Proteasome inhibition triggers tissue-specific immune responses against different pathogens in C. elegans.

Protein quality control pathways play important roles in resistance against pathogen infection. For example, the conserved transcription factor SKN-1/NRF up-regulates proteostasis capacity after blockade of the proteasome and also promotes resistance against bacterial infection in the nematode Caenorhabditis elegans. SKN-1/NRF has 3 isoforms, and the SKN-1A/NRF1 isoform, in particular, regulates proteasomal gene expression upon proteasome dysfunction as part of a conserved bounce-back response. We report here that, in contrast to the previously reported role of SKN-1 in promoting resistance against bacterial infection, loss-of-function mutants in skn-1a and its activating enzymes ddi-1 and png-1 show constitutive expression of immune response programs against natural eukaryotic pathogens of C. elegans. These programs are the oomycete recognition response (ORR), which promotes resistance against oomycetes that infect through the epidermis, and the intracellular pathogen response (IPR), which promotes resistance against intestine-infecting microsporidia. Consequently, skn-1a mutants show increased resistance to both oomycete and microsporidia infections. We also report that almost all ORR/IPR genes induced in common between these programs are regulated by the proteasome and interestingly, specific ORR/IPR genes can be induced in distinct tissues depending on the exact trigger. Furthermore, we show that increasing proteasome function significantly reduces oomycete-mediated induction of multiple ORR markers. Altogether, our findings demonstrate that proteasome regulation keeps innate immune responses in check in a tissue-specific manner against natural eukaryotic pathogens of the C. elegans epidermis and intestine.

Caenorhabditis elegans RIG-I-like receptor DRH-1 signals via CARDs to activate antiviral immunity in intestinal cells.

Upon sensing viral RNA, mammalian RIG-I-like receptors (RLRs) activate downstream signals using caspase activation and recruitment domains (CARDs), which ultimately promote transcriptional immune responses that have been well studied. In contrast, the downstream signaling mechanisms for invertebrate RLRs are much less clear. For example, the Caenorhabditis elegans RLR DRH-1 lacks annotated CARDs and up-regulates the distinct output of RNA interference. Here, we found that similar to mammal RLRs, DRH-1 signals through two tandem CARDs (2CARD) to induce a transcriptional immune response. Expression of DRH-1(2CARD) alone in the intestine was sufficient to induce immune gene expression, increase viral resistance, and promote thermotolerance, a phenotype previously associated with immune activation in C. elegans. We also found that DRH-1 is required in the intestine to induce immune gene expression, and we demonstrate subcellular colocalization of DRH-1 puncta with double-stranded RNA inside the cytoplasm of intestinal cells upon viral infection. Altogether, our results reveal mechanistic and spatial insights into antiviral signaling in C. elegans, highlighting unexpected parallels in RLR signaling between C. elegans and mammals.

Multiple pals gene modules control a balance between immunity and development in Caenorhabditis elegans.

The immune system continually battles against pathogen-induced pressures, which often leads to the evolutionary expansion of immune gene families in a species-specific manner. For example, the pals gene family expanded to 39 members in the Caenorhabditis elegans genome, in comparison to a single mammalian pals ortholog. Our previous studies have revealed that two members of this family, pals-22 and pals-25, act as antagonistic paralogs to control the Intracellular Pathogen Response (IPR). The IPR is a protective transcriptional response, which is activated upon infection by two molecularly distinct natural intracellular pathogens of C. elegans-the Orsay virus and the fungus Nematocida parisii from the microsporidia phylum. In this study, we identify a previously uncharacterized member of the pals family, pals-17, as a newly described negative regulator of the IPR. pals-17 mutants show constitutive upregulation of IPR gene expression, increased immunity against intracellular pathogens, as well as impaired development and reproduction. We also find that two other previously uncharacterized pals genes, pals-20 and pals-16, are positive regulators of the IPR, acting downstream of pals-17. These positive regulators reverse the effects caused by the loss of pals-17 on IPR gene expression, immunity, and development. We show that the negative IPR regulator protein PALS-17 and the positive IPR regulator protein PALS-20 colocalize inside and at the apical side of intestinal epithelial cells, which are the sites of infection for IPR-inducing pathogens. In summary, our study demonstrates that several pals genes from the expanded pals gene family act as ON/OFF switch modules to regulate a balance between organismal development and immunity against natural intracellular pathogens in C. elegans.

