# Caenorhabditis elegans immune responses to microsporidia and viruses

Rubén González, Marie-Anne Félix

► To cite this version:

Rubén González, Marie-Anne Félix. Caenorhabditis elegans immune responses to microsporidia and viruses. Developmental and Comparative Immunology, 2024, 154, pp.105148. 10.1016/j.dci.2024.105148. hal-04756473

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# *Caenorhabditis elegans* immune responses to microsporidia and viruses

Rubén González*, Marie-Anne Félix

Institut de Biologie de l'École Normale Supérieure, CNRS, INSERM, 75005, Paris, France

## ABSTRACT

The model organism *Caenorhabditis elegans* is susceptible to infection by obligate intracellular pathogens, specifically microsporidia and viruses. These intracellular pathogens infect intestinal cells, or, for some microsporidia, epidermal cells. Strikingly, intestinal cell infections by viruses or microsporidia trigger a common transcriptional response, activated in part by the ZIP-1 transcription factor. Among the strongest activated genes in this response are ubiquitin-pathway members and members of the *pals* family, an intriguing gene family with cross-regulations of different members of genomic clusters. Some of the induced genes participate in host defense against the pathogens, for example through ubiquitin-mediated inhibition. Other mechanisms defend the host specifically against viral infections, including antiviral RNA interference and uridylation. These various immune responses are altered by environmental factors and by intraspecific genetic variation of the host. These pathogens were first isolated 15 years ago and much remains to be discovered using *C. elegans* genetics; also, other intracellular pathogens of *C. elegans* may yet to be discovered.

## 1. Introducing the host and its intracellular pathogens

### 1.1. *The animal host: Caenorhabditis elegans*

C. elegans is a free-living nematode that has been extensively used as an experimental model organism due to its small size, rapid life cycle, transparency, well-annotated genome, and tolerance to freezing and thawing (Corsi, 2015). As a transparent organism, *C. elegans* is particularly useful for studying intracellular pathogens as they can be easily visualized (Fig. 1). In its natural habitat, *C. elegans* can be found worldwide in decomposing plant material, a microbe-rich environment where the nematode feeds on bacteria (Frézal and Félix, 2015). Apart from bacteria that serve as food, *C. elegans* encounters microbes that may closely associate with it (Schulenburg and Félix, 2017). *C. elegans* is thus exposed to a wide variety of pathogens (Schulenburg et al., 2004; Jiang and Wang, 2018). This review focuses on the immune response to obligate intracellular pathogens, i.e. pathogens that invade and replicate exclusively inside host cells (Balla and Troemel, 2013).

### 1.2. Intracellular pathogens of nematodes

A summary of the known intracellular pathogens of *C. elegans* can be found in Table 1. Their degree of intracellular lifestyle varies from low to obligate. The two main obligate intracellular pathogens that have been found so far are microsporidia and viruses, first isolated from natural *C. elegans* populations in the last 15 years, on which we will focus in this review.

Microsporidia are eukaryotic organisms related to fungi, forming either a basal branch of the fungi or a sister group. Microsporidia are all obligate intracellular pathogens with a wide range of animal hosts including vertebrates, and even some protists. Outside of their host, microsporidia can only exist as environmentally resistant spores. When infecting a host, the spore everts an internally coiled tube and injects its content into the host cell. The parasite then reproduces inside the host, forming a multinucleated cell called a meront. The meront eventually cellularizes and the resulting sporonts mature into spores, which are then released from the host cell. Microsporidia rely on their host resources and have experienced an evolutionary reduction in their genome and organelles. For example, their degenerated mitochondria (called mitosomes) do not carry a genome and cannot perform oxidative phosphorylation (Balla et al., 2016; Han and Weiss, 2017; Troemel, 2016; Wadi and Reinke, 2020). *C. elegans* is naturally infected by at least eight species of *Nematocida*: the first discovered *N. parisii*, followed by *N. ausubeli*, *N. cider*, *N. displodere*, *N. ferruginous*, *N. homosporus*, *N. ironsii* and *N. major* (Troemel et al., 2008; Zhang et al., 2016; Wadi et al., 2023). Infections can be detected by observing microsporidia using differential interference contrast (DIC) microscopy (Fig. 1A). Additionally, immunostaining or in situ hybridization can be used to detect protein or RNA molecules from microsporidia (Troemel et al., 2008, Fig. 1B). Most species of *Nematocida* infect intestinal cells and are horizontally transmitted through the oral-fecal route. Some species however, such as *N. displodere*, mostly proliferate in epidermal and muscle cells after sending their polar tube apparently through the intestinal cells (Luallen et al., 2016). This tissue tropism is shared with

