Meta-Analysis of Caenorhabditis elegans Transcriptomics Implicates Hedgehog-Like Signaling in Host-Microbe Interactions.

Controlling nematode-caused diseases that affect cattle and crops world-wide remains a critical economic issue, owing to the lack of effective sustainable interventions. The interdependence of roundworms and their environmental microbes, including their microbiota, offers an opportunity for developing more targeted anthelminthic strategies. However, paucity of information and a currently narrow understanding of nematode-microbe interactions limited to specific infection contexts has precluded us from exploiting it. With the advent of omics approaches to map host-microbe genetic interactions, particularly in the model roundworm Caenorhabditis elegans, large datasets are now available across multiple models, that enable identification of nematode-microbe-specific pathways. In this work we collected 20 transcriptomic datasets documenting gene expression changes of C. elegans exposed to 20 different commensal and pathogenic microbes, performing gene enrichment analyses followed by functional testing using RNA interference directed toward genes of interest, before contrasting results from transcriptomic meta-analyses and phenomics. Differential expression analyses revealed a broad enrichment in signaling, innate immune response and (lipid) metabolism genes. Amongst signaling gene families, the nematode-divergent and expanded Hedgehog-like signaling (HHLS) pathway featured prominently. Indeed, 24/60 C. elegans Hedgehog-like proteins (HRPs) and 15/27 Patched-related receptors (PTRs) were differentially expressed in at least four microbial contexts, while up to 32/60 HRPs could be differentially expressed in a single context. interestingly, differentially expressed genes followed a microbe-specific pattern, suggestive of an adaptive microbe-specific response. To investigate this further, we knocked-down 96 individual HHLS genes by RNAi, using high-throughput assays to assess their impact on three worm-gut infection models (Pseudomonas aeruginosa, Staphylococcus aureus, and Enterococcus faecalis) and two worm-commensal paradigms (Comamonas sp., and Bacillus subtilis). We notably identified new putative infection response genes whose upregulation was required for normal pathogen resistance (i.e., grl-21 and ptr-18 protective against E. faecalis), as well as commensal-specific host-gene expression changes that are required for normal host stress handling. Importantly, interactions appeared more microbe-specific than shared. Our results thus implicate the Hedgehog-like signaling pathway in the modulation and possibly fine-tuning of nematode-microbe interactions and support the idea that interventions targeting this pathway may provide a new avenue for anthelmintic development.

High-Throughput Screening of Microbial Isolates with Impact on Caenorhabditis elegans Health.

With its small size, short lifespan, and easy genetics, Caenorhabditis elegans offers a convenient platform to study the impact of microbial isolates on host physiology. It also fluoresces in blue when dying, providing a convenient means of pinpointing death. This property has been exploited to develop high-throughput label-free C. elegans survival assays (LFASS). These involve time-lapse fluorescence recording of worm populations set in multiwell plates, from which population median time of death can be derived. The present study adopts the LFASS approach to screen multiple microbial isolates at once for the effects on C. elegans susceptibility to severe heat and oxidative stresses. Such microbial screening pipeline, which can notably be used to prescreen probiotics, using severe stress resistance as a proxy for host health is reported here. The protocol describes how to grow both C. elegans gut microbiota isolate collections and synchronous worm populations in multiwell arrays before combining them for the assays. The example provided covers the testing of 47 bacterial isolates and one control strain on two worm strains, in two stress assays in parallel. However, the approach pipeline is readily scalable and applicable to the screening of many other modalities. Thus, it provides a versatile setup to rapidly survey a multiparametric landscape of biological and biochemical conditions that impact C. elegans health.

Guard proteins keep watch at epithelial walls.

Cells can detect pathogens through guard proteins that sense disturbances in core cellular processes, but the exact mechanisms often remain elusive. In this issue of Immunity, Orzalli et al. identify Bcl-2 family members as guard proteins that detect virus-induced translational inhibition and induce pyroptosis in human keratinocytes.

The effects of nested miRNAs and their host genes on immune defense against Bacillus thuringiensis infection in Caenorhabditis elegans.

microRNAs (miRNAs) are small non-coding RNA-molecules that influence translation by binding to the target gene mRNA. Many miRNAs are found in nested arrangements within larger protein-coding host genes. miRNAs and host genes in a nested arrangement are often transcribed simultaneously, which may indicate that both have similar functions. miRNAs have been implicated in regulating defense responses against pathogen infection in C. elegans and in mammals. Here, we asked if miRNAs in nested arrangements and their host genes are involved in the C. elegans response against infection with Bacillus thuringiensis (Bt). We performed miRNA sequencing and subsequently focused on four nested miRNA-host gene arrangements for a functional genetic analysis. We identified mir-58.1 and mir-2 as negative regulators of C. elegans resistance to Bt infection. However, we did not find any miRNA/host gene pair in which both contribute to defense against Bt.

