A ribonuclease III involved in virulence of Mucorales fungi has evolved to cut exclusively single-stranded RNA.

Members of the ribonuclease III (RNase III) family regulate gene expression by processing double-stranded RNA (dsRNA). This family includes eukaryotic Dicer and Drosha enzymes that generate small dsRNAs in the RNA interference (RNAi) pathway. The fungus Mucor lusitanicus, which causes the deadly infection mucormycosis, has a complex RNAi system encompassing a non-canonical RNAi pathway (NCRIP) that regulates virulence by degrading specific mRNAs. In this pathway, Dicer function is replaced by R3B2, an atypical class I RNase III, and small single-stranded RNAs (ssRNAs) are produced instead of small dsRNA as Dicer-dependent RNAi pathways. Here, we show that R3B2 forms a homodimer that binds to ssRNA and dsRNA molecules, but exclusively cuts ssRNA, in contrast to all known RNase III. The dsRNA cleavage inability stems from its unusual RNase III domain (RIIID) because its replacement by a canonical RIIID allows dsRNA processing. A crystal structure of R3B2 RIIID resembles canonical RIIIDs, despite the low sequence conservation. However, the groove that accommodates dsRNA in canonical RNases III is narrower in the R3B2 homodimer, suggesting that this feature could be responsible for the cleavage specificity for ssRNA. Conservation of this activity in R3B2 proteins from other mucormycosis-causing Mucorales fungi indicates an early evolutionary acquisition.

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.

Genetic Manipulation in Mucorales and New Developments to Study Mucormycosis.

The study of the Mucoralean fungi physiology is a neglected field that the lack of effective genetic tools has hampered in the past. However, the emerging fungal infection caused by these fungi, known as mucormycosis, has prompted many researchers to study the pathogenic potential of Mucorales. The main reasons for this current attraction to study mucormycosis are its high lethality, the lack of effective antifungal drugs, and its recent increased incidence. The most contemporary example of the emergence character of mucormycosis is the epidemics declared in several Asian countries as a direct consequence of the COVID-19 pandemic. Fortunately, this pressure to understand mucormycosis and develop new treatment strategies has encouraged the blossoming of new genetic techniques and methodologies. This review describes the history of genetic manipulation in Mucorales, highlighting the development of methods and how they allowed the main genetic studies in these fungi. Moreover, we have emphasized the recent development of new genetic models to study mucormycosis, a landmark in the field that will configure future research related to this disease.

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.

The Evolutionary Significance of RNAi in the Fungal Kingdom.

RNA interference (RNAi) was discovered at the end of last millennium, changing the way scientists understood regulation of gene expression. Within the following two decades, a variety of different RNAi mechanisms were found in eukaryotes, reflecting the evolutive diversity that RNAi entails. The essential silencing mechanism consists of an RNase III enzyme called Dicer that cleaves double-stranded RNA (dsRNA) generating small interfering RNAs (siRNAs), a hallmark of RNAi. These siRNAs are loaded into the RNA-induced silencing complex (RISC) triggering the cleavage of complementary messenger RNAs by the Argonaute protein, the main component of the complex. Consequently, the expression of target genes is silenced. This mechanism has been thoroughly studied in fungi due to their proximity to the animal phylum and the conservation of the RNAi mechanism from lower to higher eukaryotes. However, the role and even the presence of RNAi differ across the fungal kingdom, as it has evolved adapting to the particularities and needs of each species. Fungi have exploited RNAi to regulate a variety of cell activities as different as defense against exogenous and potentially harmful DNA, genome integrity, development, drug tolerance, or virulence. This pathway has offered versatility to fungi through evolution, favoring the enormous diversity this kingdom comprises.

Generation of lycopene-overproducing strains of the fungus Mucor circinelloides reveals important aspects of lycopene formation and accumulation.

