organization as one of the four most critical fungal pathogens impacting humans. The patient population at risk to Afu infections continues to rise owing to increased utilization of immune suppression for organ transplants, viral infections and cancer chemotherapy. While our need to understand the pathogenesis of aspergillosis is high, the experimental tools for genetics in Afu are limited. Most genetic approaches rely on changing the level of the gene of interest using disruption or overproduction strategies. Doxycycline-regulated promoter systems have also been used but these also only indirectly effect the level of the target protein by altering its mRNA production. Here we describe a new approach to analysis of the transcriptional control of virulence in Afu by regulated proteolysis of transcription factors already known to be important in pathogenesis. We have expressed an anti-GFP nanobody (GFPNb: single chain antibody from camelid species) fused to a mammalian E3 ligase enzyme (Rnf4) and found this fusion protein to trigger proteolysis of GFP fusion proteins in Afu. Expression of this fusion protein is under the control of a doxycycline-inducible promoter system allowing us to trigger protein degradation with the addition of doxycycline. We propose to accomplish two goals in this application. First, we will prepare GFP fusion proteins to 8 different transcription factors already known to be required for virulence when deleted from the cell. We will test all of these fusion proteins for localization, expression, phenotype and the ability to be degraded by the GFPNb-Rnf4 fusion protein. We will select up to 4 of these fusion proteins to test in a mouse infection model based on their retention of a normal in vitro phenotype and sensitivity to the GFPNb-Rnf4-mediated degradation. Immunosuppressed mice will be infected with these strains and then treated with doxycycline at different times to determine if the acute proteolytic removal of a factor of interest has an impact on fungal burden/survival. We will also confirm loss of a given factor after doxycycline treatment by western analysis of proteins from the mouse lung. This approach of directly degrading a transcription factor of interest, coupled with the ability to do this in a time-dependent manner, represents a fundamentally different means of interrogating the progression of mammalian infection by Afu. Project Number: 5R21AI190747-02 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of Allergy and Infectious Diseases (NIAID) | Principal Investigator: W Moye-Rowley | Institution: UNIVERSITY OF IOWA, IOWA CITY, IA | Award Amount: $197,420 | Activity Code: R21 | Study Section: Pathogenic Eukaryotes Study Section[PTHE] View on NIH RePORTER: https://reporter.nih.gov/project-details/5R21AI19074702