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1 (1) Zip

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1 (1) zip

Defense against intracellular infection has been extensively studied in vertebrate hosts, but less is known about invertebrate hosts; specifically, the transcription factors that induce defense against intracellular intestinal infection in the model nematode Caenorhabditis elegans remain understudied. Two different types of intracellular pathogens that naturally infect the C. elegans intestine are the Orsay virus, which is an RNA virus, and microsporidia, which comprise a phylum of fungal pathogens. Despite their molecular differences, these pathogens induce a common host transcriptional response called the intracellular pathogen response (IPR). Here we show that zip-1 is an IPR regulator that functions downstream of all known IPR-activating and regulatory pathways. zip-1 encodes a putative bZIP transcription factor, and we show that zip-1 controls induction of a subset of genes upon IPR activation. ZIP-1 protein is expressed in the nuclei of intestinal cells, and is at least partially required in the intestine to upregulate IPR gene expression. Importantly, zip-1 promotes resistance to infection by the Orsay virus and by microsporidia in intestinal cells. Altogether, our results indicate that zip-1 represents a central hub for triggers of the IPR, and that this transcription factor has a protective function against intracellular pathogen infection in C. elegans.

Viruses and other obligate intracellular pathogens are responsible for a myriad of serious illnesses1. RNA viruses, like the single-stranded, positive-sense RNA virus SARS-CoV-2 that causes COVID-19, are detected by RIG-I-like receptors2,3,4. These receptors detect viral RNA replication products and trigger transcriptional upregulation of interferon genes to induce antiviral defense5. The nematode Caenorhabditis elegans provides a simple model host to understanding responses to RNA viruses, as a single-stranded, positive-sense RNA virus from Orsay, France infects C. elegans in the wild6. Interestingly, natural variation in drh-1, a C. elegans gene encoding a RIG-I-like receptor, was found to underlie natural variation in resistance to the Orsay virus7. Several studies indicate that detection of viral RNA by the drh-1 receptor induces an antiviral response through regulating RNA interference (RNAi)7,8,9.

In addition to regulating RNAi, drh-1 detection of viral replication products was recently shown to activate a transcriptional immune/stress response in C. elegans called the intracellular pathogen response (IPR)10. The IPR was defined as a common transcriptional response to the Orsay virus and a molecularly distinct natural intracellular pathogen of C. elegans called Nematocida parisii11,12,13. N. parisii is a species of Microsporidia, which comprise a phylum of obligate intracellular fungal pathogens that infect a large range of animal hosts including humans. It is not known which host receptors detects N. parisii infection, but the DRH-1 RIG-I-like receptor appears to detect viral RNA replication products, and to be critical for viral induction of the IPR10. Notably, C. elegans does not have clear orthologs of interferon, or the signaling factors that act downstream of RIG-I-like receptors in mammals, such as the transcription factors NF-kB and IRF3/714. It is unknown how drh-1 activates the IPR transcriptional program in C. elegans.

Several noninfection inputs can also trigger IPR gene expression. For example, proteotoxic stress, such as that caused by blockade of the proteasome, or by prolonged heat stress, will induce IPR genes. While intracellular infection by the Orsay virus or by N. parisii cause hallmarks of proteotoxic stress in C. elegans intestinal cells11, genetic and kinetic analyses indicate that proteotoxic stress is activating IPR gene expression in parallel to viral infection, and that there are several independent triggers of the IPR10. Another trigger of the IPR is mutation in the enzyme purine nucleoside phosphorylase-1, PNP-1, which acts in C. elegans intestinal epithelial cells to regulate pathogen resistance and the majority of IPR genes12,13,15. Of note, mutations in human PNP cause T-cell dysfunction, but its role in epithelial cells is less well-described15. In addition to pnp-1, analysis of another IPR repressor called pals-22, has provided insight into the regulation and function of IPR genes12,13. pals-22 belongs to the pals (protein containing ALS2cr12 signature) gene family, which has one ortholog each in mouse and human of unknown function, while this family has expanded to 39 members in C. elegans12,16,17. The biochemical functions of pals genes are unknown, but they play important roles in intracellular infection in C. elegans13. Several pals genes (e.g., pals-5) are upregulated by virus infection and the other IPR triggers mentioned above. Furthermore, two pals genes, pals-22 and pals-25, are opposing regulators of the IPR, acting as an ON/OFF switch for IPR gene expression as well as resistance to infection13. Not only do pals-22 and pals-25 control immunity, but they also control thermotolerance, a phenotype that is dependent on a subset of IPR genes that encode a newly described, multi-subunit, E3 ubiquitin ligase that promotes proteostasis13,18.