Effector and regulator: Diverse functions of C. elegans C-type lectin-like domain proteins.

In C. elegans, 283 clec genes encode a highly diverse family of C-type lectin-like domain (CTLD) proteins. Since vertebrate CTLD proteins have characterized functions in defense responses against pathogens and since expression of C. elegans clec genes is pathogen-dependent, it is generally assumed that clec genes function in C. elegans immune defenses. However, little is known about the relative contribution and exact function of CLEC proteins in C. elegans immunity. Here, we focused on the C. elegans clec gene clec-4, whose expression is highly upregulated by pathogen infection, and its paralogs clec-41 and clec-42. We found that, while mutation of clec-4 resulted in enhanced resistance to the Gram-positive pathogen Bacillus thuringiensis MYBt18247 (Bt247), inactivation of clec-41 and clec-42 by RNAi enhanced susceptibility to Bt247. Further analyses revealed that enhanced resistance of clec-4 mutants to Bt247 was due to an increase in feeding cessation on the pathogen and consequently a decrease in pathogen load. Moreover, clec-4 mutants exhibited feeding deficits also on non-pathogenic bacteria that were in part reflected in the clec-4 gene expression profile, which overlapped with gene sets affected by starvation or mutation in nutrient sensing pathways. However, loss of CLEC-4 function only mildly affected life-history traits such as fertility, indicating that clec-4 mutants are not subjected to dietary restriction. While CLEC-4 function appears to be associated with the regulation of feeding behavior, we show that CLEC-41 and CLEC-42 proteins likely function as bona fide immune effector proteins that have bacterial binding and antimicrobial capacities. Together, our results exemplify functional diversification within clec gene paralogs.

The C. elegans GATA transcription factor elt-2 mediates distinct transcriptional responses and opposite infection outcomes towards different Bacillus thuringiensis strains.

The nematode Caenorhabditis elegans has been extensively used as a model for the study of innate immune responses against bacterial pathogens. While it is well established that the worm mounts distinct transcriptional responses to different bacterial species, it is still unclear in how far it can fine-tune its response to different strains of a single pathogen species, especially if the strains vary in virulence and infection dynamics. To rectify this knowledge gap, we systematically analyzed the C. elegans response to two strains of Bacillus thuringiensis (Bt), MYBt18247 (Bt247) and MYBt18679 (Bt679), which produce different pore forming toxins (PFTs) and vary in infection dynamics. We combined host transcriptomics with cytopathological characterizations and identified both a common and also a differentiated response to the two strains, the latter comprising almost 10% of the infection responsive genes. Functional genetic analyses revealed that the AP-1 component gene jun-1 mediates the common response to both Bt strains. In contrast, the strain-specific response is mediated by the C. elegans GATA transcription factor ELT-2, a homolog of Drosophila SERPENT and vertebrate GATA4-6, and a known master regulator of intestinal responses in the nematode. elt-2 RNAi knockdown decreased resistance to Bt679, but remarkably, increased survival on Bt247. The elt-2 silencing-mediated increase in survival was characterized by reduced intestinal tissue damage despite a high pathogen burden and might thus involve increased tolerance. Additional functional genetic analyses confirmed the involvement of distinct signaling pathways in the C. elegans defense response: the p38-MAPK pathway acts either directly with or in parallel to elt-2 in mediating resistance to Bt679 infection but is not required for protection against Bt247. Our results further suggest that the elt-2 silencing-mediated increase in survival on Bt247 is multifactorial, influenced by the nuclear hormone receptors NHR-99 and NHR-193, and may further involve lipid metabolism and detoxification. Our study highlights that the nematode C. elegans with its comparatively simple immune defense system is capable of generating a differentiated response to distinct strains of the same pathogen species. Importantly, our study provides a molecular insight into the diversity of biological processes that are influenced by a single master regulator and jointly determine host survival after pathogen infection.

The putative immune recognition repertoire of the model cnidarian Hydractinia symbiolongicarpus is large and diverse.