<footnote>* Corresponding author.
E-mail address: ruben.gonzalez@bio.ens.psl.eu (R. González).</footnote>
<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Visualization of intracellular pathogens of *C. elegans*. A) FISH staining of microsporidia spores using a *Nematocida parisii*-specific probe (red). Small spores are indicated with small arrows, large spores by larger arrows. Scale bar is 10 µm. B) DIC image of *N. parisii* spores. Arrows indicate spores. Small spores indicated with smaller arrow, large spores indicated by larger arrow. Scale bar is 10 µm. C) Fluorescence in situ hybridization (FISH) staining of OrV-infected N2 animals using probes against OrV RNA2 (blue). Nuclei are DAPI-stained in grey. Scale bar is 100 µm. D) Differential Interferential Contrast (DIC) images of OrV-infected and healthy *C. elegans* animals of the JU1580 wild isolate. The intestine has been manually highlighted (in blue for the infected cells and in grey for the uninfected cells) to facilitate the visualization of the intestinal cell fusion caused by viral infection. Black arrow heads indicate lateral cell membranes. Scale bars are 10 µm. Micrographs in panels A and B were adapted from Troemel et al. (2008). Micrograph in panel C is from the Félix laboratory (acquired by Rubén González). Micrographs in panel D were adapted from Félix et al. (2011). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)</figcaption></figure>

*Pancytospora epiphaga*, a microsporidium initially found in *Caenorhabditis brenneri*, which can infect the epidermis, coelomocytes, and muscles of *C. elegans* (Zhang et al., 2016). More information about the biology of microsporidia that infect *C. elegans* can be found in Tecle and Troemel (2022).

The first virology studies on *C. elegans* used RNA viruses that do not naturally infect *C. elegans*: Vesicular stomatitis virus (VSV; a rhabdovirus), Vaccinia virus (VV; a poxvirus), and Flock House virus (FHV; a nodavirus). These pioneer studies revealed the existence of antiviral immune responses in *C. elegans* (Lu et al., 2005; Wilkins et al., 2005; Schott et al., 2005; Liu et al., 2006). To study these viruses in *C. elegans*, it is necessary to microinject them (Martin et al., 2017) or, in the case of FHV, use transgenic animals expressing the viral RNAs, capable of replication but not of re-infection (Lu et al., 2005). This limited virological research on *C. elegans*, until the discovery of the Orsay virus (OrV; similar to nodavirus, not formally classified), the first virus found to naturally infect *C. elegans*. OrV is a positive single-stranded RNA virus with a bipartite genome. OrV is horizontally transmitted between individuals, with virions entering new hosts through the mouth and infecting a few intestinal cells. Infected cells create new infectious virions that exit the cell without disruption of the host cell apical membrane (Yuan et al., 2018) and are released outside the host. Infected animals do not have a reduced lifespan but have a reduced brood size and their progeny production is delayed (Félix et al., 2011; Ashe et al., 2013). OrV infection can be detected by immunostaining of viral

<caption>**Table 1**</caption>

List of pathogens that can intracellularly infect *C. elegans*. Microsporidia and virus are obligate intracellular pathogens, while the bacterium is a facultative intracellular pathogen.

<table><thead><tr><th>Taxa</th><th>Species</th><th>Infected tissue</th><th>Reference</th></tr></thead><tbody><tr><td>Virus</td><td>Orsay virus</td><td>Intestine</td><td>Félix et al. (2011); Frézal et al. (2019)</td></tr><tr><td rowspan="7">Microsporidia</td><td>Nematocida<br>ausubeli</td><td>Intestine</td><td>Troemel et al. (2008); Zhang et al., (2016)</td></tr><tr><td>Nematocida<br>cider</td><td>Intestine, epidermis<br>and muscles</td><td>Wadi et al. (2023)</td></tr><tr><td>Nematocida<br>displorede</td><td>Epidermis and<br>muscles</td><td>Luallen et al. (2016)</td></tr><tr><td>Nematocida<br>ferruginous</td><td>Epidermis and<br>muscles</td><td>Wadi et al. (2023)</td></tr><tr><td>Nematocida<br>homosporus</td><td>Intestine</td><td>Zhang et al. (2016)</td></tr><tr><td>Nematocida<br>ironsii</td><td>Intestine</td><td>Reinke et al. (2017)</td></tr><tr><td>Nematocida<br>major</td><td>Intestine</td><td>Zhang et al. (2016)</td></tr><tr><td>Nematocida<br>parisii</td><td>Intestine</td><td>Troemel et al. (2008); Zhang et al., (2016)</td></tr><tr><td rowspan="2">Bacteria</td><td>Pancytospora<br>epiphaga</td><td>Epidermis,<br>coelomocytes, and<br>muscles</td><td>Zhang et al. (2016)</td></tr><tr><td>Pseudomonas<br>aeruginosa</td><td>Intestine (but mostly<br>in lumen)</td><td>Irazoqui et al. (2010)</td></tr></tbody></table>