OBJECTIVES: To generate lycopene-overproducing strains of the fungus Mucor circinelloides with interest for industrial production and to gain insight into the catalytic mechanism of lycopene cyclase and regulatory process during lycopene overaccumulation. RESULTS: Three lycopene-overproducing mutants were generated by classic mutagenesis techniques from a ß-carotene-overproducing strain. They carried distinct mutations in the carRP gene encoding lycopene cyclase that produced loss of enzymatic activity to different extents. In one mutant (MU616), the lycopene cyclase was completely destroyed, and a 43.8% (1.1 mg/g dry mass) increase in lycopene production was observed in comparison to that by the previously existing lycopene overproducer. In addition, feedback regulation of the end product was suggested in lycopene-overproducing strains. CONCLUSIONS: A lycopene-overaccumulating strain of the fungus M. circinelloides was generated that could be an alternative for the industrial production of lycopene. Vital catalytic residues for lycopene cyclase activity and the potential mechanism of lycopene formation and accumulation were identified.

A new regulatory mechanism controlling carotenogenesis in the fungus Mucor circinelloides as a target to generate ß-carotene over-producing strains by genetic engineering.

BACKGROUND: Carotenoids are natural pigments with antioxidant properties that have important functions in human physiology and must be supplied through the diet. They also have important industrial applications as food colourants, animal feed additives and nutraceuticals. Some of them, such as ß-carotene, are produced on an industrial scale with the use of microorganisms, including fungi. The mucoral Blakeslea trispora is used by the industry to produce ß-carotene, although optimisation of production by molecular genetic engineering is unfeasible. However, the phylogenetically closely related Mucor circinelloides, which is also able to accumulate ß-carotene, possesses a vast collection of genetic tools with which to manipulate its genome. RESULTS: This work combines classical forward and modern reverse genetic techniques to deepen the regulation of carotenoid synthesis and generate candidate strains for biotechnological production of ß-carotene. Mutagenesis followed by screening for mutants with altered colour in the dark and/or in light led to the isolation of 26 mutants that, together with eight previously isolated mutants, have been analysed in this work. Although most of the mutants harboured mutations in known structural and regulatory carotenogenic genes, eight of them lacked mutations in those genes. Whole-genome sequencing of six of these strains revealed the presence of many mutations throughout their genomes, which makes identification of the mutation that produced the phenotype difficult. However, deletion of the crgA gene, a well-known repressor of carotenoid biosynthesis in M. circinelloides, in two mutants (MU206 and MU218) with high levels of ß-carotene resulted in a further increase in ß-carotene content to differing extents with respect to the crgA single-null strain; in particular, one strain derived from MU218 was able to accumulate up to 4 mg/g of ß-carotene. The additive effect of crgA deletion and the mutations present in MU218 suggests the existence of a previously unknown regulatory mechanism that represses carotenoid biosynthesis independently and in parallel to crgA. CONCLUSIONS: The use of a mucoral model such as M. circinelloides can allow the identification of the regulatory mechanisms that control carotenoid biosynthesis, which can then be manipulated to generate tailored strains of biotechnological interest. Mutants in the repressor crgA and in the newly identified regulatory mechanism generated in this work accumulate high levels of ß-carotene and are candidates for further improvements in biotechnological ß-carotene production.

Molecular mechanisms that govern infection and antifungal resistance in Mucorales.

SUMMARYThe World Health Organization has established a fungal priority pathogens list that includes species critical or highly important to human health. Among them is the order Mucorales, a fungal group comprising at least 39 species responsible for the life-threatening infection known as mucormycosis. Despite the continuous rise in cases and the poor prognosis due to innate resistance to most antifungal drugs used in the clinic, Mucorales has received limited attention, partly because of the difficulties in performing genetic manipulations. The COVID-19 pandemic has further escalated cases, with some patients experiencing the COVID-19-associated mucormycosis, highlighting the urgent need to increase knowledge about these fungi. This review addresses significant challenges in treating the disease, including delayed and poor diagnosis, the lack of accurate global incidence estimation, and the limited treatment options. Furthermore, it focuses on the most recent discoveries regarding the mechanisms and genes involved in the development of the disease, antifungal resistance, and the host defense response. Substantial advancements have been made in identifying key fungal genes responsible for invasion and tissue damage, host receptors exploited by the fungus to invade tissues, and mechanisms of antifungal resistance. This knowledge is expected to pave the way for the development of new antifungals to combat mucormycosis. In addition, we anticipate significant progress in characterizing Mucorales biology, particularly the mechanisms involved in pathogenesis and antifungal resistance, with the possibilities offered by CRISPR-Cas9 technology for genetic manipulation of the previously intractable Mucorales species.

