Antifungal drug resistance evoked via RNAi-dependent epimutations.

Microorganisms evolve via a range of mechanisms that may include or involve sexual/parasexual reproduction, mutators, aneuploidy, Hsp90 and even prions. Mechanisms that may seem detrimental can be repurposed to generate diversity. Here we show that the human fungal pathogen Mucor circinelloides develops spontaneous resistance to the antifungal drug FK506 (tacrolimus) via two distinct mechanisms. One involves Mendelian mutations that confer stable drug resistance; the other occurs via an epigenetic RNA interference (RNAi)-mediated pathway resulting in unstable drug resistance. The peptidylprolyl isomerase FKBP12 interacts with FK506 forming a complex that inhibits the protein phosphatase calcineurin. Calcineurin inhibition by FK506 blocks M. circinelloides transition to hyphae and enforces yeast growth. Mutations in the fkbA gene encoding FKBP12 or the calcineurin cnbR or cnaA genes confer FK506 resistance and restore hyphal growth. In parallel, RNAi is spontaneously triggered to silence the fkbA gene, giving rise to drug-resistant epimutants. FK506-resistant epimutants readily reverted to the drug-sensitive wild-type phenotype when grown without exposure to the drug. The establishment of these epimutants is accompanied by generation of abundant fkbA small RNAs and requires the RNAi pathway as well as other factors that constrain or reverse the epimutant state. Silencing involves the generation of a double-stranded RNA trigger intermediate using the fkbA mature mRNA as a template to produce antisense fkbA RNA. This study uncovers a novel epigenetic RNAi-based epimutation mechanism controlling phenotypic plasticity, with possible implications for antimicrobial drug resistance and RNAi-regulatory mechanisms in fungi and other eukaryotes.

A non-canonical RNA degradation pathway suppresses RNAi-dependent epimutations in the human fungal pathogen Mucor circinelloides.

Mucorales are a group of basal fungi that includes the casual agents of the human emerging disease mucormycosis. Recent studies revealed that these pathogens activate an RNAi-based pathway to rapidly generate drug-resistant epimutant strains when exposed to stressful compounds such as the antifungal drug FK506. To elucidate the molecular mechanism of this epimutation pathway, we performed a genetic analysis in Mucor circinelloides that revealed an inhibitory role for the non-canonical RdRP-dependent Dicer-independent silencing pathway, which is an RNAi-based mechanism involved in mRNA degradation that was recently identified. Thus, mutations that specifically block the mRNA degradation pathway, such as those in the genes r3b2 and rdrp3, enhance the production of drug resistant epimutants, similar to the phenotype previously described for mutation of the gene rdrp1. Our genetic analysis also revealed two new specific components of the epimutation pathway related to the quelling induced protein (qip) and a Sad-3-like helicase (rnhA), as mutations in these genes prevented formation of drug-resistant epimutants. Remarkably, drug-resistant epimutant production was notably increased in M. circinelloides f. circinelloides isolates from humans or other animal hosts. The host-pathogen interaction could be a stressful environment in which the phenotypic plasticity provided by the epimutant pathway might provide an advantage for these strains. These results evoke a model whereby balanced regulation of two different RNAi pathways is determined by the activation of the RNAi-dependent epimutant pathway under stress conditions, or its repression when the regular maintenance of the mRNA degradation pathway operates under non-stress conditions.

Antimicrobial resistance profiles of microorganisms isolated from hospitalized patients in Dominican Republic.

