Alternative ergosterol biosynthetic pathways confer antifungal drug resistance in the human pathogens within the Mucor species complex.

Mucormycoses are emerging fungal infections caused by a variety of heterogeneous species within the order Mucorales. Among the Mucor species complex, Mucor circinelloides is the most frequently isolated pathogen in mucormycosis patients and despite its clinical significance, there is an absence of established genome manipulation techniques to conduct molecular pathogenesis studies. In this study, we generated a spontaneous uracil auxotrophic strain and developed a genetic transformation procedure to analyze molecular mechanisms conferring antifungal drug resistance. With this new model, phenotypic analyses of gene deletion mutants were conducted to define Erg3 and Erg6a as key biosynthetic enzymes in the M. circinelloides ergosterol pathway. Erg3 is a C-5 sterol desaturase involved in growth, sporulation, virulence, and azole susceptibility. In other fungal pathogens, erg3 mutations confer azole resistance because Erg3 catalyzes the production of a toxic diol upon azole exposure. Surprisingly, M. circinelloides produces only trace amounts of this toxic diol and yet, it is still susceptible to posaconazole and isavuconazole due to alterations in membrane sterol composition. These alterations are severely aggravated by erg3 Δ mutations, resulting in ergosterol depletion and consequently, hypersensitivity to azoles. We also identified Erg6a as the main C-24 sterol methyltransferase, whose activity may be partially rescued by the paralogs Erg6b and Erg6c. Loss of Erg6a function diverts ergosterol synthesis to the production of cholestatype sterols, resulting in resistance to amphotericin B. Our findings suggest that mutations or epimutations causing loss of Erg6 function may arise during human infections, resulting in antifungal drug resistance to first-line treatments against mucormycoses. Importance: The Mucor species complex includes a variety of opportunistic pathogens known to cause mucormycosis, a potentially lethal fungal infection with limited therapeutic options. The only effective first-line treatments against mucormycosis consist of liposomal formulations of amphotericin B and the triazoles posaconazole and isavuconazole, all of which target components within the ergosterol biosynthetic pathway. This study uncovered M. circinelloides Erg3 and Erg6a as key enzymes to produce ergosterol, a vital constituent of fungal membranes. Absence of any of those enzymes leads to decreased ergosterol and consequently, resistance to ergosterol-binding polyenes such as amphotericin B. Particularly, losing Erg6a function pose a higher threat as the ergosterol pathway is channeled into alternative sterols similar to cholesterol, which maintain membrane permeability. As a result, erg6a mutants survive within the host and disseminate the infection, indicating that Erg6a deficiency may arise during human infections and confer resistance to the most effective treatment against mucormycoses.

Heterochromatin and RNAi act independently to ensure genome stability in Mucorales human fungal pathogens.

Chromatin modifications play a fundamental role in controlling transcription and genome stability and yet despite their importance, are poorly understood in early-diverging fungi. We present a comprehensive study of histone lysine and DNA methyltransferases across the Mucoromycota, emphasizing heterochromatin formation pathways that rely on the Clr4 complex involved in H3K9-methylation, the Polycomb-repressive complex 2 driving H3K27-methylation, or DNMT1-like methyltransferases that catalyze 5mC DNA methylation. Our analysis uncovered H3K9-methylated heterochromatin as the major chromatin modification repressing transcription in these fungi, which lack both Polycomb silencing and cytosine methylation. Although small RNAs generated by RNA interference (RNAi) pathways facilitate the formation of heterochromatin in many eukaryotic organisms, we show that RNAi is not required to maintain either genomic or centromeric heterochromatin in Mucor. H3K9-methylation and RNAi act independently to control centromeric regions, suggesting a functional subspecialization. Whereas the H3K9 methyltransferase Clr4 and heterochromatin formation are essential for cell viability, RNAi is dispensable for viability yet acts as the main epigenetic, regulatory force repressing transposition of centromeric GremLINE1 elements. Mutations inactivating canonical RNAi lead to rampant transposition and insertional inactivation of targets resulting in antimicrobial drug resistance. This fine-tuned, Rdrp2-dependent RNAi activity is critical for genome stability, restricting GremLINE1 retroelements to the centromeres where they occupy long heterochromatic islands. Taken together, our results suggest that RNAi and heterochromatin formation are independent genome defense and regulatory mechanisms in the Mucorales, contributing to a paradigm shift from the cotranscriptional gene silencing observed in fission yeasts to models in which heterochromatin and RNAi operate independently in early-diverging fungi.

