Browsing by Subject "Mucormycosis"

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    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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    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.