TENT5 cytoplasmic noncanonical poly(A) polymerases regulate the innate immune response in animals.

Innate immunity is the first line of host defense against pathogens. Here, through global transcriptome and proteome analyses, we uncover that newly described cytoplasmic poly(A) polymerase TENT-5 (terminal nucleotidyltransferase 5) enhances the expression of secreted innate immunity effector proteins in Caenorhabditis elegans. Direct RNA sequencing revealed that multiple mRNAs with signal peptide-encoding sequences have shorter poly(A) tails in tent-5-deficient worms. Those mRNAs are translated at the endoplasmic reticulum where a fraction of TENT-5 is present, implying that they represent its direct substrates. Loss of tent-5 makes worms more susceptible to bacterial infection. Notably, the role of TENT-5 in innate immunity is evolutionarily conserved. Its orthologs, TENT5A and TENT5C, are expressed in macrophages and induced during their activation. Analysis of macrophages devoid of TENT5A/C revealed their role in the regulation of secreted proteins involved in defense response. In summary, our study reveals cytoplasmic polyadenylation to be a previously unknown component of the posttranscriptional regulation of innate immunity in animals.

C. elegans: out on an evolutionary limb.

The natural environment of the free-living nematode Caenorhabditis elegans is rich in pathogenic microbes. There is now ample evidence to indicate that these pathogens exert a strong selection pressure on C. elegans, and have shaped its genome, physiology, and behaviour. In this short review, we concentrate on how C. elegans stands out from other animals in terms of its immune repertoire and innate immune signalling pathways. We discuss how C. elegans often detects pathogens because of their effects on essential cellular processes, or organelle integrity, in addition to direct microbial recognition. We illustrate the extensive molecular plasticity that is characteristic of immune defences in C. elegans and highlight some remarkable instances of lineage-specific innovation in innate immune mechanisms.

An integrated view of innate immune mechanisms in C. elegans.

The simple notion 'infection causes an immune response' is being progressively refined as it becomes clear that immune mechanisms cannot be understood in isolation, but need to be considered in a more global context with other cellular and physiological processes. In part, this reflects the deployment by pathogens of virulence factors that target diverse cellular processes, such as translation or mitochondrial respiration, often with great molecular specificity. It also reflects molecular cross-talk between a broad range of host signalling pathways. Studies with the model animal C. elegans have uncovered a range of examples wherein innate immune responses are intimately connected with different homeostatic mechanisms, and can influence reproduction, ageing and neurodegeneration, as well as various other aspects of its biology. Here we provide a short overview of a number of such connections, highlighting recent discoveries that further the construction of a fully integrated view of innate immunity.

Antagonistic fungal enterotoxins intersect at multiple levels with host innate immune defences.

Animals and plants need to defend themselves from pathogen attack. Their defences drive innovation in virulence mechanisms, leading to never-ending cycles of co-evolution in both hosts and pathogens. A full understanding of host immunity therefore requires examination of pathogen virulence strategies. Here, we take advantage of the well-studied innate immune system of Caenorhabditis elegans to dissect the action of two virulence factors from its natural fungal pathogen Drechmeria coniospora. We show that these two enterotoxins have strikingly different effects when expressed individually in the nematode epidermis. One is able to interfere with diverse aspects of host cell biology, altering vesicle trafficking and preventing the key STAT-like transcription factor STA-2 from activating defensive antimicrobial peptide gene expression. The second increases STA-2 levels in the nucleus, modifies the nucleolus, and, potentially as a consequence of a host surveillance mechanism, causes increased defence gene expression. Our results highlight the remarkably complex and potentially antagonistic mechanisms that come into play in the interaction between co-evolved hosts and pathogens.

Ubiquitin-related processes and innate immunity in C. elegans.

Innate immunity is an evolutionary ancient defence strategy that serves to eliminate infectious agents while maintaining host health. It involves a complex network of sensors, signaling proteins and immune effectors that detect the danger, then relay and execute the immune programme. Post-translational modifications relying on conserved ubiquitin and ubiquitin-like proteins are an integral part of the system. Studies using invertebrate models of infection, such as the nematode Caenorhabditis elegans, have greatly contributed to our understanding of how ubiquitin-related processes act in immune sensing, regulate immune signaling pathways, and participate to host defence responses. This review highlights the interest of working with a genetically tractable model organism and illustrates how C. elegans has been used to identify ubiquitin-dependent immune mechanisms, discover novel ubiquitin-based resistance strategies that mediate pathogen clearance, and unravel the role of ubiquitin-related processes in tolerance, preserving host fitness during pathogen attack. Special emphasis is placed on processes that are conserved in mammals.

Comparison of lipidome profiles of Caenorhabditis elegans-results from an inter-laboratory ring trial.