proteins or in situ hybridization of viral RNA (Franz et al., 2014, Fig. 1C). The virions can be visualized by electron microscopy (Félix et al., 2011; Jiang et al., 2014). The cellular symptoms the viruses causes, i.e. lateral epithelial cell fusions, can be detected using DIC microscopy (Félix et al., 2011, Fig. 1D). Remarkably, Jiang et al. (2014) created a *C. elegans* line that expresses ORV RNA1 and RNA2 under a heat shock promoter. This engineered mutant produces endogenous and infectious OrV virions upon a heat shock, capable of infecting new animals. To date, OrV is the only known virus that naturally infects *C. elegans*. Other nodavirus-like viruses can infect *Caenorhabditis brigg-sae*, a close relative of *C. elegans*; these viruses (Santeuil, Le Blanc, and Mělník virus) also infect intestinal cells (Franz et al., 2014; Frézal et al., 2019). More information about viruses that infect *Caenorhabditis* species can be found in Félix and Wang (2019).

In addition to these obligate intracellular pathogens, nematophagous oomycetes and fungi first invade *C. elegans* cells, then mostly invade the nematode body cavity, killing cells and host, and feeding off cell contents. The oomycetes *Myzocytiopsis humicola* (Osman et al., 2018) or *Haptoglossa zoospora* (Grover et al., 2021) and nematophagous fungi, such as *Drechmeria coniospora* (Dijksterhuis et al., 1990; Couillault et al., 2004), penetrate the epidermis through the cuticle or, for some fungi, the gut (Engelmann et al., 2011; Félix and Duveau, 2012). We will not consider them further here.

Concerning bacterial pathogens, while many remain extracellular, some can eventually invade host cells and may be considered facultative intracellular pathogens. An example is the Gram-negative bacterium *Pseudomonas aeruginosa*, which mostly resides in the intestinal lumen but has been observed inside intestinal cells (Irazoqui et al., 2010). Another bacterium, *Bordetella atropi*, has been recently described as an intracellular pathogen of the rhabditid nematode *Oscheius tipulae* (Tran et al., 2022). *B. atropi* parasitizes internally the intestinal cells and spreads laterally between them using filamentation. This bacterium can also grow outside its nematode host. Such an intracellular bacterium infecting *C. elegans* remains to be found.

In this review we will discuss the immune response to microsporidia and viruses, the obligate intracellular pathogens that require *C. elegans* cells to complete their lifecycle. In the following sections, we will first
review the common immune responses of *C. elegans* to both microsporidia and viruses, studied using transcriptomic studies and genetic screens in the host. The second section will then specifically concern antiviral defense and host interaction with the virus. The third section will review modulatory effects on the immune response against intracellular pathogens: parental effects, environmental factors, and natural genetic variation of the host. The last section will address the broad evolutionary conservation of the immune defense against these intracellular pathogens. Finally, we will suggest promising directions for future research.

### 1.3. *C. elegans* common immune responses to microsporidia and viruses

Despite lacking an adaptive immune system or mobile immune cells, *C. elegans* employs innate and nonclassical adaptive immune responses that are evolutionary conserved among metazoans (Ewbank and Pujol, 2016). Recent comprehensive reviews on *C. elegans* innate immunity have been published (Martineau et al., 2021; Tran and Luallen, 2023); here we will focus on the immunity against viruses and microsporidia (Fig. 2).