Transformation and CRISPR-Cas9-mediated homologous recombination in the fungus Rhizopus microsporus.

Here, we describe a reliable approach for targeted DNA integrations in the genome of R. microsporus, one of the main causal agents of mucormycosis. We provide a strategy for stable, targeted integration of DNA templates by homologous recombination (HR) based on the CRISPR-Cas9 technology. This strategy opens a wide range of possibilities for the genetic modification of R. microsporus and will be useful for the study of mucormycosis. For complete details on the use and execution of this protocol, please refer to Lax et al. (2021).

Role of the Non-Canonical RNAi Pathway in the Antifungal Resistance and Virulence of Mucorales.

Mucorales are the causal agents for the lethal disease known as mucormycosis. Mortality rates of mucormycosis can reach up to 90%, due to the mucoralean antifungal drug resistance and the lack of effective therapies. A concerning urgency among the medical and scientific community claims to find targets for the development of new treatments. Here, we reviewed different studies describing the role and machinery of a novel non-canonical RNAi pathway (NCRIP) only conserved in Mucorales. Its non-canonical features are the independence of Dicer and Argonaute proteins. Conversely, NCRIP relies on RNA-dependent RNA Polymerases (RdRP) and an atypical ribonuclease III (RNase III). NCRIP regulates the expression of mRNAs by degrading them in a specific manner. Its mechanism binds dsRNA but only cuts ssRNA. NCRIP exhibits a diversity of functional roles. It represses the epimutational pathway and the lack of NCRIP increases the generation of drug resistant strains. NCRIP also regulates the control of retrotransposons expression, playing an essential role in genome stability. Finally, NCRIP regulates the response during phagocytosis, affecting the multifactorial process of virulence. These critical NCRIP roles in virulence and antifungal drug resistance, along with its exclusive presence in Mucorales, mark this pathway as a promising target to fight against mucormycosis.

The RNAi Mechanism Regulates a New Exonuclease Gene Involved in the Virulence of Mucorales.

Mucormycosis is a lethal disease caused by Mucorales, which are emerging as human causes that explain the high mortality for this disease. Consequently, the research community is searching for virulence determinants that could be repurposed as targets to develop new treatments against mucormycosis. Our work explores an RNA interference (RNAi)-based approach to find targets involved in the virulence of Mucorales. A transcriptomewide analysis compared sRNAs and their target mRNAs in two Mucor lusitanicus different pathotypes, virulent and avirulent, generating a list of 75 loci selected by their differential sRNA accumulation in these strains. As a proof of concept and validity, an experimental approach characterized two loci showing opposite behavior, confirming that RNAi activity causes their differential expression in the two pathotypes. We generated deletion mutants for two loci and a knockin-strain overexpressing for one of these loci. Their functional analysis in murine virulence assays identified the gene wex1, a putative DEDDy exonuclease with RNase domains, as an essential factor for virulence. The identification of wex1 showed the potential of our approach to discover virulence factors not only in Mucorales but also in any other fungal model with an active RNAi machinery. More importantly, it adds a new layer to the biological processes controlled by RNAi in M. lusitanicus, confirming that the Dicer-dependent RNAi pathway can silence gene expression to promote virulence.

A Mucoralean White Collar-1 Photoreceptor Controls Virulence by Regulating an Intricate Gene Network during Host Interactions.

