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Lax Molina, Carlos

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Lax Molina, Carlos
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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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    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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    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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