### 1.4. A common transcriptional Intracellular Pathogen Response to microsporidia and viruses

Strikingly, *C. elegans* activates a similar transcriptional response when its intestinal cells are infected by virus or microsporidia, and this response is distinct from that activated by extracellular pathogens remaining in the intestinal lumen (Sarkies et al., 2013; Bakowski et al., 2014; Yang et al., 2016; Chen et al., 2017a). This transcriptional response has thus been named the Intracellular Pathogen Response (IPR) (Reddy et al., 2017). Of the approximately 20,000 genes in *C. elegans* (The C. elegans Sequencing Consortium, 1998), the IPR encompasses a core of 80 highly upregulated genes, in addition to hundreds of other genes that are upregulated to a lesser extent and in different conditions (Reddy et al., 2017). Activation of these genes has been observed as soon as 8 h post inoculation (hpi) for *N. parisii* and 12 hpi for OrV (Bakowski et al., 2014; Chen et al., 2017a). A large proportion of IPR genes is formed by a subset of the *pals* gene family, whose biochemical activity is currently unknown. One of these genes, named *pals-5*, was used to engineer a transcriptional reporter driving the expression of a fluorescent protein, allowing to screen for animals responding to intracellular infection (Fig. 3). The IPR is also enriched for genes encoding

<figure><img src="image_4.png" /><figcaption>**Fig. 3.** Visualization of a *pals-5p:gfp* fluorescent reporter for intracellular infection in *C. elegans*. Overlap of bright-field, green fluorescence, and red fluorescence channels to image *rde-1* mutants expressing a green fluorescent reporter for intracellular infections and a constitutive pharynx reporter in red [pals-5p: gfp; myo-2p: mCherry]. Mock-inoculated animals show no activation of the reporter (left panel). Animals show activation of the reporter after being challenged with Orsay virus (central panel) or *N. parisii* (right panel). Scale bars are 200 µm. Micrographs were adapted from Jiang et al. (2017). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)</figcaption></figure>

components of ubiquitin ligase complexes (such as the cullin CUL-6, SKP1-related), which mediate a defense response and thermotolerance (see the section on Ubiquitin-mediated responses). The core set of genes is regulated by both microsporidia and viral infections (see dataset comparisons in Chen et al., 2017a; Lažetić et al., 2022). Beyond this canonical response, further genes are upregulated depending on host genetics, pathogen inoculum, laboratory conditions, and stage of infection (Sarkies et al., 2013; Bakowski et al., 2014; Chen et al., 2017a; Castiglioni et al., 2023).

Further work will be needed to define whether the IPR defined above is specific to the response of intestinal cells to intracellular pathogens or applies to other parasitized cells. The epidermal infection by the microsporidia *N. displodere* appears to trigger the same transcriptional response in the intestinal cells that it traverses but not in the epidermal cells where it reproduces (Luallen et al., 2016). Of note however are similarities between the IPR and the transcriptional response to oomycete infection of the epidermis, called the Oomycete Recognition Response (ORR) (Gang et al., 2022; Drury et al., 2023).

<figure><img src="image_5.png" /><figcaption>**Fig. 2.** Schematic representation of known immune responses to intracellular intestinal infection of *C. elegans* by A) virus and B) microsporidia. Host genes and proteins are represented in white rectangles, while pathogen factors are colored in blue for viruses and red for microsporidia. For more detailed information on the microsporidian life cycle, see Tecle and Troemel (2022). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)</figcaption></figure>
## 1.5. The IPR is an immune response against microsporidia and virus and partially relies on the ZIP-1 transcription factor

The IPR as a global response is an immune response, as artificial activation of the IPR prior to infection makes the animals resistant to infection by microsporidia or virus (Reddy et al., 2019). IPR activation can be achieved by: i) microsporidian or viral infections, ii) environmental conditions such as a prolonged heat stress or chemical induction of protein stress by bortezomib, or iii) mutations in repressors of the IPR obtained in a genetic screen for animals with constitutive *pals-5p:GFP* activation (Reddy et al., 2019).

Specifically, a genetic screen for constitutive *pals-5p:GFP* activation yielded mutations in another gene of the *pals* family, *pals-22*, which is not part of the IPR gene set and instead represses this transcriptional response (Fig. 2). *pals-22* mutants have an increased resistance to infection by *N. parisii* or Orsay virus (Reddy et al., 2017). Further genetic screening for constitutive *pals-5p:GFP* activation yielded mutations in the *pnp-1* gene, encoding a purine nucleoside phosphorylase acting in purine salvage pathway (Tecle et al., 2021).