OBJECTIVE: To define the antimicrobial resistance profiles of the microorganisms most commonly isolated from hospitalized adult patients in Dominican Republic (DR). METHODS: A retrospective, cross-sectional study of phenotypic antimicrobial susceptibility patterns was conducted using data from 3 802 clinical microbiology reports specifying positive bacterial cultures in samples collected from patients admitted to the clinical, surgery, and intensive care units (ICU) at three tertiary-level care hospitals in the city of Santiago de los Caballeros from 1 January 2016 - 31 December 2017. Descriptive statistics and chi-square test (P ≤ 0.05) were used to analyze the qualitative variables. RESULTS: At the three hospitals, there were 932, 1 090, and 1 780 microbiology reports analyzed. Of the total, 1274 were from the ICU, 1 042 from the surgery unit, and 1 486 from the clinical unit. Methicillin resistance was found in 57.3% of the Staphylococcus aureus isolates and 75.3% of the coagulase-negative staphylococci. Third-generation cephalosporin resistance was detected in 54.4% of isolates identified as members of the Enterobacteriaceae family, 67.3% of the Acinetobacter spp., and 91.7% of the Pseudomonas, while carbapenem resistance was shown by 8.0%, 23.8%, and 51.0% of these, respectively. Most of the resistant Acinetobacter spp. isolates were found in just one hospital and the prevalence of Enterobacteriaceae resistant to carbapenems was highest in the ICU. CONCLUSION: Antimicrobial resistance levels are high among hospitalized patients in Dominican Republic and may cause enhanced risk factors that impact clinical outcomes. Urgent measures are needed to address antimicrobial resistance in DR.

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.

A non-canonical RNA silencing pathway promotes mRNA degradation in basal Fungi.

The increasing knowledge on the functional relevance of endogenous small RNAs (esRNAs) as riboregulators has stimulated the identification and characterization of these molecules in numerous eukaryotes. In the basal fungus Mucor circinelloides, an emerging opportunistic human pathogen, esRNAs that regulate the expression of many protein coding genes have been described. These esRNAs share common machinery for their biogenesis consisting of an RNase III endonuclease Dicer, a single Argonaute protein and two RNA-dependent RNA polymerases. We show in this study that, besides participating in this canonical dicer-dependent RNA interference (RNAi) pathway, the rdrp genes are involved in a novel dicer-independent degradation process of endogenous mRNAs. The analysis of esRNAs accumulated in wild type and silencing mutants demonstrates that this new rdrp-dependent dicer-independent regulatory pathway, which does not produce sRNA molecules of discrete sizes, controls the expression of target genes promoting the specific degradation of mRNAs by a previously unknown RNase. This pathway mainly regulates conserved genes involved in metabolism and cellular processes and signaling, such as those required for heme biosynthesis, and controls responses to specific environmental signals. Searching the Mucor genome for candidate RNases to participate in this pathway, and functional analysis of the corresponding knockout mutants, identified a new protein, R3B2. This RNase III-like protein presents unique domain architecture, it is specifically found in basal fungi and, besides its relevant role in the rdrp-dependent dicer-independent pathway, it is also involved in the canonical dicer-dependent RNAi pathway, highlighting its crucial role in the biogenesis and function of regulatory esRNAs. The involvement of RdRPs in RNA degradation could represent the first evolutionary step towards the development of an RNAi mechanism and constitutes a genetic link between mRNA degradation and post-transcriptional gene silencing.

Calcineurin orchestrates dimorphic transitions, antifungal drug responses and host-pathogen interactions of the pathogenic mucoralean fungus Mucor circinelloides.

Calcineurin plays essential roles in virulence and growth of pathogenic fungi and is a target of the natural products FK506 and Cyclosporine A. In the pathogenic mucoralean fungus Mucor circinelloides, calcineurin mutation or inhibition confers a yeast-locked phenotype indicating that calcineurin governs the dimorphic transition. Genetic analysis in this study reveals that two calcineurin A catalytic subunits (out of three) are functionally diverged. Homology modeling illustrates modes of resistance resulting from amino substitutions in the interface between each calcineurin subunit and the inhibitory drugs. In addition, we show how the dimorphic transition orchestrated by calcineurin programs different outcomes during host-pathogen interactions. For example, when macrophages phagocytose Mucor yeast, subsequent phagosomal maturation occurs, indicating host cells respond appropriately to control the pathogen. On the other hand, upon phagocytosis of spores, macrophages fail to form mature phagosomes. Cytokine production from immune cells differs following exposure to yeast versus spores (which germinate into hyphae). Thus, the morphogenic transition can be targeted as an efficient treatment option against Mucor infection. In addition, genetic analysis (including gene disruption and mutational studies) further strengthens the understanding of calcineurin and provides a foundation to develop antifungal agents targeting calcineurin to deploy against Mucor and other pathogenic fungi.