Similarities in the induction of the intracellular pathogen response in Caenorhabditis elegans and the type I interferon response in mammals.

Although the type-I interferon (IFN-I) response is considered vertebrate-specific, recent findings about the Intracellular Pathogen Response (IPR) in nematode Caenorhabditis elegans indicate that there are similarities between these two transcriptional immunological programs. The IPR is induced during infection with natural intracellular fungal and viral pathogens of the intestine and promotes resistance against these pathogens. Similarly, the IFN-I response is induced by viruses and other intracellular pathogens and promotes resistance against infection. Whether the IPR and the IFN-I response evolved in a divergent or convergent manner is an unanswered and exciting question, which could be addressed by further studies of immunity against intracellular pathogens in C. elegans and other simple host organisms. Here we highlight similar roles played by RIG-I-like receptors, purine metabolism enzymes, proteotoxic stressors, and transcription factors to induce the IPR and IFN-I response, as well as the similar consequences of these defense programs on organismal development.

A pals-25 gain-of-function allele triggers systemic resistance against natural pathogens of C. elegans.

Regulation of immunity throughout an organism is critical for host defense. Previous studies in the nematode Caenorhabditis elegans have described an "ON/OFF" immune switch comprised of the antagonistic paralogs PALS-25 and PALS-22, which regulate resistance against intestinal and epidermal pathogens. Here, we identify and characterize a PALS-25 gain-of-function mutant protein with a premature stop (Q293*), which we find is freed from physical repression by its negative regulator, the PALS-22 protein. PALS-25(Q293*) activates two related gene expression programs, the Oomycete Recognition Response (ORR) against natural pathogens of the epidermis, and the Intracellular Pathogen Response (IPR) against natural intracellular pathogens of the intestine. A subset of ORR/IPR genes is upregulated in pals-25(Q293*) mutants, and they are resistant to oomycete infection in the epidermis, and microsporidia and virus infection in the intestine, but without compromising growth. Surprisingly, we find that activation of PALS-25 seems to primarily stimulate the downstream bZIP transcription factor ZIP-1 in the epidermis, with upregulation of gene expression in both the epidermis and in the intestine. Interestingly, we find that PALS-22/25-regulated epidermal-to-intestinal signaling promotes resistance to the N. parisii intestinal pathogen, demonstrating cross-tissue protective immune induction from one epithelial tissue to another in C. elegans.

Correction: The purine nucleoside phosphorylase pnp-1 regulates epithelial cell resistance to infection in C. elegans.

[This corrects the article DOI: 10.1371/journal.ppat.1009350.].

The purine nucleoside phosphorylase pnp-1 regulates epithelial cell resistance to infection in C. elegans.

Intestinal epithelial cells are subject to attack by a diverse array of microbes, including intracellular as well as extracellular pathogens. While defense in epithelial cells can be triggered by pattern recognition receptor-mediated detection of microbe-associated molecular patterns, there is much to be learned about how they sense infection via perturbations of host physiology, which often occur during infection. A recently described host defense response in the nematode C. elegans called the Intracellular Pathogen Response (IPR) can be triggered by infection with diverse natural intracellular pathogens, as well as by perturbations to protein homeostasis. From a forward genetic screen, we identified the C. elegans ortholog of purine nucleoside phosphorylase pnp-1 as a negative regulator of IPR gene expression, as well as a negative regulator of genes induced by extracellular pathogens. Accordingly, pnp-1 mutants have resistance to both intracellular and extracellular pathogens. Metabolomics analysis indicates that C. elegans pnp-1 likely has enzymatic activity similar to its human ortholog, serving to convert purine nucleosides into free bases. Classic genetic studies have shown how mutations in human purine nucleoside phosphorylase cause immunodeficiency due to T-cell dysfunction. Here we show that C. elegans pnp-1 acts in intestinal epithelial cells to regulate defense. Altogether, these results indicate that perturbations in purine metabolism are likely monitored as a cue to promote defense against epithelial infection in the nematode C. elegans.

A cullin-RING ubiquitin ligase promotes thermotolerance as part of the intracellular pathogen response in Caenorhabditis elegans.