Conversely, genetic screens for lack of *pals-5p:GFP* activation in genetic or environmental situations where it should be activated yielded *zip-1*, encoding a transcription factor found in intestinal and epidermal nuclei. ZIP-1 controls the induction of a subset of genes during the IPR, whether induced by microsporidia, virus or other triggers, which have a protective function against both types of intracellular pathogens in *C. elegans* (Lažetić et al., 2022) (Fig. 2). This further demonstrates that the IPR has an immune activity, thus that at least some genes that are transcriptionally activated are involved in immunity against intracellular pathogens.

## 1.6. The intriguing *pals* gene family

As mentioned, the *pals* gene family is overrepresented in the transcriptional response to intracellular pathogens, with several family members, including *pals-5*, being strongly upregulated. In *C. elegans*, the family contains 39 members in tight genomic clusters. Analysis of other *Caenorhabditis* species indicates that local duplication events occurred within the genus *Caenorhabditis*, including the *pals-22/pals-25* pair (Leyva-Díaz et al., 2017). The IPR is constitutively activated in *pals-22* mutants while loss of function of the adjacent gene *pals-25* abolishes this constitutive activation. Unlike the *zip-1* mutation, *pals-25* mutation does not have a noticeable impact in a wild-type background (Reddy et al., 2019). PALS-22 and PALS-25 proteins bind each other in a complex and a *pals-25* mutation with a premature stop codon avoids repression by PALS-22 (Gang et al., 2022). This protein complex affects IPR gene expression through the ZIP-1 transcription factor. In addition, it is involved in the detection of oomycete infection in the epidermal cells (Gang et al., 2022). The (over)expression of PALS-25 in the epidermis also signals to the intestine to enhance resistance against *N. parisii*. This cell non-autonomous signaling does not occur if the levels of PALS-25 are increased in neurons (Gang et al., 2022). A second example of cross-regulation within a *pals* gene genomic cluster is the *pals-16/17/20* cluster. A screen for constitutive *pals-5p:GFP* activation in the background of a *pals-22/pals-25* mutant yielded a hypomorphic mutation in *pals-17*, an essential gene. The IPR induction phenotype of *pals-17* mutants was suppressed by *pals-16* and *pals-20* mutations (Lažetić et al., 2023a) (Fig. 2). Intriguingly, *pals-22* mutants were also found by screening for lack of expression of a repeated transgene in neurons (Leyva-Díaz et al., 2017). As *pals-22* mutants instead are able to activate the *pals-5p:GFP* repeated transgene in intestinal cells (as well as endogenous genes), there may be some tissue specificity at stake in the *pals-22* effect on repeated transgenes.

## 1.7. Ubiquitin-mediated responses

Ubiquitin, a small protein, can be attached to other proteins in a

process called ubiquitylation. The attachment of ubiquitin to a protein can alter its stability, activity, or destination within the cell (Haglund and Dikic, 2005). Once a protein is ubiquitin-marked it can be degraded by the proteasome or by a process called autophagy; or have a diversity of other fates (Welchman et al., 2005; Shaid et al., 2013). The current model of protein complex that promotes protein degradation includes F-box adaptors (FBXA-75 and FBXA-158), SKR proteins (SKR-3, SKR-4, or SKR-5), a RING domain protein (RCS-1) and a cullin protein (CUL-6). The cullin protein needs to be neddylated by NED-8 (Panek et al., 2020) (Fig. 2). The components of this complex are induced upon infection by *N. parisii* or OrV. RNAi against ubiquitin ligase components (*skr-3*, *skr-4*, *skr-5*, and *cul-6*) corroborated that these elements mediate a defense to *N. parisii* and viral infection. However, less than 10 % of *N. parisii* cells are targeted by ubiquitin. This low fraction of targeted cells may be the result of pathogen evasion of ubiquitin targeting (Bakowski et al., 2014).

## 1.8. The detection of intracellular pathogens

How are the intracellular pathogens detected? In the case of microsporidia, this point remains unclear. Given the effect of the purine phosphorylase mutation, and of protein stress induction by bortezomib or heat stress, it is possible that depletion of nucleotides (Dean et al., 2016, 2018) and/or protein stress is required for microsporidia detection. Alternatively, a more specific upstream trigger remains to be discovered. Of note, *N. parisii* requires secretion of the host AAIM-1 protein into the intestinal lumen for its spores to be properly positioned in the lumen and successfully infect the animal (El Jarkass et al., 2022). Furthermore, *C. elegans* transcription factors of the Myc family regulate the production of chemicals that affect sporulation of *N. parisii* within intestinal cells (Botts et al., 2016).