Calcineurin plays key roles in the dimorphic transition and virulence of the human pathogenic zygomycete Mucor circinelloides.

Many pathogenic fungi are dimorphic and switch between yeast and filamentous states. This switch alters host-microbe interactions and is critical for pathogenicity. However, in zygomycetes, whether dimorphism contributes to virulence is a central unanswered question. The pathogenic zygomycete Mucor circinelloides exhibits hyphal growth in aerobic conditions but switches to multi-budded yeast growth under anaerobic/high CO2 conditions. We found that in the presence of the calcineurin inhibitor FK506, Mucor exhibits exclusively multi-budded yeast growth. We also found that M. circinelloides encodes three calcineurin catalytic A subunits (CnaA, CnaB, and CnaC) and one calcineurin regulatory B subunit (CnbR). Mutations in the latch region of CnbR and in the FKBP12-FK506 binding domain of CnaA result in hyphal growth of Mucor in the presence of FK506. Disruption of the cnbR gene encoding the sole calcineurin B subunit necessary for calcineurin activity yielded mutants locked in permanent yeast phase growth. These findings reveal that the calcineurin pathway plays key roles in the dimorphic transition from yeast to hyphae. The cnbR yeast-locked mutants are less virulent than the wild-type strain in a heterologous host system, providing evidence that hyphae or the yeast-hyphal transition are linked to virulence. Protein kinase A activity (PKA) is elevated during yeast growth under anaerobic conditions, in the presence of FK506, or in the yeast-locked cnbR mutants, suggesting a novel connection between PKA and calcineurin. cnaA mutants lacking the CnaA catalytic subunit are hypersensitive to calcineurin inhibitors, display a hyphal polarity defect, and produce a mixture of yeast and hyphae in aerobic culture. The cnaA mutants also produce spores that are larger than wild-type, and spore size is correlated with virulence potential. Our results demonstrate that the calcineurin pathway orchestrates the yeast-hyphal and spore size dimorphic transitions that contribute to virulence of this common zygomycete fungal pathogen.

RNAi function, diversity, and loss in the fungal kingdom.

RNAi is conserved and has been studied in a broad cross-section of the fungal kingdom, including Neurospora crassa, Schizosaccharomyces pombe, Cryptococcus neoformans, and Mucor circinelloides. And yet well known species, including the model yeast Saccharomyces cerevisiae and the plant pathogen Ustilago maydis, have lost RNAi, providing insights and opportunities to illuminate benefits conferred both by the presence of RNAi and its loss. Some of the earliest studies of RNAi were conducted in Neurospora, contemporaneously with the elucidation of RNAi in Caenorhabditis elegans. RNAi is a key epigenetic mechanism for maintaining genomic stability and integrity, as well as to defend against viruses, and given its ubiquity was likely present in the last eukaryotic common ancestor. In this review, we describe the diversity of RNAi mechanisms found in the fungi, highlighting recent work in Neurospora, S. pombe, and Cryptococcus. Finally, we consider frequent, independent losses of RNAi in diverse fungal lineages and both review and speculate on evolutionary forces that may drive the losses or result therefrom.

The Use of Lightweight Aggregates in Geopolymeric Mortars: The Effect of Liquid Absorption on the Physical/Mechanical Properties of the Mortar.