Intracellular pathogen infection leads to proteotoxic stress in host organisms. Previously we described a physiological program in the nematode Caenorhabditis elegans called the intracellular pathogen response (IPR), which promotes resistance to proteotoxic stress and appears to be distinct from canonical proteostasis pathways. The IPR is controlled by PALS-22 and PALS-25, proteins of unknown biochemical function, which regulate expression of genes induced by natural intracellular pathogens. We previously showed that PALS-22 and PALS-25 regulate the mRNA expression of the predicted ubiquitin ligase component cullin cul-6, which promotes thermotolerance in pals-22 mutants. However, it was unclear whether CUL-6 acted alone, or together with other cullin-ring ubiquitin ligase components, which comprise a greatly expanded gene family in C. elegans Here we use coimmunoprecipitation studies paired with genetic analysis to define the cullin-RING ligase components that act together with CUL-6 to promote thermotolerance. First, we identify a previously uncharacterized RING domain protein in the TRIM family we named RCS-1, which acts as a core component with CUL-6 to promote thermotolerance. Next, we show that the Skp-related proteins SKR-3, SKR-4, and SKR-5 act redundantly to promote thermotolerance with CUL-6. Finally, we screened F-box proteins that coimmunoprecipitate with CUL-6 and find that FBXA-158 and FBXA-75 promote thermotolerance. In summary, we have defined the three core components and two F-box adaptors of a cullin-RING ligase complex that promotes thermotolerance as part of the IPR in C. elegans, which adds to our understanding of how organisms cope with proteotoxic stress.

The Caenorhabditis elegans RIG-I Homolog DRH-1 Mediates the Intracellular Pathogen Response upon Viral Infection.

Mammalian retinoic acid-inducible gene I (RIG-I)-like receptors detect viral double-stranded RNA (dsRNA) and 5'-triphosphorylated RNA to activate the transcription of interferon genes and promote antiviral defense. The Caenorhabditis elegans RIG-I-like receptor DRH-1 promotes defense through antiviral RNA interference (RNAi), but less is known about its role in regulating transcription. Here, we describe a role for DRH-1 in directing a transcriptional response in C. elegans called the intracellular pathogen response (IPR), which is associated with increased pathogen resistance. The IPR includes a set of genes induced by diverse stimuli, including intracellular infection and proteotoxic stress. Previous work suggested that the proteotoxic stress caused by intracellular infections might be the common trigger of the IPR, but here, we demonstrate that different stimuli act through distinct pathways. Specifically, we demonstrate that DRH-1/RIG-I is required for inducing the IPR in response to Orsay virus infection but not in response to other triggers like microsporidian infection or proteotoxic stress. Furthermore, DRH-1 appears to be acting independently of its known role in RNAi. Interestingly, expression of the replication-competent Orsay virus RNA1 segment alone is sufficient to induce most of the IPR genes in a manner dependent on RNA-dependent RNA polymerase activity and on DRH-1. Altogether, these results suggest that DRH-1 is a pattern recognition receptor that detects viral replication products to activate the IPR stress/immune program in C. elegansIMPORTANCEC. elegans lacks homologs of most mammalian pattern recognition receptors, and how nematodes detect pathogens is poorly understood. We show that the C. elegans RIG-I homolog DRH-1 mediates the induction of the intracellular pathogen response (IPR), a novel transcriptional defense program, in response to infection by the natural C. elegans viral pathogen Orsay virus. DRH-1 appears to act as a pattern recognition receptor to induce the IPR transcriptional defense program by sensing the products of viral RNA-dependent RNA polymerase activity. Interestingly, this signaling role of DRH-1 is separable from its previously known role in antiviral RNAi. In addition, we show that there are multiple host pathways for inducing the IPR, shedding light on the regulation of this novel transcriptional immune response.

Antagonistic paralogs control a switch between growth and pathogen resistance in C. elegans.

