Candida albicans selection for human commensalism results in substantial within-host diversity without decreasing fitness for invasive disease.

Candida albicans is a frequent colonizer of human mucosal surfaces as well as an opportunistic pathogen. C. albicans is remarkably versatile in its ability to colonize diverse host sites with differences in oxygen and nutrient availability, pH, immune responses, and resident microbes, among other cues. It is unclear how the genetic background of a commensal colonizing population can influence the shift to pathogenicity. Therefore, we examined 910 commensal isolates from 35 healthy donors to identify host niche-specific adaptations. We demonstrate that healthy people are reservoirs for genotypically and phenotypically diverse C. albicans strains. Using limited diversity exploitation, we identified a single nucleotide change in the uncharacterized ZMS1 transcription factor that was sufficient to drive hyper invasion into agar. We found that SC5314 was significantly different from the majority of both commensal and bloodstream isolates in its ability to induce host cell death. However, our commensal strains retained the capacity to cause disease in the Galleria model of systemic infection, including outcompeting the SC5314 reference strain during systemic competition assays. This study provides a global view of commensal strain variation and within-host strain diversity of C. albicans and suggests that selection for commensalism in humans does not result in a fitness cost for invasive disease.

N6-Methyladenosine Regulates Host Responses to Viral Infection.

Recent discoveries have revealed that, during viral infection, the presence of the RNA modification N6-methyladenosine (m6A) on viral and cellular RNAs has profound impacts on infection outcome. Although m6A directly regulates many viral RNA processes, its effects on cellular RNAs and pathways during infection have only recently begun to be elucidated. Disentangling the effects of m6A on viral and host RNAs remains a challenge for the field. m6A has been found to regulate host responses such as viral RNA sensing, cytokine responses, and immune cell functions. We highlight recent findings describing how m6A modulates host responses to viral infection and discuss future directions that will lead to a synergistic understanding of the processes by which m6A regulates viral infection.

Prospective, randomized, double blind comparison of suture materials with and without triclosan in dogs undergoing tibial plateau leveling osteotomy.

Objectives: To determine if triclosan-impregnated suture decreases surgical site infection rates after tibial plateau leveling osteotomy (TPLO) in dogs. Sample population: There were 116 dogs with naturally occurring cranial cruciate ligament disease presenting for treatment with TPLO. Procedures: Written consent was obtained by all clients in order to be included in this study. Dogs were randomly assigned a suture type immediately before the start of anesthesia. Infection rates were compared between the suture groups, as were the gender, duration of anesthesia, duration of surgery, age of dog, weight, length of incision, and stifle side. Direct examination by a veterinarian was conducted at 24 h, 10 to 14 d, and 8 to 12 wk after surgery. If the dogs did not return for direct examination, owners were contacted by a veterinarian and phone interviews were conducted. Results: Overall, 12.9% of the incisions were diagnosed with a surgical site infection (SSI). The SSI rate for dogs that received the triclosan suture was 5.35% (3/56), and the rate for dogs that received the regular suture was 19.64% (11/56), with P = 0.016. The duration of anesthesia, duration of surgery, age, weight, length of incision, and right versus left stifle did not show a significant difference in infection rates. The suture type did have a significant effect, and triclosan-impregnated suture had a decreased infection rate when compared to regular suture. Gender also had a significant effect, with P = 0.032. Conclusion: Triclosan-impregnated suture decreased SSI when used for closure in dogs undergoing TPLO. Triclosan-impregnated suture may be considered a material of choice to close surgical wounds at risk of SSI when implants are used.