Mucolares are an ancient group of fungi encompassing the causal agents for the lethal infection mucormycosis. The high lethality rates, the emerging character of this disease, and the broad antifungal resistance of its causal agents are mucormycosis features that are alarming clinicians and researchers. Thus, the research field around mucormycosis is currently focused on finding specific weaknesses and targets in Mucorales for developing new treatments. In this work, we tested the role of the white-collar genes family in the virulence potential of Mucor lusitanicus. Study of the three genes of this family, mcwc-1a, mcwc-1b, and mcwc-1c, resulted in a marked functional specialization, as only mcwc-1a was essential to maintain the virulence potential of M. lusitanicus. The traditional role of wc-1 genes regulating light-dependent responses is a thoroughly studied field, whereas their role in virulence remains uncharacterized. In this work, we investigated the mechanism involving mcwc-1a in virulence from an integrated transcriptomic and functional approach during the host-pathogen interaction. Our results revealed mcwc-1a as a master regulator controlling an extensive gene network. Further dissection of this gene network clustering its components by type of regulation and functional criteria disclosed a multifunctional mechanism depending on diverse pathways. In the absence of phagocytic cells, mcwc-1a controlled pathways related to cell motility and the cytoskeleton that could be associated with the essential tropism during tissue invasion. After phagocytosis, several oxidative response pathways dependent on mcwc-1a were activated during the germination of M. lusitanicus spores inside phagocytic cells, which is the first stage of the infection. The third relevant group of genes involved in virulence and regulated by mcwc-1a belonged to the "unknown function," indicating that new and hidden pathways are involved in virulence. The unknown function category is especially pertinent in the study of mucormycosis, as it is highly enriched in specific fungal genes that represent the most promising targets for developing new antifungal compounds. These results unveil a complex multifunctional mechanism used by wc-1 genes to regulate the pathogenic potential in Mucorales that could also apply to other fungal pathogens.

Stable and reproducible homologous recombination enables CRISPR-based engineering in the fungus Rhizopus microsporus.

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.

Mucorales Species and Macrophages.

Mucormycosis is an emerging fungal infection caused by Mucorales with an unacceptable high mortality rate. Mucorales is a complex fungal group, including eleven different genera that can infect humans. This heterogeneity is associated with species-specific invasion pathways and responses to the host defense mechanisms. The host innate immune system plays a major role in preventing Mucorales growth and host invasion. In this system, macrophages are the main immune effector cells in controlling these fungi by rapid and efficient phagocytosis of the spores. However, Mucorales have evolved mechanisms to block phagosomal maturation and species-specific mechanisms to either survive as dormant spores inside the macrophage, as Rhizopus species, or geminate and escape, as Mucor species. Classical fungal models of mucormycosis, mostly Rhizopus, have made important contributions to elucidate key aspects of the interaction between Mucorales and macrophages, but they lack robust tools for genetic manipulation. The recent introduction of the genetically tractable Mucor circinelloides as a model of mucormycosis offers the possibility to analyze gene function. This has allowed the identification of regulatory pathways that control the fungal response to phagocytosis, including a non-canonical RNAi pathway (NCRIP) that regulates the expression of most genes regulated by phagocytosis.

A non-canonical RNAi pathway controls virulence and genome stability in Mucorales.

Epimutations in fungal pathogens are emerging as novel phenomena that could explain the fast-developing resistance to antifungal drugs and other stresses. These epimutations are generated by RNA interference (RNAi) mechanisms that transiently silence specific genes to overcome stressful stimuli. The early-diverging fungus Mucor circinelloides exercises a fine control over two interacting RNAi pathways to produce epimutants: the canonical RNAi pathway and a new RNAi degradative pathway. The latter is considered a non-canonical RNAi pathway (NCRIP) because it relies on RNA-dependent RNA polymerases (RdRPs) and a novel ribonuclease III-like named R3B2 to degrade target transcripts. Here in this work, we uncovered the role of NCRIP in regulating virulence processes and transposon movements through key components of the pathway, RdRP1 and R3B2. Mutants in these genes are unable to launch a proper virulence response to macrophage phagocytosis, resulting in a decreased virulence potential. The transcriptomic profile of rdrp1Δ and r3b2Δ mutants revealed a pre-exposure adaptation to the stressful phagosomal environment even when the strains are not confronted by macrophages. These results suggest that NCRIP represses key targets during regular growth and releases its control when a stressful environment challenges the fungus. NCRIP interacts with the RNAi canonical core to protect genome stability by controlling the expression of centromeric retrotransposable elements. In the absence of NCRIP, these retrotransposons are robustly repressed by the canonical RNAi machinery; thus, supporting the antagonistic role of NCRIP in containing the epimutational pathway. Both interacting RNAi pathways might be essential to govern host-pathogen interactions through transient adaptations, contributing to the unique traits of the emerging infection mucormycosis.