Immune genes are under intense, pathogen-induced pressure, which causes these genes to diversify over evolutionary time and become species-specific. Through a forward genetic screen we recently described a C. elegans-specific gene called pals-22 to be a repressor of "Intracellular Pathogen Response" or IPR genes. Here we describe pals-25, which, like pals-22, is a species-specific gene of unknown biochemical function. We identified pals-25 in a screen for suppression of pals-22 mutant phenotypes and found that mutations in pals-25 suppress all known phenotypes caused by mutations in pals-22. These phenotypes include increased IPR gene expression, thermotolerance, and immunity against natural pathogens, including Nematocida parisii microsporidia and the Orsay virus. Mutations in pals-25 also reverse the reduced lifespan and slowed growth of pals-22 mutants. Transcriptome analysis indicates that pals-22 and pals-25 control expression of genes induced not only by natural pathogens of the intestine, but also by natural pathogens of the epidermis. Indeed, in an independent forward genetic screen we identified pals-22 as a repressor and pals-25 as an activator of epidermal defense gene expression. In summary, the species-specific pals-22 and pals-25 genes act as a switch to regulate a program of gene expression, growth, and defense against diverse natural pathogens in C. elegans.

A Large Collection of Novel Nematode-Infecting Microsporidia and Their Diverse Interactions with Caenorhabditis elegans and Other Related Nematodes.

Microsporidia are fungi-related intracellular pathogens that may infect virtually all animals, but are poorly understood. The nematode Caenorhabditis elegans has recently become a model host for studying microsporidia through the identification of its natural microsporidian pathogen Nematocida parisii. However, it was unclear how widespread and diverse microsporidia infections are in C. elegans or other related nematodes in the wild. Here we describe the isolation and culture of 47 nematodes with microsporidian infections. N. parisii is found to be the most common microsporidia infecting C. elegans in the wild. In addition, we further describe and name six new species in the Nematocida genus. Our sampling and phylogenetic analysis further identify two subclades that are genetically distinct from Nematocida, and we name them Enteropsectra and Pancytospora. Interestingly, unlike Nematocida, these two genera belong to the main clade of microsporidia that includes human pathogens. All of these microsporidia are horizontally transmitted and most specifically infect intestinal cells, except Pancytospora epiphaga that replicates mostly in the epidermis of its Caenorhabditis host. At the subcellular level in the infected host cell, spores of the novel genus Enteropsectra show a characteristic apical distribution and exit via budding off of the plasma membrane, instead of exiting via exocytosis as spores of Nematocida. Host specificity is broad for some microsporidia, narrow for others: indeed, some microsporidia can infect Oscheius tipulae but not its sister species Oscheius sp. 3, and conversely some microsporidia found infecting Oscheius sp. 3 do not infect O. tipulae. We also show that N. ausubeli fails to strongly induce in C. elegans the transcription of genes that are induced by other Nematocida species, suggesting it has evolved mechanisms to prevent induction of this host response. Altogether, these newly isolated species illustrate the diversity and ubiquity of microsporidian infections in nematodes, and provide a rich resource to investigate host-parasite coevolution in tractable nematode hosts.

Discovery of a Natural Microsporidian Pathogen with a Broad Tissue Tropism in Caenorhabditis elegans.

Microbial pathogens often establish infection within particular niches of their host for replication. Determining how infection occurs preferentially in specific host tissues is a key aspect of understanding host-microbe interactions. Here, we describe the discovery of a natural microsporidian parasite of the nematode Caenorhabditis elegans that displays a unique tissue tropism compared to previously described parasites of this host. We characterize the life cycle of this new species, Nematocida displodere, including pathogen entry, intracellular replication, and exit. N. displodere can invade multiple host tissues, including the epidermis, muscle, neurons, and intestine of C. elegans. Despite robust invasion of the intestine very little replication occurs there, with the majority of replication occurring in the muscle and epidermis. This feature distinguishes N. displodere from two closely related microsporidian pathogens, N. parisii and N. sp. 1, which exclusively invade and replicate in the intestine. Comparison of the N. displodere genome with N. parisii and N. sp. 1 reveals that N. displodere is the earliest diverging species of the Nematocida genus. Over 10% of the proteins encoded by the N. displodere genome belong to a single species-specific family of RING-domain containing proteins of unknown function that may be mediating interactions with the host. Altogether, this system provides a powerful whole-animal model to investigate factors responsible for pathogen growth in different tissue niches.

A wild C. elegans strain has enhanced epithelial immunity to a natural microsporidian parasite.