In the case of viruses, an upstream element required for the transcriptional response is the helicase DRH-1 of the retinoic acid-inducible gene I (RIG-I) family (Sowa et al., 2020). The RIG-I-like receptors induce a signaling cascade upon the presence of unusual nucleic acids, leading to the transcriptional induction of immune genes (Rehwinkel and Gack, 2020). As in mammals (Yoneyama et al., 2005), DRH-1 appears to function as a pattern recognition receptor that can detect viral replication products and trigger a transcriptional response. Introduction of the OrV RNA1 segment coding for the viral RNA polymerase is sufficient for IPR activation, whereas introduction of an inactivating mutation (D601A) in the OrV RNA1 segment abolishes IPR activation (Sowa et al., 2020). This DRH-1 dependent activation of the IPR is specific for viral infections; microsporidia infections and proteotoxic stresses do not require DRH-1 to trigger the IPR. In the absence of DRH-1, neither the IPR nor the small RNA immune response are activated and viral infections are severe. Viral infection induces in this case a more general transcriptional response that overlaps with that induced by extracellular pathogens. This general 'biotic stress response' is likely caused by the severe infection, which leads to disruption of the integrity and homeostasis of the host cells (Sarkies et al., 2013; Le Pen et al., 2018).

# 2. Specific antiviral defense

The immune defense that are part of the transcriptional response (IPR) appear common to both microsporidia and viruses. However, other defenses are specific in targeting viral RNA.

## 2.1. RNA interference in viral defense

In addition to mediating the IPR upon viral infection, DRH-1 has a role in promoting defense through antiviral RNA interference (RNAi). RNAi is a post-transcriptional gene silencing mechanism that uses small RNA molecules to specifically suppress gene expression. By silencing specific RNA molecules, RNAi contributes to protection against exogenous RNA. One source of exogenous RNA are viruses, especially RNA
viruses that include a double-stranded RNA intermediate during replication. RNAi responses against pathogens typically recognize the pathogen-associated patterns at the 5' termini. The use of the RNAi pathway in antiviral immune responses is present in a variety of host species (Ding, 2010).

RNAi triggered by artificial exogenous double-stranded RNAs was extensively studied in *C. elegans* (Fire et al., 1998; Timmons et al., 2001; Tabara et al., 2002). *C. elegans* antiviral response is partially similar: mutants with impaired RNAi are more sensitive to infection to OrV (Ashe et al., 2013) and artificially associated viruses (Lu et al., 2005; Schott et al., 2005; Wilkins et al., 2005; Gammon et al., 2017). However, the upstream trigger DRH-1 is dispensable for response to exogenous double-stranded RNAs while the antiviral small RNA response starts when DRH-1 recruits DCR-1 and RDE-4 to target viral double-stranded RNA (dsRNA) replication intermediates. The viral dsRNA is cut by the enzyme DCR-1 into 23 nucleotide-long small interfering RNAs (siRNAs). These primary siRNAs are then amplified by a host-encoded RNA-dependent RNA polymerase recruited by RDE-1. These secondary viral siRNAs tend to start with a guanine and be 22 nucleotides in length and are therefore called 22G 2° siRNAs. Viral infection of *C. elegans* thus results in a partial destruction of the viral genome by small RNAs complementary to the viral genome, which bind specific Argonautes such as SAGO-2, resulting in viral RNA destruction (Ashe et al., 2013; Guo et al., 2013) (Fig. 2).

The exogenous RNAi pathway in *C. elegans* is systemic (Winston et al., 2002), yet oddly the small RNA response to OrV infection is not (Ashe et al., 2015). When artificially injecting VSV particles in muscle cells, the viral small RNA response can restrict the spread of the infection. In animals lacking *drh-1*, the VSV can spread to the embryos and be vertically transmitted (Gammon et al., 2017). In *Caenorhabditis* species closely related to *C. elegans*, nematodes that are incompetent for exogenous RNAi can transmit natural endogenous viral RNAs vertically through the germline (Richaud et al., 2019). The non-systemic nature of the RNAi response against OrV contrasts with the response in other animals such as *Drosophila*, where the systemic spread of viral dsRNA is essential for an effective antiviral immunity (Saleh et al., 2009), or mammals that can use extracellular vesicles for systemic dissemination of virus-derived siRNAs (Zhang et al., 2022).