Genes, Pathways, and Mechanisms Involved in the Virulence of Mucorales.

The order Mucorales is a group of ancient fungi with limited tools for gene manipulation. The main consequence of this manipulation unwillingness is the limited knowledge about its biology compared to other fungal groups. However, the emerging of mucormycosis, a fungal infection caused by Mucorales, is attracting the medical spotlight in recent years because the treatments available are not efficient in reducing the high mortality associated with this disease. The result of this renewed interest in Mucorales and mucormycosis is an extraordinarily productive effort to unveil their secrets during the last decade. In this review, we describe the most compelling advances related to the genetic study of virulence factors, pathways, and molecular mechanisms developed in these years. The use of a few genetic study models has allowed the characterization of virulence factors in Mucorales that were previously described in other pathogens, such as the uptake iron systems, the mechanisms of dimorphism, and azole resistances. More importantly, recent studies are identifying new genes and mechanisms controlling the pathogenic potential of Mucorales and their interactions with the host, offering new alternatives to develop specific strategies against mucormycosis.

Early Diverging Fungus Mucor circinelloides Lacks Centromeric Histone CENP-A and Displays a Mosaic of Point and Regional Centromeres.

Centromeres are rapidly evolving across eukaryotes, despite performing a conserved function to ensure high-fidelity chromosome segregation. CENP-A chromatin is a hallmark of a functional centromere in most organisms. Due to its critical role in kinetochore architecture, the loss of CENP-A is tolerated in only a few organisms, many of which possess holocentric chromosomes. Here, we characterize the consequence of the loss of CENP-A in the fungal kingdom. Mucor circinelloides, an opportunistic human pathogen, lacks CENP-A along with the evolutionarily conserved CENP-C but assembles a monocentric chromosome with a localized kinetochore complex throughout the cell cycle. Mis12 and Dsn1, two conserved kinetochore proteins, were found to co-localize to a short region, one in each of nine large scaffolds, composed of an ∼200-bp AT-rich sequence followed by a centromere-specific conserved motif that echoes the structure of budding yeast point centromeres. Resembling fungal regional centromeres, these core centromere regions are embedded in large genomic expanses devoid of genes yet marked by Grem-LINE1s, a novel retrotransposable element silenced by the Dicer-dependent RNAi pathway. Our results suggest that these hybrid features of point and regional centromeres arose from the absence of CENP-A, thus defining novel mosaic centromeres in this early-diverging fungus.

Mucor circinelloides Thrives inside the Phagosome through an Atf-Mediated Germination Pathway.

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.IMPORTANCE Mucorales are a group of ancient saprophytic fungi that cause neglected infectious diseases collectively known as mucormycoses. The molecular processes underlying the establishment and progression of this disease are largely unknown. Our work presents a transcriptomic study to unveil the Mucor circinelloides genetic network triggered in fungal spores in response to phagocytosis by macrophages and the transcriptional response of the host cells. Functional characterization of differentially expressed fungal genes revealed three transcription factors and three extracellular proteins essential for the fungus to survive and germinate inside the phagosome and to cause disease in mice. Two of the transcription factors, highly similar to activating transcription factors (ATFs), coordinate a complex secondary gene response involved in pathogenesis. The significance of our research is in characterizing the initial stages that lead to evasion of the host innate immune response and, in consequence, the dissemination of the infection. This genetic study offers possible targets for novel antifungal drugs against these opportunistic human pathogens.

Generation of A Mucor circinelloides Reporter Strain-A Promising New Tool to Study Antifungal Drug Efficacy and Mucormycosis.