INTRODUCTION: Lipidomic profiling allows 100s if not 1000s of lipids in a sample to be detected and quantified. Modern lipidomics techniques are ultra-sensitive assays that enable the discovery of novel biomarkers in a variety of fields and provide new insight in mechanistic investigations. Despite much progress in lipidomics, there remains, as for all high throughput "omics" strategies, the need to develop strategies to standardize and integrate quality control into studies in order to enhance robustness, reproducibility, and usability of studies within specific fields and beyond. OBJECTIVES: We aimed to understand how much results from lipid profiling in the model organism Caenorhabditis elegans are influenced by different culture conditions in different laboratories. METHODS: In this work we have undertaken an inter-laboratory study, comparing the lipid profiles of N2 wild type C. elegans and daf-2(e1370) mutants lacking a functional insulin receptor. Sample were collected from worms grown in four separate laboratories under standardized growth conditions. We used an UPLC-UHR-ToF-MS system allowing chromatographic separation before MS analysis. RESULTS: We found common qualitative changes in several marker lipids in samples from the individual laboratories. On the other hand, even in this controlled experimental system, the exact fold-changes for each marker varied between laboratories. CONCLUSION: Our results thus reveal a serious limitation to the reproducibility of current lipid profiling experiments and reveal challenges to the integration of such data from different laboratories.

Reduced peroxisomal import triggers peroxisomal retrograde signaling.

Maintaining organelle function in the face of stress is known to involve organelle-specific retrograde signaling. Using Caenorhabditis elegans, we present evidence of the existence of such retrograde signaling for peroxisomes, which we define as the peroxisomal retrograde signaling (PRS). Specifically, we show that peroxisomal import stress caused by knockdown of the peroxisomal matrix import receptor prx-5/PEX5 triggers NHR-49/peroxisome proliferator activated receptor alpha (PPARα)- and MDT-15/MED15-dependent upregulation of the peroxisomal Lon protease lonp-2/LONP2 and the peroxisomal catalase ctl-2/CAT. Using proteomic and transcriptomic analyses, we show that proteins involved in peroxisomal lipid metabolism and immunity are also upregulated upon prx-5(RNAi). While the PRS can be triggered by perturbation of peroxisomal ß-oxidation, we also observed hallmarks of PRS activation upon infection with Pseudomonas aeruginosa. We propose that the PRS, in addition to a role in lipid metabolism homeostasis, may act as a surveillance mechanism to protect against pathogens.

Increased Pathogenicity of the Nematophagous Fungus Drechmeria coniospora Following Long-Term Laboratory Culture.

Domestication provides a window into adaptive change. Over the course of 2 decades of laboratory culture, a strain of the nematode-specific fungus Drechmeria coniospora became more virulent during its infection of Caenorhabditis elegans. Through a close comparative examination of the genome sequences of the original strain and its more pathogenic derivative, we identified a small number of non-synonymous mutations in protein-coding genes. In one case, the mutation was predicted to affect a gene involved in hypoxia resistance and we provide direct corroborative evidence for such an effect. The mutated genes with functional annotation were all predicted to impact the general physiology of the fungus and this was reflected in an increased in vitro growth, even in the absence of C. elegans. While most cases involved single nucleotide substitutions predicted to lead to a loss of function, we also observed a predicted restoration of gene function through deletion of an extraneous tandem repeat. This latter change affected the regulatory subunit of a cAMP-dependent protein kinase. Remarkably, we also found a mutation in a gene for a second protein of the same, protein kinase A, pathway. Together, we predict that they result in a stronger repression of the pathway for given levels of ATP and adenylate cyclase activity. Finally, we also identified mutations in a few lineage-specific genes of unknown function that are candidates for factors that influence virulence in a more direct manner.

Innate Immunity Promotes Sleep through Epidermal Antimicrobial Peptides.

Wounding and infection trigger a protective innate immune response that includes the production of antimicrobial peptides in the affected tissue as well as increased sleep. Little is known, however, how peripheral wounds or innate immunity signal to the nervous system to increase sleep. We found that, during C. elegans larval molting, an epidermal tolloid/bone morphogenic protein (BMP)-1-like protein called NAS-38 promotes sleep. NAS-38 is negatively regulated by its thrombospondin domain and acts through its astacin protease domain to activate p38 mitogen-activated protein (MAP)/PMK-1 kinase and transforming growth factor ß (TGF-ß)-SMAD/SMA-3-dependent innate immune pathways in the epidermis that cause STAT/STA-2 and SLC6 (solute carrier)/SNF-12-dependent expression of antimicrobial peptide (AMP) genes. We show that more than a dozen epidermal AMPs act as somnogens, signaling across tissues to promote sleep through the sleep-active RIS neuron. In the adult, epidermal injury activates innate immunity and turns up AMP production to trigger sleep, a process that requires epidermal growth factor receptor (EGFR) signaling that is known to promote sleep following cellular stress. We show for one AMP, neuropeptide-like protein (NLP)-29, that it acts through the neuropeptide receptor NPR-12 in locomotion-controlling neurons that are presynaptic to RIS and that depolarize this neuron to induce sleep. Sleep in turn increases the chance of surviving injury. Thus, we found a novel mechanism by which peripheral wounds signal to the nervous system to increase protective sleep. Such a cross-tissue somnogen-signaling function of AMPs might also boost sleep in other animals, including humans.