Microbial pathogens impose selective pressures on their hosts, and combatting these pathogens is fundamental to the propagation of a species. Innate immunity is an ancient system that provides the foundation for pathogen resistance, with epithelial cells in humans increasingly appreciated to play key roles in innate defense. Here, we show that the nematode C. elegans displays genetic variation in epithelial immunity against intestinal infection by its natural pathogen, Nematocida parisii. This pathogen belongs to the microsporidia phylum, which comprises a large phylum of over 1400 species of fungal-related parasites that can infect all animals, including humans, but are poorly understood. Strikingly, we find that a wild C. elegans strain from Hawaii is able to clear intracellular infection by N. parisii, with this ability restricted to young larval animals. Notably, infection of older larvae does not impair progeny production, while infection of younger larvae does. The early-life immunity of Hawaiian larvae enables them to produce more progeny later in life, providing a selective advantage in a laboratory setting--in the presence of parasite it is able to out-compete a susceptible strain in just a few generations. We show that enhanced immunity is dominant to susceptibility, and we use quantitative trait locus mapping to identify four genomic loci associated with resistance. Furthermore, we generate near-isogenic strains to directly demonstrate that two of these loci influence resistance. Thus, our findings show that early-life immunity of C. elegans against microsporidia is a complex trait that enables the host to produce more progeny later in life, likely improving its evolutionary success.

Small GTPases promote actin coat formation on microsporidian pathogens traversing the apical membrane of Caenorhabditis elegans intestinal cells.

Many intracellular pathogens co-opt actin in host cells, but little is known about these interactions in vivo. We study the in vivo trafficking and exit of the microsporidian Nematocida parisii, which is an intracellular pathogen that infects intestinal cells of the nematode Caenorhabditis elegans. We recently demonstrated that N. parisii uses directional exocytosis to escape out of intestinal cells into the intestinal tract. Here, we show that an intestinal-specific isoform of C. elegans actin called ACT-5 forms coats around membrane compartments that contain single exocytosing spores, and that these coats appear to form after fusion with the apical membrane. We performed a genetic screen for host factors required for actin coat formation and identified small GTPases important for this process. Through analysis of animals defective in these factors, we found that actin coats are not required for pathogen exit although they may boost exocytic output. Later during infection, we find that ACT-5 also forms coats around membrane-bound vesicles that contain multiple spores. These vesicles are likely formed by clathrin-dependent compensatory endocytosis to retrieve membrane material that has been trafficked to the apical membrane as part of the exocytosis process. These findings provide insight into microsporidia interaction with host cells, and provide novel in vivo examples of the manner in which intracellular pathogens co-opt host actin during their life cycle.

The small GTPase RAB-11 directs polarized exocytosis of the intracellular pathogen N. parisii for fecal-oral transmission from C. elegans.

Pathogen exit is a key stage in the spread and propagation of infectious disease, with the fecal-oral route being a common mode of disease transmission. However, it is poorly understood which molecular pathways provide the major modes for intracellular pathogen exit and fecal-oral transmission in vivo. Here, we use the transparent nematode Caenorhabditis elegans to investigate intestinal cell exit and fecal-oral transmission by the natural intracellular pathogen Nematocida parisii, which is a recently identified species of microsporidia. We show that N. parisii exits from polarized host intestinal cells by co-opting the host vesicle trafficking system and escaping into the lumen. Using a genetic screen, we identified components of the host endocytic recycling pathway that are required for N. parisii spore exit via exocytosis. In particular, we show that the small GTPase RAB-11 localizes to apical spores, is required for spore-containing compartments to fuse with the apical plasma membrane, and is required for spore exit. In addition, we find that RAB-11-deficient animals exhibit impaired contagiousness, supporting an in vivo role for this host trafficking factor in microsporidia disease transmission. Altogether, these findings provide an in vivo example of the major mode of exit used by a natural pathogen for disease spread via fecal-oral transmission.

Ubiquitin-mediated response to microsporidia and virus infection in C. elegans.