Geopolymers have been proposed as a green alternative to Portland cement with lowered carbon footprints. In this work, a geopolymeric mortar obtained using waste materials is studied. Fly ash, a waste generated by coal combustion, is used as one of the precursors, and waste glass as lightweight aggregates (LWAs) to improve the thermal performance of the mortar. The experimental study investigates the effect of varying the alkali activating solution (AAS) amount on the workability, compressive strength, and thermal conductivity of the mortar. Indeed, AAS represents the most expensive component in geopolymer production and is the highest contributor to the environmental footprint of these materials. This research starts by observing that LWA absorbs part of the activating solution during mixing, suggesting that only a portion of the solution effectively causes the geopolymerization reactions, the remaining part wetting the aggregates. Three mixes were investigated to clarify these aspects: a reference mix with a solution content calibrated to have a plastic consistency and two others with the activating solution reduced by the amount absorbed by aggregates. In these cases, the reduced workability was solved by adding the aggregates in a saturated surface dry state in one mix and free water in the other. The experimental results evidenced that free water addiction in place of a certain amount of the solution may be an efficient way to improve thermal performance without compromising the resistance of the mortar. The maximum compressive strength reached by the mortars was about 10 MPa at 48 days, a value in line with those of repair mortars. Another finding of the experimental research is that UPV was used to follow the curing stages of materials. Indeed, the instrument was sensitive to microstructural changes in the mortars with time. The field of reference of the research is the rehabilitation of existing buildings, as the geopolymeric mortars were designed for thermal and structural retrofitting.

Two distinct RNA-dependent RNA polymerases are required for initiation and amplification of RNA silencing in the basal fungus Mucor circinelloides.

RNA-dependent RNA polymerases (RdRPs) play key roles in the RNA silencing pathway in a number of organisms. They have been involved in the production of double-stranded RNA (dsRNA) molecules that initiate the silencing mechanism as well as in the amplification of the silencing signal. The roles of RdRPs from fungi in these processes are poorly described compared with other eukaryotes. RNA silencing in the zygomycete Mucor circinelloides exhibits uncommon features, such as induction by self-replicative sense transgenes and an amplification process associated with two size classes of antisense small interfering RNAs (siRNAs). To investigate the function of fungal RdRP proteins in initiation and amplification of silencing we have cloned and characterized two different rdrp genes in M. circinelloides. Functional analysis of rdrp(-) disruption mutants indicates that rdrp-1 is essential for initiation of silencing by sense transgenes by producing antisense RNA transcripts derived from the transgene, but it is not necessary for amplification of the silencing signal, whereas rdrp-2 is required for efficient accumulation of the two different classes of secondary siRNAs regardless the nature of the trigger. Our results provide evidence for a functional diversification of M. circinelloides rdrp genes in different steps of the same RNA silencing pathway.

Endogenous short RNAs generated by Dicer 2 and RNA-dependent RNA polymerase 1 regulate mRNAs in the basal fungus Mucor circinelloides.

Endogenous short RNAs (esRNAs) play diverse roles in eukaryotes and usually are produced from double-stranded RNA (dsRNA) by Dicer. esRNAs are grouped into different classes based on biogenesis and function but not all classes are present in all three eukaryotic kingdoms. The esRNA register of fungi is poorly described compared to other eukaryotes and it is not clear what esRNA classes are present in this kingdom and whether they regulate the expression of protein coding genes. However, evidence that some dicer mutant fungi display altered phenotypes suggests that esRNAs play an important role in fungi. Here, we show that the basal fungus Mucor circinelloides produces new classes of esRNAs that map to exons and regulate the expression of many protein coding genes. The largest class of these exonic-siRNAs (ex-siRNAs) are generated by RNA-dependent RNA Polymerase 1 (RdRP1) and dicer-like 2 (DCL2) and target the mRNAs of protein coding genes from which they were produced. Our results expand the range of esRNAs in eukaryotes and reveal a new role for esRNAs in fungi.

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.

A single dicer gene is required for efficient gene silencing associated with two classes of small antisense RNAs in Mucor circinelloides.