Long-read only assembly of Drechmeria coniospora genomes reveals widespread chromosome plasticity and illustrates the limitations of current nanopore methods.

BACKGROUND: Long-read sequencing is increasingly being used to determine eukaryotic genomes. We used nanopore technology to generate chromosome-level assemblies for 3 different strains of Drechmeria coniospora, a nematophagous fungus used extensively in the study of innate immunity in Caenorhabditis elegans. RESULTS: One natural geographical isolate demonstrated high stability over decades, whereas a second isolate not only had a profoundly altered genome structure but exhibited extensive instability. We conducted an in-depth analysis of sequence errors within the 3 genomes and established that even with state-of-the-art tools, nanopore methods alone are insufficient to generate eukaryotic genome sequences of sufficient accuracy to merit inclusion in public databases. CONCLUSIONS: Although nanopore long-read sequencing is not accurate enough to produce publishable eukaryotic genomes, in our case, it has revealed new information about genome plasticity in D. coniospora and provided a backbone that will permit future detailed study to characterize gene evolution in this important model fungal pathogen.

Non-Canonical Caspase Activity Antagonizes p38 MAPK Stress-Priming Function to Support Development.

Recent studies have revealed non-canonical activities of apoptotic caspases involving specific modulation of gene expression, such as limiting asymmetric divisions of stem-like cell types. Here we report that CED-3 caspase negatively regulates an epidermal p38 stress-responsive MAPK pathway to promote larval development in C. elegans. We show that PMK-1 (p38 MAPK) primes animals for encounters with hostile environments at the expense of retarding post-embryonic development. CED-3 counters this function by directly cleaving PMK-1 to promote development. Moreover, we found that CED-3 and PMK-1 oppose each other to balance developmental and stress-responsive gene expression programs. Specifically, expression of more than 300 genes is inversely regulated by CED-3 and PMK-1. Analyses of these genes showed enrichment for epidermal stress-responsive factors, including the fatty acid synthase FASN-1, anti-microbial peptides, and genes involved in lethargus states. Our findings demonstrate a non-canonical role for a caspase in promoting development by limiting epidermal stress response programs.

Fabrication of sharp silicon arrays to wound Caenorhabditis elegans.

Understanding how animals respond to injury and how wounds heal remains a challenge. These questions can be addressed using genetically tractable animals, including the nematode Caenorhabditis elegans. Given its small size, the current methods for inflicting wounds in a controlled manner are demanding. To facilitate and accelerate the procedure, we fabricated regular arrays of pyramidal features ("pins") sharp enough to pierce the tough nematode cuticle. The pyramids were made from monocrystalline silicon wafers that were micro-structured using optical lithography and alkaline wet etching. The fabrication protocol and the geometry of the pins, determined by electron microscopy, are described in detail. We also used electron microscopy to characterize the different types of injury caused by these pins. Upon wounding, C. elegans expresses genes encoding antimicrobial peptides. A comparison of the induction of antimicrobial peptide gene expression using traditional needles and the pin arrays demonstrates the utility of this new method.

Microtubule plus-end dynamics link wound repair to the innate immune response.

The skin protects animals from infection and physical damage. In Caenorhabditis elegans, wounding the epidermis triggers an immune reaction and a repair response, but it is not clear how these are coordinated. Previous work implicated the microtubule cytoskeleton in the maintenance of epidermal integrity (Chuang et al., 2016). Here, by establishing a simple wounding system, we show that wounding provokes a reorganisation of plasma membrane subdomains. This is followed by recruitment of the microtubule plus end-binding protein EB1/EBP-2 around the wound and actin ring formation, dependent on ARP2/3 branched actin polymerisation. We show that microtubule dynamics are required for the recruitment and closure of the actin ring, and for the trafficking of the key signalling protein SLC6/SNF-12 toward the injury site. Without SNF-12 recruitment, there is an abrogation of the immune response. Our results suggest that microtubule dynamics coordinate the cytoskeletal changes required for wound repair and the concomitant activation of innate immunity.

