Zygomycosis is the third most common invasive fungal infection that causes devastating diseases in individuals with immune deficiency, such as those with neutropenia or diabetic ketoacidosis. Mortality due to zygomycosis is very high, with a nearly 50% fatality rate even when appropriately treated. Due to the increasing prevalence of diabetic ketoacidosis, cancer, and organ transplantation, the number of patients at risk is dramatically increasing, such as in the case of a recent outbreak of hospital acquired Rhizopus delemar infection in New Orleans, which resulted in the death of at least six children. Despite such importance, very limited research resources are available to investigate R. delemar pathogenesis. This is partially due to the organism being not as genetically tractable as other pathogenic fungi. For example, there often exist multiple gene copies due to whole-genome duplication, and few mitotically stable genetic transformants can be obtained with the existing methodology. We propose to develop a CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated) system that allows efficient gene specific manipulation and custom genome editing in R. delemar. We will begin by characterizing unique clinical strains in comparison to the archetypical strain for characteristics relevant to pathogenicity, auxotrophy, and resistance to antifungal compounds. We will construct vectors for Cas9 nuclease and guide RNA (gRNA) expression using the endogenous promoter and terminator sequences. We will then test the utility of the CRISPR-Cas system through manipulation of the marker PYRF (URA5) gene encoding orotidine phosphate decarboxylase, and the CNBR and CNA(A-C) genes encoding regulatory and catalytic subunits of calcineurin whose homologs play critical roles in fungal growth, morphogenesis, drug resistance, and pathogenesis. Successful implementation of our research plan will provide a revolutionary tool in promoting genetic studies of R. delemar pathogenesis mechanism.

Public Health Relevance

Rhizopus delemar is an emerging fungus pathogen that causes devastating diseases in those with suppressed immune status, but very limited research resources are available for studying its disease mechanisms. We here propose to develop a CRISPR-Cas system for precise gene modification and genome editing that will greatly facilitate Rhizopus pathogenesis studies and discovery of potential targets for antifungal therapy.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21AI121451-02
Application #
9304959
Study Section
Pathogenic Eukaryotes Study Section (PTHE)
Program Officer
Love, Dona
Project Start
2016-07-01
Project End
2019-06-30
Budget Start
2017-07-01
Budget End
2019-06-30
Support Year
2
Fiscal Year
2017
Total Cost
Indirect Cost
Name
Louisiana State Univ Hsc New Orleans
Department
Pediatrics
Type
Schools of Medicine
DUNS #
782627814
City
New Orleans
State
LA
Country
United States
Zip Code
70112
Yin, Ziyi; Zhang, Xiaofang; Wang, Jingzhen et al. (2018) MoMip11, a MoRgs7-interacting protein, functions as a scaffolding protein to regulate cAMP signaling and pathogenicity in the rice blast fungus Magnaporthe oryzae. Environ Microbiol 20:3168-3185
Kang, Xue; Kirui, Alex; Muszy?ski, Artur et al. (2018) Molecular architecture of fungal cell walls revealed by solid-state NMR. Nat Commun 9:2747
Yang, Lina; Ru, Yanyan; Cai, Xingjia et al. (2018) MoImd4 mediates crosstalk between MoPdeH-cAMP signaling and purine metabolism to govern growth and pathogenicity in Magnaporthe oryzae. Mol Plant Pathol :
Liu, Xinyu; Yang, Jie; Qian, Bin et al. (2018) MoYvh1 subverts rice defense through functions of ribosomal protein MoMrt4 in Magnaporthe oryzae. PLoS Pathog 14:e1007016
Liu, Muxing; Bruni, Gillian O; Taylor, Christopher M et al. (2018) Comparative genome-wide analysis of extracellular small RNAs from the mucormycosis pathogen Rhizopus delemar. Sci Rep 8:5243
Yang, Jie; Liu, Muxing; Liu, Xinyu et al. (2018) Heat-Shock Proteins MoSsb1, MoSsz1, and MoZuo1 Attenuate MoMkk1-Mediated Cell-Wall Integrity Signaling and Are Important for Growth and Pathogenicity of Magnaporthe oryzae. Mol Plant Microbe Interact 31:1211-1221
Tang, Wei; Gao, Chuyun; Wang, Jingzhen et al. (2018) Disruption of actin motor function due to MoMyo5 mutation impairs host penetration and pathogenicity in Magnaporthe oryzae. Mol Plant Pathol 19:689-699
Wang, Ping (2018) Two Distinct Approaches for CRISPR-Cas9-Mediated Gene Editing in Cryptococcus neoformans and Related Species. mSphere 3:
Zhang, Shengpei; Liu, Xiu; Li, Lianwei et al. (2017) The ArfGAP protein MoGlo3 regulates the development and pathogenicity of Magnaporthe oryzae. Environ Microbiol 19:3982-3996
Bruni, Gillian O; Battle, Blake; Kelly, Ben et al. (2017) Comparative proteomic analysis of Gib2 validating its adaptor function in Cryptococcus neoformans. PLoS One 12:e0180243

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