Comparaison prospective, randomisée, en double aveugle des matériaux de suture avec et sans triclosan chez les chiens subissant une ostéotomie de nivellement du plateau tibial. Objectifs: Déterminer si la suture imprégnée de triclosan diminue les taux d'infection du site opératoire après une ostéotomie de nivellement du plateau tibial (TPLO) chez le chien. Échantillon de population: Il y avait 116 chiens avec une pathologie naturelle du ligament croisé crânial se présentant pour un traitement avec TPLO. Procédures: Un consentement écrit a été obtenu par tous les clients afin d'être inclus dans cette étude. Les chiens ont été répartis au hasard à un type de suture immédiatement avant le début de l'anesthésie. Les taux d'infection ont été comparés entre les groupes de suture, de même que le sexe, la durée de l'anesthésie, la durée de la chirurgie, l'âge du chien, le poids, la longueur de l'incision et le côté du grasset. Un examen direct par un vétérinaire a été effectué à 24 h, 10 à 14 j et 8 à 12 semaines après la chirurgie. Si les chiens ne revenaient pas pour un examen direct, les propriétaires étaient contactés par un vétérinaire et des entretiens téléphoniques étaient menés. Résultats: Dans l'ensemble, 12,9 % des incisions ont été diagnostiquées avec une infection du site opératoire (SSI). Le taux de SSI pour les chiens ayant reçu la suture au triclosan était de 5,35 % (3/56) et le taux pour les chiens ayant reçu la suture régulière était de 19,64 % (11/56), avec P = 0,016. La durée de l'anesthésie, la durée de la chirurgie, l'âge, le poids, la longueur de l'incision et le grasset droit versus le gauche n'ont pas montré de différence significative dans les taux d'infection. Le type de suture avait un effet significatif et la suture imprégnée de triclosan avait un taux d'infection réduit par rapport à la suture ordinaire. Le sexe avait également un effet significatif, avec P = 0,032. Conclusion: La suture imprégnée de triclosan a diminué le SSI lorsqu'elle était utilisée pour la fermeture de l'incision chez les chiens subissant une TPLO. La suture imprégnée de triclosan peut être considérée comme un matériau de choix pour fermer les plaies chirurgicales à risque de SSI lorsque des implants sont utilisés.(Traduit par Dr Serge Messier).

Pervasive tertiary structure in the dengue virus RNA genome.

RNA virus genomes are efficient and compact carriers of biological information, encoding information required for replication both in their primary sequences and in higher-order RNA structures. However, the ubiquity of RNA elements with higher-order folds-in which helices pack together to form complex 3D structures-and the extent to which these elements affect viral fitness are largely unknown. Here we used single-molecule correlated chemical probing to define secondary and tertiary structures across the RNA genome of dengue virus serotype 2 (DENV2). Higher-order RNA structures are pervasive and involve more than one-third of nucleotides in the DENV2 genomic RNA. These 3D structures promote a compact overall architecture and contribute to viral fitness. Disrupting RNA regions with higher-order structures leads to stable, nonreverting mutants and could guide the development of vaccines based on attenuated RNA viruses. The existence of extensive regions of functional RNA elements with tertiary folds in viral RNAs, and likely many other messenger and noncoding RNAs, means that there are significant regions with pocket-containing surfaces that may serve as novel RNA-directed drug targets.

The small GTPase RAB1B promotes antiviral innate immunity by interacting with TNF receptor-associated factor 3 (TRAF3).

Innate immune detection of viral nucleic acids during viral infection activates a signaling cascade that induces type I and type III IFNs as well as other cytokines, to generate an antiviral response. This signaling is initiated by pattern recognition receptors, such as the RNA helicase retinoic acid-inducible gene I (RIG-I), that sense viral RNA. These sensors then interact with the adaptor protein mitochondrial antiviral signaling protein (MAVS), which recruits additional signaling proteins, including TNF receptor-associated factor 3 (TRAF3) and TANK-binding kinase 1 (TBK1), to form a signaling complex that activates IFN regulatory factor 3 (IRF3) for transcriptional induction of type I IFNs. Here, using several immunological and biochemical approaches in multiple human cell types, we show that the GTPase-trafficking protein RAB1B up-regulates RIG-I pathway signaling and thereby promotes IFN-ß induction and the antiviral response. We observed that RAB1B overexpression increases RIG-I-mediated signaling to IFN-ß and that RAB1B deletion reduces signaling of this pathway. Additionally, loss of RAB1B dampened the antiviral response, indicated by enhanced Zika virus infection of cells depleted of RAB1B. Importantly, we identified the mechanism of RAB1B action in the antiviral response, finding that it forms a protein complex with TRAF3 to facilitate the interaction of TRAF3 with mitochondrial antiviral signaling protein. We conclude that RAB1B regulates TRAF3 and promotes the formation of innate immune signaling complexes in response to nucleic acid sensing during RNA virus infection.