Evolutionary plasticity in the innate immune function of Akirin.

Eukaryotic gene expression requires the coordinated action of transcription factors, chromatin remodelling complexes and RNA polymerase. The conserved nuclear protein Akirin plays a central role in immune gene expression in insects and mammals, linking the SWI/SNF chromatin-remodelling complex with the transcription factor NFκB. Although nematodes lack NFκB, Akirin is also indispensable for the expression of defence genes in the epidermis of Caenorhabditis elegans following natural fungal infection. Through a combination of reverse genetics and biochemistry, we discovered that in C. elegans Akirin has conserved its role of bridging chromatin-remodellers and transcription factors, but that the identity of its functional partners is different since it forms a physical complex with NuRD proteins and the POU-class transcription factor CEH-18. In addition to providing a substantial step forward in our understanding of innate immune gene regulation in C. elegans, our results give insight into the molecular evolution of lineage-specific signalling pathways.

Modulatory upregulation of an insulin peptide gene by different pathogens in C. elegans.

When an animal is infected, its innate immune response needs to be tightly regulated across tissues and coordinated with other aspects of organismal physiology. Previous studies with Caenorhabditis elegans have demonstrated that insulin-like peptide genes are differentially expressed in response to different pathogens. They represent prime candidates for conveying signals between tissues upon infection. Here, we focused on one such gene, ins-11 and its potential role in mediating cross-tissue regulation of innate immune genes. While diverse bacterial intestinal infections can trigger the up-regulation of ins-11 in the intestine, we show that epidermal infection with the fungus Drechmeria coniospora triggers an upregulation of ins-11 in the epidermis. Using the Shigella virulence factor OpsF, a MAP kinase inhibitor, we found that in both cases, ins-11 expression is controlled cell autonomously by p38 MAPK, but via distinct transcription factors, STA-2/STAT in the epidermis and HLH-30/TFEB in the intestine. We established that ins-11, and the insulin signaling pathway more generally, are not involved in the regulation of antimicrobial peptide gene expression in the epidermis. The up-regulation of ins-11 in the epidermis does, however, affect intestinal gene expression in a complex manner, and has a deleterious effect on longevity. These results support a model in which insulin signaling, via ins-11, contributes to the coordination of the organismal response to infection, influencing the allocation of resources in an infected animal.

De novo assembly of the complex genome of Nippostrongylus brasiliensis using MinION long reads.

BACKGROUND: Eukaryotic genome assembly remains a challenge in part due to the prevalence of complex DNA repeats. This is a particularly acute problem for holocentric nematodes because of the large number of satellite DNA sequences found throughout their genomes. These have been recalcitrant to most genome sequencing methods. At the same time, many nematodes are parasites and some represent a serious threat to human health. There is a pressing need for better molecular characterization of animal and plant parasitic nematodes. The advent of long-read DNA sequencing methods offers the promise of resolving complex genomes. RESULTS: Using Nippostrongylus brasiliensis as a test case, applying improved base-calling algorithms and assembly methods, we demonstrate the feasibility of de novo genome assembly matching current community standards using only MinION long reads. In doing so, we uncovered an unexpected diversity of very long and complex DNA sequences repeated throughout the N. brasiliensis genome, including massive tandem repeats of tRNA genes. CONCLUSION: Base-calling and assembly methods have improved sufficiently that de novo genome assembly of large complex genomes is possible using only long reads. The method has the added advantage of preserving haplotypic variants and so has the potential to be used in population analyses.

Signaling in the innate immune response.

The nematode Caenorhabditis elegans relies on its innate immune defenses to counter infection. In this review, we focus on its response to infection by bacterial and fungal pathogens. We describe the different families of effector proteins that contribute to host defense, as well as the signal transduction pathways that regulate their expression. We discuss what is known of the activation of innate immunity in C. elegans, via pathogen recognition or sensing the damage provoked by infection. Damage causes a stress response; we review the role of stress signaling in host defense to infection. We examine examples of inter-tissue communication in innate immunity and end with a survey of post-transcriptional regulation of innate immune responses.

Polyethylene Glycol-mediated Transformation of Drechmeria coniospora.

Drechmeria coniospora is a nematophagous fungus and potential biocontrol agent. It belongs to the Ascomycota. It is related to Hirsutella minnesotensis, another nematophagous fungus but, phylogenetically, it is currently closest to the truffle parasite Tolypocladium ophioglossoides. Together with its natural host, Caenorhabditis elegans, it is used to study host-pathogen interactions. Here, we report a polyethylene glycol-mediated transformation method ( Turgeon et al., 2010 ; Ochman et al., 1988 ) for this fungus. The protocol can be used to generate both knock-in or knock-out strains ( Lebrigand et al., 2016 ).