While Orsay virus infection, N. parisii infection, proteotoxic stress, pnp-1 and pals-22 mutations all appear to act independently of each other to trigger IPR gene expression, here we show that they converge on a common downstream transcription factor. Using two RNAi screens, we find that the gene encoding a putative basic region-leucine zipper (bZIP) transcription factor called zip-1 plays a role in activating expression of the IPR gene pals-5 by all known IPR triggers. Furthermore, we use proteasome inhibition as a trigger to show that zip-1 controls induction of only a subset of IPR genes. These results demonstrate that there are at least three classes of IPR genes as defined by whether their induction is dependent on zip-1 early after proteasome inhibition, late after proteasome inhibition, or their induction after proteasome inhibition is independent of zip-1. We show that the ZIP-1::GFP protein expression is induced in intestinal and epidermal nuclei upon IPR activation, and that ZIP-1 likely functions in the intestine to activate pals-5 expression. We find that induction of ZIP-1::GFP expression in intestinal nuclei by viral infection depends on DRH-1, suggesting that the DRH-1 receptor controls activation of the ZIP-1 transcription factor. Importantly, we show that zip-1 promotes defense against viral as well as against microsporidia infection in the intestine. Altogether, our results define zip-1 as a central signaling hub, controlling induction of IPR gene expression in response to a wide range of triggers, including diverse intracellular pathogens, other stressors, and genetic regulators. Furthermore, this study describes ZIP-1 as the first transcription factor shown to promote an inducible defense response against intracellular intestinal infection in C. elegans.

To determine which transcription factor(s) activates IPR gene expression, we screened an RNAi library composed of 363 RNAi clones targeting 357 predicted transcription factors (less than half the predicted transcription factor repertoire in C. elegans) to identify RNAi clones that repress constitutive expression of the PALS-5::GFP translational reporter (jyEx191) in a pals-22(jy3) background. In parallel, we also screened this library for RNAi clones that prevent induction of the pals-5p::GFP transcriptional reporter (jyIs8) upon prolonged heat stress. In both screens, we found that zip-1(RNAi) led to a substantial decrease in GFP signal (Fig. 1a, b, Supplementary Data 1), suggesting that this putative bZIP-containing transcription factor plays a role in IPR regulation. We confirmed this zip-1(RNAi) phenotype in another pals-22 loss-of-function allele, jy1, showing that here too, RNAi against zip-1 repressed the constitutive expression of PALS-5::GFP in pals-22 mutants (Fig. 1c). To demonstrate that this phenotype is not just restricted to zip-1(RNAi), we created a full deletion allele of zip-1 called jy13 (Supplementary Fig. 1), and observed decreased expression of the pals-5p::GFP reporter following prolonged heat stress in this putative zip-1 null mutant (Fig. 1d). These results indicate that zip-1 is important for regulating expression of two different pals-5 GFP reporters by two different IPR triggers.

We also specifically examined whether RNAi against two other transcription factors implicated in intracellular infection were required to induce pals-5p::GFP expression. First, we analyzed the role of bZIP transcription factor zip-10, which is induced as part of the IPR and promotes N. parisii sporulation13,19. Here we found no effect of zip-10(RNAi) on pals-5p::GFP induction after chronic heat stress. We also analyzed the role of STAT-like transcription factor sta-2, which is important for response to Drechmeria coniospora, a fungal pathogen that penetrates and grows inside epidermal cells20,21. Here as well, we did not find an effect of sta-2(RNAi) on induction of pals-5p::GFP expression (Supplementary Fig. 2). 041b061a72


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