Use of vessel sealing system for multiple partial lung lobectomies for spontaneous pneumothorax.

A wolf hybrid dog was presented for dyspnea and tachypnea. Thoracic radiographs revealed a pneumothorax. A median sternotomy was performed, and multiple pulmonary blebs were identified on several lung lobes. Multiple partial lung lobectomies were performed using a vessel sealing system. The dog was discharged 4 days after surgery free of clinical signs related to surgery or pneumothorax. This case represents a novel utilization of a vessel sealing system to remove the apex of the lung when there are numerous pulmonary lesions present. Key clinical message: A vessel sealing system simplified multiple partial lung lobectomies in an open thoracotomy. The system reduced tissue trauma as well as the amount of normal pulmonary tissue removed while efficiently creating a seal.

Utilisation d'un système de scellement des vaisseaux lors de lobectomies pulmonaires partielles pour un pneumothorax spontané. Un hybride chien-loup fut présenté pour dyspnée et tachypnée. Des radiographies thoraciques ont révélé un pneumothorax. Une sternotomie médiane fut effectuée et de multiples vésicules furent identifiés sur plusieurs lobes pulmonies. De multiples lobectomies partielles furent effectuées en utilisant un système de scellement des vaisseaux. Le chien obtint son congé 4 jours après la chirurgie sans aucun signe clinique relié à la chirurgie ou au pneumothorax. Ce cas représente une utilisation nouvelle d'un système de scellement des vaisseaux pour retirer l'apex des poumons lorsqu'il y a de nombreuses lésions pulmonaires présentes.Message clinique clé :Un système de scellement des vaisseaux simplifia des lobectomies pulmonaires partielles multiples lors d'une thoracotomie ouverte. Le système réduisit les traumatismes tissulaires ainsi que la quantité de tissu pulmonaire normal retirée tout en créant efficacement un sceau.(Traduit par Dr Serge Messier).

Phylogenetic investigation of skin sloughing rates in frogs: relationships with skin characteristics and disease-driven declines.

Amphibian skin is highly variable in structure and function across anurans, and plays an important role in physiological homeostasis and immune defence. For example, skin sloughing has been shown to reduce pathogen loads on the skin, such as the lethal fungus Batrachochytrium dendrobatidis ( Bd), but interspecific variation in sloughing frequency is largely unknown. Using phylogenetic linear mixed models, we assessed the relationship between skin turnover rate, skin morphology, ecological traits and overall evidence of Bd-driven declines. We examined skin sloughing rates in 21 frog species from three continents, as well as structural skin characteristics measured from preserved specimens. We found that sloughing rate varies significantly with phylogenetic group, but was not associated with evidence of Bd-driven declines, or other skin characteristics examined. This is the first comparison of sloughing rate across a wide range of amphibian species, and creates the first database of amphibian sloughing behaviour. Given the strong phylogenetic signal observed in sloughing rate, approximate sloughing rates of related species may be predicted based on phylogenetic position. While not related to available evidence of declines, understanding variation in sloughing rate may help explain differences in the severity of infection in genera with relatively slow skin turnover rates (e.g. Atelopus).

The efficacy and pharmacokinetics of terbinafine against the frog-killing fungus (Batrachochytrium dendrobatidis).

Captive and wild amphibians are under threat of extinction from the deadly fungal pathogen Batrachochytrium dendrobatidis (Bd). The antifungal drug terbinafine (TBF) is used by pet owners to treat Bd-infected frogs; however, it is not widely used in academic or zoological institutions due to limited veterinary clinical trials. To assess TBF's efficacy, we undertook treatment trials and pharmacokinetic studies to investigate drug absorption and persistence in frog skin; and then we correlated these data to the minimal lethal concentrations (MLC) against Bd. Despite an initial reduction in zoospore load, the recommended treatment (five daily 5 min 0.01% TBF baths) was unable to cure experimentally infected alpine tree frogs and naturally infected common eastern froglets. In vitro and in vivo pharmacokinetics showed that absorbed TBF accumulates in frog skin with increased exposure, indicating its suitability for treating cutaneous pathogens via direct application. The MLC of TBF for zoosporangia was 100 µg/ml for 2 h, while the minimal inhibitory concentration was 2 µg/ml, suggesting that the drug concentration absorbed during 5 min treatments is not sufficient to cure high Bd burdens. With longer treatments of five daily 30 min baths, Bd clearance improved from 12.5% to 50%. A higher dose of 0.02% TBF resulted in 78% of animals cured; however, clearance was not achieved in all individuals due to low TBF skin persistence, as the half-life was less than 2 h. Therefore, the current TBF regime is not recommended as a universal treatment against Bd until protocols are optimized, such as with increased exposure frequency.