RNA silencing in the zygomycete Mucor circinelloides exhibits uncommon features, such as induction by self-replicative sense transgenes and the accumulation of two size classes of antisense small interfering RNAs (siRNAs). To investigate whether this silencing phenomenon follows the rules of a canonical RNA-silencing mechanism, we used hairpin RNA (hpRNA)-producing constructs as silencing triggers and analyzed the efficiency and stability of silencing in different genetic backgrounds. We show here that the dsRNA-induced silencing mechanism is also associated with the accumulation of two sizes of antisense siRNAs and that this mechanism is not mediated by the previously known dcl-1 (dicer-like) gene, which implies the existence of an additional dicer gene. An M. circinelloides dcl-2 gene was cloned and characterized, and the corresponding null mutant was generated by gene replacement. This mutant is severely impaired in the silencing mechanism induced by self-replicative sense or inverted-repeat transgenes, providing the first genetic evidence of a canonical silencing mechanism in this class of fungus and pointing to a role for dcl-2 in the mechanism. Moreover, a functional dcl-2 gene is required for the normal accumulation of the two sizes of antisense RNAs, as deduced from the analysis of dcl-2(-) transformants containing hpRNA-expressing plasmids. In addition to its critical role in transgene-induced silencing, the dcl-2 gene seems to play a role in the control of vegetative development, since the dcl-2 null mutants showed a significant decrease in their production of asexual spores.

A RING-finger photocarotenogenic repressor involved in asexual sporulation in Mucor circinelloides.

Mucor circinelloides responds to blue light by activating the biosynthesis of carotenoids and bending its sporangiophores towards the light source. The CrgA protein product acts as a repressor of carotene biosynthesis, as its inactivation leads to the overaccumulation of carotenoids in both the dark and the light. We show here that asexual sporulation in Mucor is also stimulated by light and that the crgA gene is involved in sporulation, given that lack of crgA function affects both carotenogenesis and the normal production of spores. A small interference RNA (siRNA) gene silencing approach was used to block the biosynthesis of carotenoids and to demonstrate that abnormal sporulation in crgA mutants is not a consequence of a defective production of carotenes. These results reveal an active role for the predicted CrgA product, a RING-finger protein, in the control of cellular light-regulated processes in Mucor.

Antibiotic resistance profile in intrahospital pediatric services at third level centers in Dominican Republic / Perfil de resistencia a antibióticos en servicios pediátricos intrahospitalarios en centros de tercer nivel en República Dominicana

Objectives: The Dominican Republic lacks reliable information on antimicrobial resistance (AMR), which would allow physicians to prescribe the best treatment for common infectious diseases. This study aimed to define the antimicrobial resistance profiles of the more common pathogens from pediatric services, where data is even more important due to the vulnerability of the population. Methods: We collected data from patients admitted in the pediatric unit of three third level hospitals in the city of Santiago de los Caballeros, Dominican Republic, showing positive bacterial cultures, during a period of two years. Results: Half of the Gram negative pathogens exhibited third generation cephalosporins (3GC) resistance, 17% were resistant to carbapenems. Serratia marcescens presented an exceptionally high proportion of resistance to 3GC (95.9%). Staphylococcus aureus showed elevated resistance to methicillin (58.4%) and even to clindamycin (35.8%). Conclusion: There are elevated levels of antimicrobial resistance among the Enterobacteriaceae family and the Staphylococcus genus isolated from the pediatric population. Necessary measures should be taken to tackle AMR in the country.

Objetivos: La República Dominicana carece de información confiable sobre las resistencias antimicrobianas en el país, lo que permitiría al personal médico prescribir los mejores tratamientos para infecciones comunes. El objetivo de este estudio es definir los perfiles de resistencia antimicrobiana de los patógenos más comunes en servicios pediátricos, donde esta información es esencial, debido a la vulnerabilidad de la población. Métodos: Se tomaron los datos de reportes microbiológicos con cultivo bacteriano positivo procedentes de pacientes admitidos en la unidad pediátrica de tres hospitales de tercer nivel en la ciudad de Santiago de los Caballeros, República Dominicana, durante un periodo de dos años. Resultados: La mitad de los patógenos Gram negativos mostraron resistencia a cefalosporinas de tercera generación (3GC), 17% eran resistentes a carbapenémicos. Serratia marcescens presentó una resistencia excepcionalmente elevada a 3GC (95.9%). Staphylococcus aureus mostró alta resistencia a meticilina (58.4%) e incluso a clindamicina (35.8%). Conclusión: Existen elevados niveles de resistencia antimicrobiana entre las enterobacterias y los estafilococos en la población pediátrica dominicana. Es necesario tomar medidas para abordar este problema en el país.