1. Abstract Rhizopus delemar is the leading casual-agent of mucormycosis, a life-threatening fungal infection in immunocompromised patients. Despite its clinical importance, R. delemar remains understudied due to its limited genetic tractability and lack of available research resources. Specifically, R. delemar produces coenocytic hyphae and multinucleated spores, resulting in a high difficulty obtaining stable gene-specific mutant strains. We have previously employed consecutive hyphal tip microdissection, and with CRISPR-Cas9 techniques, to isolate mononuclear mutant strains. However, this isolation method is laborious and inefficient. To circumvent this obstacle, we propose hyphal fragmentation and protoplast generation, coupled with fluorescence-activated cell sorting (FACS), which allows isolation of mononuclear propagules to obtain mutant clones following genetic transformation robustly. We will first optimize hyphal fragmentation and protoplast production to enrich single nucleated cells (Aim 1a) and label R. delemar strains with fluorescent nuclear markers through DNA transformation (Aim 1b). We will then sort and isolate mononuclear hyphal fragments/protoplasts through FACS analysis (Aim 2a). Additionally, we will improve the DNA plasmid for further genetic manipulation of R. delemar (Aim 2b). Our research plan's successful implementation will provide an invaluable tool to facilitate genetic studies of R. delemar-induced mucormycosis mechanisms.

Public Health Relevance

Rhizopus delemar is an important fungal pathogen that causes life-threatening mucormycosis infection in patients with immunodeficient patients; however, the fungus's multinuclear cell status severely hampers genetic analysis of the disease mechanism. We propose to develop a mononuclear propagule isolation approach that will greatly facilitate genetic studies of this important pathogen.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Small Research Grants (R03)
Project #
1R03AI156254-01A1
Application #
10221180
Study Section
Pathogenic Eukaryotes Study Section (PTHE)
Program Officer
Love, Dona
Project Start
2021-02-16
Project End
2023-01-31
Budget Start
2021-02-16
Budget End
2022-01-31
Support Year
1
Fiscal Year
2021
Total Cost
Indirect Cost
Name
Louisiana State Univ Hsc New Orleans
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
782627814
City
New Orleans
State
LA
Country
United States
Zip Code
70112