Evolution of resistance to chytridiomycosis is associated with a robust early immune response.

Potentiating the evolution of immunity is a promising strategy for addressing biodiversity diseases. Assisted selection for infection resistance may enable the recovery and persistence of amphibians threatened by chytridiomycosis, a devastating fungal skin disease threatening hundreds of species globally. However, knowledge of the mechanisms involved in the natural evolution of immunity to chytridiomycosis is limited. Understanding the mechanisms of such resistance may help speed-assisted selection. Using a transcriptomics approach, we examined gene expression responses of endangered alpine tree frogs (Litoria verreauxii alpina) to subclinical infection, comparing two long-exposed populations with a naïve population. We performed a blinded, randomized and controlled exposure experiment, collecting skin, liver and spleen tissues at 4, 8 and 14 days postexposure from 51 wild-caught captively reared infection-naïve adult frogs for transcriptome assembly and differential gene expression analyses. We analysed our results in conjunction with infection intensity data, and the results of a large clinical survival experiment run concurrently with individuals from the same clutches. Here, we show that frogs from an evolutionarily long-exposed and phenotypically more resistant population of the highly susceptible alpine tree frog demonstrate a more robust innate and adaptive immune response at the critical early subclinical stage of infection when compared with two more susceptible populations. These results are consistent with the occurrence of evolution of resistance against chytridiomycosis, help to explain underlying resistance mechanisms, and provide genes of potential interest and sequence data for future research. We recommend further investigation of cell-mediated immunity pathways, the role of interferons and mechanisms of lymphocyte suppression.

Hormone-induced spawning of the critically endangered northern corroboree frog Pseudophryne pengilleyi.

Fundamental knowledge of the optimal hormone concentrations required to stimulate amplexus and spawning in breeding pairs of amphibians is currently lacking, hindering our understanding of the proximate mechanisms underpinning mating behaviour. The present study investigated the effects of: (1) the dose of a gonadotropin-releasing hormone analogue (GnRH-a) administered; (2) male-female hormone administration interval; and (3) topical application of GnRH-a, on spawning success in the northern corroboree frog. Administration of GnRH-a at doses of 0.5, 1.0 and 2.0µgg-1 were highly successful, with a significantly greater proportion of hormone-treated pairs ovipositing (89-100%) compared with the 0µgg-1 treatment (22%). Of the hormone-treated pairs, those receiving 0.5µgg-1 GnRH-a exhibited the highest fertilisation success (61%). Administration of GnRH-a to males and females simultaneously (0h) was more effective than injecting males either 48 or 24h before the injection of females. Overall, administration of GnRH-a was highly successful at inducing spawning in northern corroboree frogs. For the first time, we also effectively induced spawning following the topical application of GnRH-a to the ventral pelvic region. Topical application of GnRH-a eliminates the need for specialised training in amphibian injection, and will allow assisted reproductive technologies to be adopted by a greater number of captive facilities globally.

Planning for ex situ conservation in the face of uncertainty.