## 2.2. Uridylation pathway in viral defense

Uridylation is an RNA modification that plays a role in modulating RNA degradation (Zigáčková and Vaňáčová, 2018). Uridylation involves the addition of one or two uridine residues to the 3' termini of RNA molecules. The tagged RNA molecules are then degraded by the host machinery (Zigáčková and Vaňáčová, 2018). Uridine addition is catalyzed by terminal uridylyltransferases. In *C. elegans* a terminal uridylyltransferase is encoded by *cde-1* (Fig. 2). The catalytic activity of CDE-1 is necessary for the immune response to viruses, as mutants lacking this enzyme display a higher viral load. Although we know that the uridylation response acts at early stages of virus infection, the mechanisms by which CDE-1 recognizes viral RNAs as a target in ин-testinal cells are not yet understood (Le Pen et al., 2018). CDE-1 also uridylates genomically-encoded small RNAs (van Wolfswinkel et al., 2009).

The nematode terminal uridylyltransferase CDE-1 has two mammalian orthologues called TUT4 and TUT7. These proteins have been shown to play a role in the antiviral response to influenza A virus (IAV) in mouse cells. When both TUT4 and TUT7 are absent, mutant murine embryonic fibroblasts are more susceptible to IAV infection (Le Pen et al., 2018). TUT4 and TUT7 uridylation target the mRNA of IAV, but not the host genome (Lim et al., 2014). This demonstrates the evolutionary conservation in antiviral activity of terminal uridylyltransferases.

## 2.3. Host factors required for the viral cycle

Certain host factors can affect how it defends against infection. *C. elegans* likely uses physical barriers to prevent OrV entry as mutants lacking components of these intestinal barriers show increased susceptibility to infection. These components include collagens (Zhou et al., 2024).

Once inside the cell, the intracellular pathogens require particular cellular environments to thrive. Certain cell states constitute an unfavorable environment for the pathogen. Factors providing such a mean of defense were found in a screen for mutants failing to activate *pals-5p*: *GFP* upon OrV infection in a sensitized *rde-1* background, where the RNAi immune pathway was defective. This screen yielded factors likely required at early steps of OrV infection, including a kinase signaling pathway (*nck-1*, *viro-2*, and *sid-3*) (Jiang et al., 2017), *drl-1* (Sandoval et al., 2019) and *hipr-1* (Jiang et al., 2020) and others at a later step of OrV infection, such as host cell lipids (Casorla-Perez et al., 2022). In the latter case, when the nematodes lack the master regulator *sbp-1*, the lipid content of the animals is depleted and the virus RNA levels are largely reduced during infection. Addition of specific lipids allow the infection to proceed, suggesting that OrV relies on host lipids for replication.

Another host cell factor relevant for viral infections is the state of the endoplasmic reticulum (ER), as disruptions in ER homeostasis also reduce OrV accumulation (Efstathiou et al., 2022). These disruptions (induced with tunicamycin) activate two processes, ER-associated degradation (ERAD) and ER-associated RNA silencing (ERAS), both of which work together to maintain ER protein homeostasis. However, the underlying mechanisms of ERAS and ERAD are distinct. ERAS functions by utilizing the cellular RNA silencing machinery to initiate RNA degradation, while ERAD employs the ubiquitin/proteasome system to degrade proteins.

# 3. Modulation OF *C. elegans* interaction with microsporidia and viruses

## 3.1. Parental effect on immune responses

*C. elegans* exhibits innate immune priming in response to both natural and human pathogens (Willis et al., 2021a). For example, infection by *N. parisii* microsporidia induces an inherited immunity in the progeny, depending on the dose of spores the parents were challenged with (Willis et al., 2021b). The induced transcriptional response confers immunity to the next generation and restricts the presence of spores in the F1 progeny intestine. This immunity lasts a single generation. Mutants (i.e. *pals-22* or *lin-35*) that mimic the transcriptional response are also able to induce immunity in the offspring (Willis et al., 2021b).