Microsporidia comprise a phylum of over 1400 species of obligate intracellular pathogens that can infect almost all animals, but little is known about the host response to these parasites. Here we use the whole-animal host C. elegans to show an in vivo role for ubiquitin-mediated response to the microsporidian species Nematocida parisii, as well to the Orsay virus, another natural intracellular pathogen of C. elegans. We analyze gene expression of C. elegans in response to N. parisii, and find that it is similar to response to viral infection. Notably, we find an upregulation of SCF ubiquitin ligase components, such as the cullin ortholog cul-6, which we show is important for ubiquitin targeting of N. parisii cells in the intestine. We show that ubiquitylation components, the proteasome, and the autophagy pathway are all important for defense against N. parisii infection. We also find that SCF ligase components like cul-6 promote defense against viral infection, where they have a more robust role than against N. parisii infection. This difference may be due to suppression of the host ubiquitylation system by N. parisii: when N. parisii is crippled by anti-microsporidia drugs, the host can more effectively target pathogen cells for ubiquitylation. Intriguingly, inhibition of the ubiquitin-proteasome system (UPS) increases expression of infection-upregulated SCF ligase components, indicating that a trigger for transcriptional response to intracellular infection by N. parisii and virus may be perturbation of the UPS. Altogether, our results demonstrate an in vivo role for ubiquitin-mediated defense against microsporidian and viral infections in C. elegans.

Microsporidian genome analysis reveals evolutionary strategies for obligate intracellular growth.

Microsporidia comprise a large phylum of obligate intracellular eukaryotes that are fungal-related parasites responsible for widespread disease, and here we address questions about microsporidia biology and evolution. We sequenced three microsporidian genomes from two species, Nematocida parisii and Nematocida sp1, which are natural pathogens of Caenorhabditis nematodes and provide model systems for studying microsporidian pathogenesis. We performed deep sequencing of transcripts from a time course of N. parisii infection. Examination of pathogen gene expression revealed compact transcripts and a dramatic takeover of host cells by Nematocida. We also performed phylogenomic analyses of Nematocida and other microsporidian genomes to refine microsporidian phylogeny and identify evolutionary events of gene loss, acquisition, and modification. In particular, we found that all microsporidia lost the tumor-suppressor gene retinoblastoma, which we speculate could accelerate the parasite cell cycle and increase the mutation rate. We also found that microsporidia acquired transporters that could import nucleosides to fuel rapid growth. In addition, microsporidian hexokinases gained secretion signal sequences, and in a functional assay these were sufficient to export proteins out of the cell; thus hexokinase may be targeted into the host cell to reprogram it toward biosynthesis. Similar molecular changes appear during formation of cancer cells and may be evolutionary strategies adopted independently by microsporidia to proliferate rapidly within host cells. Finally, analysis of genome polymorphisms revealed evidence for a sexual cycle that may provide genetic diversity to alleviate problems caused by clonal growth. Together these events may explain the emergence and success of these diverse intracellular parasites.

Genome analysis and polar tube firing dynamics of mosquito-infecting microsporidia.

Microsporidia are highly divergent fungi that are obligate intracellular pathogens of a wide range of host organisms. Here we review recent findings from the genome sequences of mosquito-infecting microsporidian species Edhazardia aedis and Vavraia culicis, which show large differences in genome size, although similar numbers of predicted genes. We also show a video of E. aedis polar tube firing, which is the dramatic mechanism used by microsporidia to deliver the germ cell (sporoplasm) into the host cell to initiate intracellular infection.

Caenorhabditis elegans as a model for intracellular pathogen infection.

The genetically tractable nematode Caenorhabditis elegans is a convenient host for studies of pathogen infection. With the recent identification of two types of natural intracellular pathogens of C. elegans, this host now provides the opportunity to examine interactions and defence against intracellular pathogens in a whole-animal model for infection. C. elegans is the natural host for a genus of microsporidia, which comprise a phylum of fungal-related pathogens of widespread importance for agriculture and medicine. More recently, C. elegans has been shown to be a natural host for viruses related to the Nodaviridae family. Both microsporidian and viral pathogens infect the C. elegans intestine, which is composed of cells that share striking similarities to human intestinal epithelial cells. Because C. elegans nematodes are transparent, these infections provide a unique opportunity to visualize differentiated intestinal cells in vivo during the course of intracellular infection. Together, these two natural pathogens of C. elegans provide powerful systems in which to study microbial pathogenesis and host responses to intracellular infection.