Ex situ conservation strategies for threatened species often require long-term commitment and financial investment to achieve management objectives. We present a framework that considers the decision to adopt ex situ management for a target species as the end point of several linked decisions. We used a decision tree to intuitively represent the logical sequence of decision making. The first decision is to identify the specific management actions most likely to achieve the fundamental objectives of the recovery plan, with or without the use of ex-situ populations. Once this decision has been made, one decides whether to establish an ex situ population, accounting for the probability of success in the initial phase of the recovery plan, for example, the probability of successful breeding in captivity. Approaching these decisions in the reverse order (attempting to establish an ex situ population before its purpose is clearly defined) can lead to a poor allocation of resources, because it may restrict the range of available decisions in the second stage. We applied our decision framework to the recovery program for the threatened spotted tree frog (Litoria spenceri) of southeastern Australia. Across a range of possible management actions, only those including ex situ management were expected to provide >50% probability of the species' persistence, but these actions cost more than use of in situ alternatives only. The expected benefits of ex situ actions were predicted to be offset by additional uncertainty and stochasticity associated with establishing and maintaining ex situ populations. Naïvely implementing ex situ conservation strategies can lead to inefficient management. Our framework may help managers explicitly evaluate objectives, management options, and the probability of success prior to establishing a captive colony of any given species.

Vascularized pedicle jejunal graft for closure of large duodenal defect in a dog.

A Labrador retriever dog was presented for intestinal obstruction resulting in devitalization of portions of the duodenum. A severe perforation, accounting for 70% duodenal circumference, was present at the level of the duodenal papilla. A vascularized jejunal graft was used to close the perforation, representing novel utilization of this grafting technique.

Greffe jéjunale d'un pédicule vascularisé pour la fermeture d'un grand défaut duodénal chez un canidé. Un Labrador retriever a été présenté pour un blocage intestinal qui produisait la dévitalisation de portions du duodénum. Une perforation grave, représentant 70 % de la circonférence duodénale, était présente au niveau de la papille duodénale. Une greffe jéjunale vascularisée a été utilisée pour fermer la perforation, ce qui représente une nouvelle utilisation de cette technique de greffe.(Traduit par Isabelle Vallières).

Susceptibility of amphibians to chytridiomycosis is associated with MHC class II conformation.

The pathogenic chytrid fungus Batrachochytrium dendrobatidis (Bd) can cause precipitous population declines in its amphibian hosts. Responses of individuals to infection vary greatly with the capacity of their immune system to respond to the pathogen. We used a combination of comparative and experimental approaches to identify major histocompatibility complex class II (MHC-II) alleles encoding molecules that foster the survival of Bd-infected amphibians. We found that Bd-resistant amphibians across four continents share common amino acids in three binding pockets of the MHC-II antigen-binding groove. Moreover, strong signals of selection acting on these specific sites were evident among all species co-existing with the pathogen. In the laboratory, we experimentally inoculated Australian tree frogs with Bd to test how each binding pocket conformation influences disease resistance. Only the conformation of MHC-II pocket 9 of surviving subjects matched those of Bd-resistant species. This MHC-II conformation thus may determine amphibian resistance to Bd, although other MHC-II binding pockets also may contribute to resistance. Rescuing amphibian biodiversity will depend on our understanding of amphibian immune defence mechanisms against Bd. The identification of adaptive genetic markers for Bd resistance represents an important step forward towards that goal.

Interventions for reducing extinction risk in chytridiomycosis-threatened amphibians.

Wildlife diseases pose an increasing threat to biodiversity and are a major management challenge. A striking example of this threat is the emergence of chytridiomycosis. Despite diagnosis of chytridiomycosis as an important driver of global amphibian declines 15 years ago, researchers have yet to devise effective large-scale management responses other than biosecurity measures to mitigate disease spread and the establishment of disease-free captive assurance colonies prior to or during disease outbreaks. We examined the development of management actions that can be implemented after an epidemic in surviving populations. We developed a conceptual framework with clear interventions to guide experimental management and applied research so that further extinctions of amphibian species threatened by chytridiomycosis might be prevented. Within our framework, there are 2 management approaches: reducing Batrachochytrium dendrobatidis (the fungus that causes chytridiomycosis) in the environment or on amphibians and increasing the capacity of populations to persist despite increased mortality from disease. The latter approach emphasizes that mitigation does not necessarily need to focus on reducing disease-associated mortality. We propose promising management actions that can be implemented and tested based on current knowledge and that include habitat manipulation, antifungal treatments, animal translocation, bioaugmentation, head starting, and selection for resistance. Case studies where these strategies are being implemented will demonstrate their potential to save critically endangered species.