H3K4 methylation regulates development, DNA repair, and virulence in Mucorales.

Mucorales are basal fungi that opportunistically cause a potentially fatal infection known as mucormycosis (black fungus disease), which poses a significant threat to human health due to its high mortality rate and its recent association with SARS-CoV-2 infections. On the other hand, histone methylation is a regulatory mechanism with pleiotropic effects, including the virulence of several pathogenic fungi. However, the role of epigenetic changes at the histone level never has been studied in Mucorales. Here, we dissected the functional role of Set1, a histone methyltransferase that catalyzes the methylation of H3K4, which is associated with the activation of gene transcription and virulence. A comparative analysis of the Mucor lusitanicus genome (previously known as Mucor circinelloides f. lusitanicus) identified only one homolog of Set1 from Candida albicans and Saccharomyces cerevisiae that contains the typical SET domain. Knockout strains in the gene set1 lacked H3K4 monomethylation, dimethylation, and trimethylation enzymatic activities. These strains also showed a significant reduction in vegetative growth and sporulation. Additionally, set1 null strains were more sensitive to SDS, EMS, and UV light, indicating severe impairment in the repair process of the cell wall and DNA lesions and a correlation between Set1 and these processes. During pathogen-host interactions, strains lacking the set1 gene exhibited shortened polar growth within the phagosome and attenuated virulence both in vitro and in vivo. Our findings suggest that the histone methyltransferase Set1 coordinates several cell processes related to the pathogenesis of M. lusitanicus and may be an important target for future therapeutic strategies against mucormycosis.

Symmetric and asymmetric DNA N6-adenine methylation regulates different biological responses in Mucorales.

DNA N6-adenine methylation (6mA) has recently gained importance as an epigenetic modification in eukaryotes. Its function in lineages with high levels, such as early-diverging fungi (EDF), is of particular interest. Here, we investigated the biological significance and evolutionary implications of 6mA in EDF, which exhibit divergent evolutionary patterns in 6mA usage. The analysis of two Mucorales species displaying extreme 6mA usage reveals that species with high 6mA levels show symmetric methylation enriched in highly expressed genes. In contrast, species with low 6mA levels show mostly asymmetric 6mA. Interestingly, transcriptomic regulation throughout development and in response to environmental cues is associated with changes in the 6mA landscape. Furthermore, we identify an EDF-specific methyltransferase, likely originated from endosymbiotic bacteria, as responsible for asymmetric methylation, while an MTA-70 methylation complex performs symmetric methylation. The distinct phenotypes observed in the corresponding mutants reinforced the critical role of both types of 6mA in EDF.

A white collar 1-like protein mediates opposite regulatory functions in Mucor circinelloides.

Protein ubiquitylation plays a major role in the regulation of many cellular processes by altering the stability, localization or function of target proteins. CrgA is a protein of Mucor circinelloides that shows the characteristics of ubiquitin ligases and is involved in the regulation of carotenogenesis and asexual sporulation in this fungus. CrgA, which belongs to a poorly characterized group of proteins present in almost all eukaryotes, represses carotenogenesis through the proteolysis-independent mono- and di-ubiquitylation of Mcwc-1b, a White Collar-1-like protein which, when it is non-ubiquitylated, activates carotenogenesis. Using a proteomic approach, this work shows that the regulation of M. circinelloides vegetative development by CrgA is also mediated by Mcwc-1b, although, in this case, the non-ubiquitylated Mcwc-1b form acts as a repressor. High levels of a protein that contains a classical Rossmann-fold NAD(P)H/NAD(P)(+) binding domain for NAD(P)H binding and is similar to NmrA NADP(H) sensor-like proteins occur when Mcwc-1b is inactivated by ubiquitylation. A role for this protein in the regulation of sporulation is suggested because its over-expression suppresses the sporulation defect in a crgAΔ mutant. NmrA-like proteins are repressors that interact with GATA transcription factors and have been shown to be related to cell differentiation in Magnaporthe oryzae and Dictyostelium discoideum. This proteomic approach also revealed that CrgA regulates the carbon and energy metabolism and that Mcwc-1b is the main, but not the only, target of CrgA.

