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Isolation of mononuclear propagules from coenocytic hyphae of the mucormycosis pathogen Rhizopus delemar

Award Number: R03AI156254
ORGANIZATION: NATIONAL INSTITUTE OF ALLERGY & INFECTIOUS DISEASES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

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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.


Issue Date FY	Funding FY	Legal Entity Name	Legal Entity Address	Legal Entity City	Legal Entity State	Legal Entity Zip Code	Legal Entity COUNTY	Legal Entity COUNTRY	Assistance Listing	Award Code	Budget Year	Action Date	Action Type	Action Amount

Issue Date FY: 2022 ( Subtotal = $73,500 )
2022	2022	LOUISIANA STATE UNIVERSITY SYSTEM	433 BOLIVAR ST STE 815	NEW ORLEANS	LA	70112	ORLEANS	USA	Allergy and Infectious Diseases Research	000	2	2/1/2022	NON-COMPETING CONTINUATION	$73,500
														Subtotal = $73,500

Issue Date FY: 2021 ( Subtotal = $73,500 )
2021	2021	LOUISIANA STATE UNIVERSITY SYSTEM	433 BOLIVAR ST STE 815	NEW ORLEANS	LA	70112	ORLEANS	USA	Allergy and Infectious Diseases Research	000	1	2/16/2021	NEW	$73,500
														Subtotal = $73,500

Grand Total All Awards = $147,000

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Isolation of mononuclear propagules from coenocytic hyphae of the mucormycosis pathogen Rhizopus delemar

Award Number: R03AI156254

ORGANIZATION: NATIONAL INSTITUTE OF ALLERGY & INFECTIOUS DISEASES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

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