Knot security and tensile strength of suture materials.

OBJECTIVE: To evaluate knot security and tensile failure load of suture tied in simple interrupted, beginning continuous, and ending continuous patterns for 11 suture materials commonly used in small animal surgery. STUDY DESIGN: Mechanical study. METHODS: For each of 11 suture material types, and 5 knot sizes (2, 3, 4, 5, and 6 throws) 2 surgeons each tied 6 knots (n = 12 for each knot size in 11 suture materials). Three types of patterns were evaluated: a simple interrupted square knot, a square knot beginning a simple continuous pattern, and the knot ending a simple continuous pattern. All knots were incubated in healthy canine donor plasma at 40°C for a minimum of 24 hours. Sutures were evaluated for knot security (knots untied, suture failed by breaking, suture slipped from the clamps, or suture untied before testing) and maximum load carried before knot slippage or knot failure (termed tensile failure load). RESULTS: Significant differences were found in knot security and tensile failure load among suture types. There was no significant difference between the simple interrupted knots and the knots at the beginning of a simple continuous pattern; however, both were significantly less likely to fail than the knots tied at the end of a simple continuous pattern. The number of throws per knot had a significant effect for knot security and tensile failure load. Surgeon experience had a significant effect on failure mode and tensile failure load. CONCLUSIONS: Suture type, number of throws per knot (knot size), suture pattern, and surgeon experience play an important role in knot security and should be considered when performing surgery.

Type I interferon governs immunometabolic checkpoints that coordinate inflammation during Staphylococcal infection.

Macrophage metabolic plasticity is central to inflammatory programming, yet mechanisms of coordinating metabolic and inflammatory programs during infection are poorly defined. Here, we show that type I interferon (IFN) temporally guides metabolic control of inflammation during methicillin-resistant Staphylococcus aureus (MRSA) infection. We find that staggered Toll-like receptor and type I IFN signaling in macrophages permit a transient energetic state of combined oxidative phosphorylation (OXPHOS) and aerobic glycolysis followed by inducible nitric oxide synthase (iNOS)-mediated OXPHOS disruption. This disruption promotes type I IFN, suppressing other pro-inflammatory cytokines, notably interleukin-1ß. Upon infection, iNOS expression peaks at 24 h, followed by lactate-driven Nos2 repression via histone lactylation. Type I IFN pre-conditioning prolongs infection-induced iNOS expression, amplifying type I IFN. Cutaneous MRSA infection in mice constitutively expressing epidermal type I IFN results in elevated iNOS levels, impaired wound healing, vasculopathy, and lung infection. Thus, kinetically regulated type I IFN signaling coordinates immunometabolic checkpoints that control infection-induced inflammation.

Cryopreservation Cooling Rate Impacts Post-Thaw Sperm Motility and Survival in Litoria booroolongensis.

The cryopreservation and storage of gametes (biobanking) can provide a long-term, low-cost option for the preservation of population genetic diversity and is particularly impactful when applied to manage selective breeding within conservation breeding programs (CBPs). This study aimed to develop a sperm cryopreservation protocol for the critically endangered Booroolong frog (Litoria booroolongensis) to capture founder genetics within the recently established (est. 2019) CBP for this species. Hormone-induced sperm release was achieved using established protocols, and spermic urine samples were collected over a 6-h period. Pooled spermic urine samples (n = 3 males) were divided equally between two cryoprotectant (CPA) treatments and diluted by 1:5 (sperm:CPA) with either 15% (v/v) dimethyl sulfoxide + 1% (w/v) sucrose in simplified amphibian Ringer's (SAR; CPAA) or 10% (v/v) dimethylformamide + 10% (w/v) trehalose dihydrate in SAR (CPAB). The samples were cryopreserved in 0.25 mL straws using either a programmable freezer (FrA) or an adapted dry shipper method (FrB). The thawed samples were activated via dilution in water and assessed for viability and motility using both manual assessment and computer-assisted sperm analysis (CASA; 0 h, 0.5 h post-thaw). Upon activation, the survival and recovery of motility (total motility, forward progression and velocity) of cryopreserved sperm suspensions were higher for sperm preserved using FrB than FrA, regardless of CPA composition. This work supports our long-term goal to pioneer the integration of biobanked cryopreserved sperm with population genetic management to maximize restoration program outcomes for Australian amphibian species.

