Identificación de factores de virulencia en la mucormicosis

Abstract

Mucormycosis is an emergent life-threatening infection caused by fungi of the order Mucorales. This infectious disease is characterized by high morbidity and mortality rates, mainly because of the scarcity of successful clinical treatments. This infection affects predominantly immunocompromised patients, including those with cancer, chemotherapy, or immunosuppression for organ or bone marrow transplants. Other patients at increased risk of infection are those with diabetes, iron overload syndromes, trauma, burns, or neutropenia. The fungal agents of mucormycosis are resistant to most of the antifungal drugs available; thus, clinical treatments are limited by toxicity and resistance. Overall, this emergent infection is becoming a challenge to public health, hence the need to develop new therapies to treat this disease. Mucorales are a group of understudied basal fungi, mainly because they are reluctant to classical molecular tools. Mucor circinelloides is one of the causal agents of mucormycosis and is the only model to properly study the infection at a molecular level. In this work, we propose the use of M. circinelloides to identify new virulent factors in mucormycosis, generate new tools to characterize virulent phenotypes, and deeply analyze critical mechanisms involved in the infection. The characterization of the gene silencing mechanism in M. circinelloides has allowed the design of an RNAi-based functional genomics platform to study virulence processes. This library consists of high-throughput plasmids that synthesize double-stranded RNAs targeting any possible gene in the fungal genome. Thanks to this new genetic tool, we obtained a collection of silenced transformants that represented the whole genome, showing a broad variety of phenotypes related to virulence processes. Using this platform, two genes involved in M. circinelloides pathogenesis were identified: mcplD, which encodes a Phospholipase D; and mcmyo5, encoding a cargo transporter of the Myosin V family. Iron acquisition during mucormycosis is a critical factor that shapes the outcome of the infection. The high-affinity system is the most important iron uptake mechanism during the host invasion and consists of a permease-ferroxidase complex. In this work, three genes coding for ferroxidases, named fet3a, fet3b, and fet3c, have been identified as part of this high-affinity iron uptake mechanism. This family of ferroxidases is expressed both under iron-depleted conditions and during in vivo infection in a mouse model. A combination of single and double mutants of the three genes revealed that fet3c is the most important for virulence, although there is a functional redundancy among the members of the gene family during both in vitro growth with low iron and mouse infection. Also, fet3a is only expressed during yeast growth, suggesting a functional specialization of the high-affinity iron uptake mechanism in the dimorphic yeast-mycelium transition. Aiming at the improvement of the molecular tools to study Mucorales pathogenesis, this work produced a reporter strain of M. circinelloides for in vivo monitoring of the infection. For the first time, we generated a strain expressing the firefly luciferase driven by a strong promoter that ensured a stable light emission. The two reporter strains obtained had no phenotypical differences in virulence and antifungal sensitivity compared to the wild-type control. Altogether, our strategy to identify virulence factors followed three key steps: first, the development of an RNAi-based functional genomics platform that allows the screening of phenotypes related to pathogenesis; second, the deep analysis of the iron uptake mechanism during the infection using M. circinelloides as a model for the genetic characterization of three ferroxidases genes; and third, the generation of the first bioluminescent reporter strain in M. circinelloides that facilitates in vivo monitorization of the infection in live animal models, allowing future functional studies related to pathogenesis and drug susceptibility.