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Navarro Mendoza, María Isabel

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Navarro Mendoza, María Isabel
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Universidad de Murcia. Departamento de Genética y Microbiología
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    Stable and reproducible homologous recombination enables CRISPR-based engineering in the fungus Rhizopus microsporus
    (Cell Press, 2021-12-20) Lax Molina, Carlos; Navarro Mendoza, María Isabel; Pérez Arques, Carlos; Navarro Ros, Eusebio; Nicolás Molina, Francisco Esteban; Garre Mula, Victoriano; Genética y Microbiología; Facultades de la UMU::Facultad de Biología
    Mucormycosis is a lethal and emerging disease that has lacked a genetic model fulfilling both high virulence and the possibility of performing stable and reproducible gene manipulation by homologous recombination (HR). Here, we developed a new methodology to successfully perform HR in Rhizopus microsporus. We isolated an uracil auxotrophic recipient strain and optimized the critical steps in the genetic transformation of this fungus. This was followed by an adaptation of a plasmid-free CRISPR-Cas9 system coupled with microhomology repair templates. We reproducibly generated stable mutants in the genes leuA and crgA, encoding a 3-isopropylmalate dehydratase and an ubiquitin ligase, respectively. Our new genetic model showed that mutations in the gene pyrF, a key virulence gene in several bacterial and fungal pathogens, correlated with an avirulent phenotype in an immunocompetent murine host. This was reverted by gene complementation, showing the broad possibilities of our methodology.
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    Components of a new gene family of ferroxidases involved in virulence are functionally specialized in fungal dimorphism
    (Springer Nature, 2018-05-16) Navarro Mendoza, María Isabel; Pérez Arques, Carlos; Murcia, Laura; Martínez-García, Pablo; Lax Molina, Carlos; Sanchis, Marta; Capilla, Javier; Nicolás Molina, Francisco Esteban; Garre Mula, Victoriano; Genética y Microbiología; Facultades de la UMU::Facultad de Biología
    Mucormycosis is an emerging angio-invasive infection caused by Mucorales that presents unacceptable mortality rates. Iron uptake has been related to mucormycosis, since serum iron availability predisposes the host to suffer this infection. In addition, iron uptake has been described as a limiting factor that determines virulence in other fungal infections, becoming a promising field to study virulence in Mucorales. Here, we identified a gene family of three ferroxidases in Mucor circinelloides, fet3a, fet3b and fet3c, which are overexpressed during infection in a mouse model for mucormycosis, and their expression in vitro is regulated by the availability of iron in the culture media and the dimorphic state. Thus, only fet3a is specifically expressed during yeast growth under anaerobic conditions, whereas fet3b and fet3c are specifically expressed in mycelium during aerobic growth. A deep genetic analysis revealed partially redundant roles of the three genes, showing a predominant role of fet3c, which is required for virulence during in vivo infections, and shared functional roles with fet3b and fet3c during vegetative growth in media with low iron concentration. These results represent the first described functional specialization of an iron uptake system during fungal dimorphism.
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    Open Access
    Mucor circinelloides thrives inside the phagosome through an Atf-mediated germination pathway
    (ASM Journals, 2019-02-05) Pérez Arques, Carlos; Navarro Mendoza, María Isabel; Murcia, Laura; Lax Molina, Carlos; Martínez-García, Pablo; Heitman, Joseph; Nicolás Molina, Francisco Esteban; Garre Mula, Victoriano; Genética y Microbiología; Facultades de la UMU::Facultad de Biología
    Mucormycosis is an emerging fungal infection that is often lethal due to the ineffectiveness of current therapies. Here, we have studied the first stage of this infection-the germination of Mucor circinelloides spores inside phagocytic cells-from an integrated transcriptomic and functional perspective. A relevant fungal gene network is remodeled in response to phagocytosis, being enriched in crucial functions to survive and germinate inside the phagosome, such as nutritional adaptation and response to oxidative stress. Correspondingly, the phagocytic cells induced a specific proinflammatory and apoptotic response to the pathogenic strain. Deletion of fungal genes encoding putative transcription factors (atf1, atf2, and gcn4), extracellular proteins (chi1 and pps1), and an aquaporin (aqp1) revealed that these genes perform important roles in survival following phagocytosis, germination inside the phagosome, and virulence in mice. atf1 and atf2 play a major role in these pathogenic processes, since their mutants showed the strongest phenotypes and both genes control a complex gene network of secondarily regulated genes, including chi1 and aqp1 These new insights into the initial phase of mucormycosis define genetic regulators and molecular processes that could serve as pharmacological targets.
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    Open Access
    RNAi-Based Functional Genomics Identifies New Virulence Determinants in Mucormycosis
    (Public Library of Science, 2017-01-20) Trieu, Trung Anh; Navarro Mendoza, María Isabel; Pérez Arques, Carlos; Sanchis, Marta; Capilla, Javier; Navarro Rodríguez, Patricia; López Fernández, Loida; Torres Martínez, Santiago; Garre Mula, Victoriano; Ruiz Vázquez, Rosa María; Nicolás Molina, Francisco Esteban; Genética y Microbiología
    Mucorales are an emerging group of human pathogens that are responsible for the lethal disease mucormycosis. Unfortunately, functional studies on the genetic factors behind the virulence of these organisms are hampered by their limited genetic tractability, since they are reluctant to classical genetic tools like transposable elements or gene mapping. Here, we describe an RNAi-based functional genomic platform that allows the identification of new virulence factors through a forward genetic approach firstly described in Mucorales. This platform contains a whole-genome collection of Mucor circinelloides silenced transformants that presented a broad assortment of phenotypes related to the main physiological processes in fungi, including virulence, hyphae morphology, mycelial and yeast growth, carotenogenesis and asexual sporulation. Selection of transformants with reduced virulence allowed the identification of mcplD, which encodes a Phospholipase D, and mcmyo5, encoding a probably essential cargo transporter of the Myosin V family, as required for a fully virulent phenotype of M. circinelloides. Knock-out mutants for those genes showed reduced virulence in both Galleria mellonella and Mus musculus models, probably due to a delayed germination and polarized growth within macrophages. This study provides a robust approach to study virulence in Mucorales and as a proof of concept identified new virulence determinants in M. circinelloides that could represent promising targets for future antifungal therapies.
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