Non-canonical activation of IRE1α during Candida albicans infection enhances macrophage fungicidal activity.

Infection by intracellular pathogens can trigger activation of the IRE1α branch of the unfolded protein response (UPR), which then modulates innate immunity and infection outcomes during bacterial or viral infection. However, the mechanisms by which infection activates IRE1α have not been fully elucidated. While recognition of microbe-associated molecular patterns can activate IRE1α, it is unclear whether this depends on the canonical role of IRE1α in detecting misfolded proteins. Here, we report that Candida albicans infection of macrophages results in IRE1α activation through C-type lectin receptor signaling, reinforcing a role for IRE1α as a central regulator of host responses to infection by a broad range of pathogens. However, IRE1α activation was not preceded by protein misfolding in response to either C. albicans infection or lipopolysaccharide treatment, implicating a non-canonical mode of IRE1α activation after recognition of microbial patterns. Investigation of the phenotypic consequences of IRE1α activation in macrophage antimicrobial responses revealed that IRE1α activity enhances the fungicidal activity of macrophages. Macrophages lacking IRE1α activity displayed inefficient phagolysosomal fusion, enabling C. albicans to evade fungal killing and escape the phagosome. Together, these data provide mechanistic insight for the non-canonical activation of IRE1α during infection, and reveal central roles for IRE1α in macrophage antifungal responses.

FTO Suppresses STAT3 Activation and Modulates Proinflammatory Interferon-Stimulated Gene Expression.

Signaling initiated by type I interferon (IFN) results in the induction of hundreds of IFN-stimulated genes (ISGs). The type I IFN response is important for antiviral restriction, but aberrant activation of this response can lead to inflammation and autoimmunity. Regulation of this response is incompletely understood. We previously reported that the mRNA modification m6A and its deposition enzymes, METTL3 and METTL14 (METTL3/14), promote the type I IFN response by directly modifying the mRNA of a subset of ISGs to enhance their translation. Here, we determined the role of the RNA demethylase fat mass and obesity-associated protein (FTO) in the type I IFN response. FTO, which can remove either m6A or cap-adjacent m6Am RNA modifications, has previously been associated with obesity and body mass index, type 2 diabetes, cardiovascular disease, and inflammation. We found that FTO suppresses the transcription of a distinct set of ISGs, including many known pro-inflammatory genes, and that this regulation requires its catalytic activity but is not through the actions of FTO on m6Am. Interestingly, depletion of FTO led to activation of the transcription factor STAT3, whose role in the type I IFN response is not well understood. This activation of STAT3 increased the expression of a subset of ISGs. Importantly, this increased ISG induction resulting from FTO depletion was partially ablated by depletion of STAT3. Together, these results reveal that FTO negatively regulates STAT3-mediated signaling that induces proinflammatory ISGs during the IFN response, highlighting an important role for FTO in suppression of inflammatory genes.

Post-transcriptional regulation of antiviral gene expression by N6-methyladenosine.

Type I interferons (IFNs) induce hundreds of IFN-stimulated genes (ISGs) in response to viral infection. Induction of these ISGs must be regulated for an efficient and controlled antiviral response, but post-transcriptional controls of these genes have not been well defined. Here, we identify a role for the RNA base modification N6-methyladenosine (m6A) in the regulation of ISGs. Using ribosome profiling and quantitative mass spectrometry, coupled with m6A-immunoprecipitation and sequencing, we identify a subset of ISGs, including IFITM1, whose translation is enhanced by m6A and the m6A methyltransferase proteins METTL3 and METTL14. We further determine that the m6A reader YTHDF1 increases the expression of IFITM1 in an m6A-binding-dependent manner. Importantly, we find that the m6A methyltransferase complex promotes the antiviral activity of type I IFN. Thus, these studies identify m6A as having a role in post-transcriptional control of ISG translation during the type I IFN response for antiviral restriction.