Invasive fungal infections caused by Mucorales (mucormycosis) have increased worldwide. These life-threatening infections affect mainly, but not exclusively, immunocompromised patients, and are characterized by rapid progression, severe tissue damage and an unacceptably high rate of mortality. Still, little is known about this disease and its successful therapy. New tools to understand mucormycosis and a screening method for novel antimycotics are required. Bioluminescent imaging is a powerful tool for in vitro and in vivo approaches. Hence, the objective of this work was to generate and functionally analyze bioluminescent reporter strains of Mucor circinelloides, one mucormycosis-causing pathogen. Reporter strains were constructed by targeted integration of the firefly luciferase gene under control of the M. circinelloides promoter Pzrt1. The luciferase gene was sufficiently expressed, and light emission was detected under several conditions. Phenotypic characteristics, virulence potential and antifungal susceptibility were indifferent to the wild-type strains. Light intensity was dependent on growth conditions and biomass, being suitable to determine antifungal efficacy in vitro. This work describes for the first time the generation of reporter strains in a basal fungus that will allow real-time, non-invasive infection monitoring in insect and murine models, and the testing of antifungal efficacy by means other than survival.

Molecular Tools for Carotenogenesis Analysis in the Mucoral Mucor circinelloides.

The carotene producer Mucor circinelloides is the fungus within the Mucoromycota phylum with the widest repertoire of molecular tools to manipulate its genome. The initial development of an effective procedure for genetic transformation and later improvements have resulted in an expansion of available tools, which include gene replacement, inactivation of gene expression by RNA silencing, gene overexpression, and functional genomics. Moreover, sequencing of its genome has given a definitive boost to these techniques making attainable the study of genes involved in many physiological or developmental processes, including carotenoid biosynthesis. Here, we describe in detail the latest molecular techniques currently used in M. circinelloides that have made it a valuable model for studying gene function within its phylum.

Mucor circinelloides: Growth, Maintenance, and Genetic Manipulation.

Mucor circinelloides is a fungus that belongs to the order Mucorales. It grows as mold in the environment and can cause mucormycosis, a potentially fatal infection in immunocompromised patients. M. circinelloides is a biodiesel producer and serves as a model organism for studying several biological processes, such as light responses and RNA interference-mediated gene silencing. Over the past decade, the increasing number of molecular tools has also allowed us to manipulate the genome of this fungus. This article outlines the fundamental protocols for the in vitro growth, maintenance, and genetic manipulation of M. circinelloides in the laboratory. © 2018 by John Wiley & Sons, Inc.

Components of a new gene family of ferroxidases involved in virulence are functionally specialized in fungal dimorphism.

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.

Understanding Mucor circinelloides pathogenesis by comparative genomics and phenotypical studies.

The increasing number of infections by species of Mucorales and their high mortality constitute an important concern for public health. This study aims to decipher the genetic basis of Mucor circinelloides pathogenicity, which displays virulence in a strain dependent manner. Assuming that genetic differences between strains may be linked to different pathotypes, we have conducted a study to explore genes responsible for virulence in M. circinelloides by whole genome sequencing of the avirulent strain NRRL3631 and comparison with the virulent strain CBS277.49. This genome analysis revealed 773 truncated, discontiguous and absent genes in the NRRL3631 strain. We also examined phenotypic traits resulting in reduced heat stress tolerance, chitosan content and lower susceptibility to toxic compounds (calcofluor white and sodium dodecyl sulphate) in the virulent strain, suggesting the influence of cell wall on pathogenesis. Based on these results, we focused on studying extracellular protein-coding genes by gene deletion and further pathotype characterization of mutants in murine models of pulmonary and systemic infection. Deletion of gene ID112092, which codes for a hypothetical extracellular protein of unknown function, resulted in significant reduction of virulence. Although pathogenesis is a multifactorial process, these findings highlight the crucial role of surface and secreted proteins in M. circinelloides virulence and should promote further studies of other differential genes.

RNAi-Based Functional Genomics Identifies New Virulence Determinants in Mucormycosis.

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.