Mucorales and Mucormycosis: Recent Insights and Future Prospects.

The classification of Mucorales encompasses a collection of basal fungi that have traditionally demonstrated an aversion to modern genetic manipulation techniques. This aversion led to a scarcity of knowledge regarding their biology compared to other fungal groups. However, the emergence of mucormycosis, a fungal disease caused by Mucorales, has attracted the attention of the clinical field, mainly because available therapies are ineffective for decreasing the fatal outcome associated with the disease. This revitalized curiosity about Mucorales and mucormycosis, also encouraged by the recent COVID-19 pandemic, has spurred a significant and productive effort to uncover their mysteries in recent years. Here, we elaborate on the most remarkable breakthroughs related to the recently discovered genetic advances in Mucorales and mucormycosis. The utilization of a few genetic study models has enabled the identification of virulence factors in Mucorales that were previously described in other pathogens. More notably, recent investigations have identified novel genes and mechanisms controlling the pathogenic potential of Mucorales and their interactions with the host, providing fresh avenues to devise new strategies against mucormycosis. Finally, new study models are allowing virulence studies that were previously hampered in Mucorales, predicting a prolific future for the field.

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).

DNA Methylation on N6-Adenine Regulates the Hyphal Development during Dimorphism in the Early-Diverging Fungus Mucor lusitanicus.

The epigenetic modifications control the pathogenicity of human pathogenic fungi, which have been poorly studied in Mucorales, causative agents of mucormycosis. This order belongs to a group referred to as early-diverging fungi that are characterized by high levels of N6-methyldeoxy adenine (6mA) in their genome with dense 6mA clusters associated with actively expressed genes. AlkB enzymes can act as demethylases of 6mA in DNA, with the most remarkable eukaryotic examples being mammalian ALKBH1 and Caenorhabditis elegans NMAD-1. The Mucor lusitanicus (formerly M. circinelloides f. lusitanicus) genome contains one gene, dmt1, and two genes, dmt2 and dmt3, encoding proteins similar to C. elegans NMAD-1 and ALKBH1, respectively. The function of these three genes was analyzed by the generation of single and double deletion mutants for each gene. Multiple processes were studied in the mutants, but defects were only found in single and double deletion mutants for dmt1. In contrast to the wild-type strain, dmt1 mutants showed an increase in 6mA levels during the dimorphic transition, suggesting that 6mA is associated with dimorphism in M. lusitanicus. Furthermore, the spores of dmt1 mutants challenged with macrophages underwent a reduction in polar growth, suggesting that 6mA also has a role during the spore-macrophage interaction that could be important in the infection process.

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.

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.

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.

Light regulates a Phycomyces blakesleeanus gene family similar to the carotenogenic repressor gene of Mucor circinelloides.

The transcription of about 5-10 % of the genes in Phycomyces blakesleeanus is regulated by light. Among the most up-regulated, we have identified four genes, crgA-D, with similarity to crgA of Mucor circinelloides, a gene encoding a repressor of light-inducible carotenogenesis. The four proteins have the same structure with two RING RING Finger domains and a LON domain, suggesting that they could act as ubiquitin ligases, as their M. circinelloides homolog. The expression of these genes is induced by light with different thresholds as in other Mucoromycotina fungi like Blakeslea trispora and M. circinelloides. Only the P. blakesleeanus crgD gene could restore the wild type phenotype in a M. circinelloides null crgA mutant suggesting that P. blakesleeanus crgD is the functional homolog of crgA in M. circinelloides. Despite their sequence similarity it is possible that the P. blakesleeanus Crg proteins do not participate in the regulation of beta-carotene biosynthesis since none of the carotene-overproducing mutants of P. blakesleeanus had mutations in any of the crg genes. Our results provide further support of the differences in the regulation of the biosynthesis of beta-carotene in these two Mucoromycotina fungi.

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.