Immune recognition of molecular patterns from microorganisms or self-altered cells activate effector responses that neutralize and eliminate these potentially harmful agents. In virtually every metazoan group the process is carried out by pattern recognition receptors, typically constituted by immunoglobulin (Ig), leucine rich repeat (LRR), and/or lectin domains. In order to get insights into the ancestral immune recognition repertoire of animals, we have sequenced the transcriptome of bacterially challenged colonies of the model cnidarian Hydractinia symbiolongicarpus using the Illumina platform. Over 116,000 assembled contigs were annotated by sequence similarity, domain architecture, and functionally. From these, a subset of 315 unique transcripts was predicted as the putative immune recognition repertoire of H. symbiolongicarpus. Interestingly, canonical Toll-like receptors (TLR) were not predicted, nor any transmembrane protein with the Toll/interleukine-1 receptor (TIR) domain. Yet, a variety of predicted proteins with transmembrane domains associated with LRR ectodomains were identified, as well as homologs of the key transduction factor NF-kB, and its associated regulatory proteins. This also has been documented in Hydra, and suggests that recognition and signaling initiation has been decoupled in the TLR system of hydrozoans. In contrast, both canonical and non-canonical NOD-like receptors were identified in H. symbiolongicarpus, showing a higher diversity than the TLR system and perhaps a wider functional landscape. The collection of Ig-like containing putative immune recognition molecules was diverse, and included at least 26 unique membrane-bound predicted proteins and 88 cytoplasmic/secreted predicted molecules. In addition, 25 and 5 transcripts encoding the Ig-like containing allorecognition determinants ALR1 and ALR2, respectively, were identified. Sequence and phylogenetic analyses suggested the presence of various transcriptionally active alr loci, and the action of recombination-based mechanisms diversifying them. Transcripts encoding at least six lectin families with putative roles in immune recognition were found, including 19 unique C-type lectins and 21 unique rhamnose-binding lectins. Other predicted immune recognition receptors included scavenger receptors from three families, lipopolysaccharide-binding proteins, cell-adhesion molecules and thioester-bond containing proteins. This analysis demonstrated that the putative immune recognition repertoire of H. symbiolongicarpus is large and diverse.

High Innate Immune Specificity through Diversified C-Type Lectin-Like Domain Proteins in Invertebrates.

A key question in current immunity research is how the innate immune system can generate high levels of specificity. Evidence is accumulating that invertebrates, which exclusively rely on innate defense mechanisms, can differentiate between pathogens on the species and even strain level. In this review, we identify and discuss the particular potential of C-type lectin-like domain (CTLD) proteins to generate high immune specificity. Whilst several CTLD proteins are known to act as pattern recognition receptors in the vertebrate innate immune system, the exact role of CTLD proteins in invertebrate immunity is much less understood. We show that CTLD genes are highly abundant in most metazoan genomes and summarize the current state of knowledge on CTLD protein function in insect, crustacean and nematode immune systems. We then demonstrate extreme CTLD gene diversification in the genomes of Caenorhabditis nematodes and provide an update of data from CTLD gene function studies in C. elegans, which indicate that the diversity of CTLD genes could contribute to immune specificity. In spite of recent achievements, the exact functions of the diversified invertebrate CTLD genes are still largely unknown. Our review therefore specifically discusses promising research approaches to rectify this knowledge gap.

The immunotranscriptome of the Caribbean reef-building coral Pseudodiploria strigosa.

The viability of coral reefs worldwide has been seriously compromised in the last few decades due in part to the emergence of coral diseases of infectious nature. Despite important efforts to understand the etiology and the contribution of environmental factors associated to coral diseases, the mechanisms of immune response in corals are just beginning to be studied systematically. In this study, we analyzed the set of conserved immune response genes of the Caribbean reef-building coral Pseudodiploria strigosa by Illumina-based transcriptome sequencing and annotation of healthy colonies challenged with whole live Gram-positive and Gram-negative bacteria. Searching the annotated transcriptome with immune-related terms yielded a total of 2782 transcripts predicted to encode conserved immune-related proteins that were classified into three modules: (a) the immune recognition module, containing a wide diversity of putative pattern recognition receptors including leucine-rich repeat-containing proteins, immunoglobulin superfamily receptors, representatives of various lectin families, and scavenger receptors; (b) the intracellular signaling module, containing components from the Toll-like receptor, transforming growth factor, MAPK, and apoptosis signaling pathways; and (3) the effector module, including the C3 and factor B complement components, a variety of proteases and protease inhibitors, and the melanization-inducing phenoloxidase. P. strigosa displays a highly variable and diverse immune recognition repertoire that has likely contributed to its resilience to coral diseases.