For viral infections, studies have yielded mixed conclusions, likely in part due to differences in experimental setups. Initially it was reported that the RNAi response against OrV can provide parental protection against viral infection of the F1 progeny (Sterken et al., 2014). A later study showed that the RNAi response upon OrV infection is not systemic and that the effectors of RNAi triggered by the virus were not inherited by the offspring (Ashe et al., 2015). Other studies used artificial administration of viral RNA molecules. The progeny of nematodes microinjected with VSV DNA were more resistant to VSV infection. This parental inherited response was no longer dependent on DRH-1 but on RDE-3, a factor involved in small RNA responses (Gammon et al., 2017). Using repeated transgenes expressing FHV sequences under heat shock, parental and multigenerational effects were observed (Rechavi et al., 2011). Silencing of the FHV transgene is consistent with what is known about transgenerational transgene silencing in *C. elegans*. Indeed, such repeated transgenes are prone to heritable silencing when expressed in the germline (Frolows and Ashe, 2021; Devanapally et al., 2021). Transgene-based studies are artificial and do not allow to conclude that the small RNA response towards viruses is heritable for multiple generations in *C. elegans*.
In summary, *C. elegans* generates intergenerational immune priming against microsporidia, with parental exposure conferring immunity to offspring for one generation, while evidence for similar viral immunity is inconclusive.

## 3.2. Interplay with abiotic and biotic environment, including other pathogens

Abiotic environments influence the immune response against intracellular pathogens. Because heat (or proteotoxic) stress also trigger the IPR (Sowa et al., 2020), it reduces the susceptibility of *C. elegans* to OrV (Huang et al., 2021). Conversely, OrV infection induces heat tolerance in the animals (Castiglioni and Elena 2023). Exposure to cadmium also activates a transcriptional response similar to the response to microsporidia (Willis et al., 2021b). Conversely, genes involved in the immune response are also involved in tolerance to abiotic stressors (Reddy et al., 2019; Tecle et al., 2021). Specifically, the interaction between thermotolerance and immunity could be explained by a common regulation of ubiquitin ligase complexes, which enable the nematodes to deal with both pathogenic and thermal stressors (Panek et al., 2020).

In addition, other biotic factors can impact sensitivity to intracellular pathogens. Due to their habitat in decomposing vegetal matter, *Caenorhabditis* nematodes are constantly exposed to a rich microbial environment. Libraries of naturally associated bacteria have been built (Berg et al., 2016; Dirksen et al., 2016, 2020; Samuel et al., 2016). Some of these naturally-associated bacteria induce viral resistance in *C. elegans* (Vassallo et al., 2023; González and Félix, 2024). Because the nematodes feed on their microbial environment, bacteria may regulate immunity to intracellular pathogens at the metabolic, immune, or sensory levels.

Tradeoffs in the immunity against different pathogens can be found. For example, while *aaim-1* mutants are more resistant to *N. parisii*, they display higher levels of infection by the extracellular bacterial pathogen *Pseudomonas aeruginosa*; the overexpression of AAIM-1 can reduce *P. aeruginosa* colonization (El Jarkass et al., 2022). The intergenerational immunity induced by microsporidia enhances survival to *P. aeruginosa* (Willis et al., 2021b). As another example, OrV and *Yersinia pestis*, a bacterial pathogen, trigger opposite gene expression patterns (Yang et al., 2016).

In other cases, genes regulating the IPR have a role in the defense against other non-intracellular pathogens, since animals lacking *pnp-1* are resistant to both intracellular pathogens and *P. aeruginosa* (Tecle et al., 2021). Other IPR regulators have a role in extracellular pathogen infection. Despite *pals-22* mutants showing an increased resistance against intracellular pathogens, they have an increased susceptibility to *P. aeruginosa* (Reddy et al., 2019). A gain-of-function mutant in PALS-25 activates the Oomycete Recognition Response (ORR) and is resistant to infection by the oomycete *Myzocytiopsis humicola* (Gang et al., 2022).

## 3.3. Natural genetic variation among *C. elegans* wild isolates

Given the strong selection pressures that pathogens may exert, susceptibility to pathogens is likely to vary rapidly within a species. *C. elegans* research has been traditionally performed using the laboratory-adapted strain N2 (Brenner, 1974; Sterken et al., 2015). However, in the last 20 years, sampling efforts have led to the isolation of many wild *C. elegans* (Barriè; re and Félix, 2014; Crombie et al., 2019).

As in other hosts and for other *C. elegans* pathogens, immune effectors against microsporidia and viruses appear to evolve particularly rapidly. *C. elegans* mostly reproduces by selfing and has low levels of overall genomic diversity (Barrière and Félix, 2005a, 2005b; Cutter, 2006; Andersen et al., 2012), except in Hawaiian/Pacific rim strains (Crombie et al., 2019). However, their genomes harbor regions that are hyperdivergent among wild strains and these hyperdivergent regions are enriched in putative immune effectors (Lee et al., 2021). At the level of the *Caenorhabditis* genus, fast-evolving gene families also include putative immune effectors such as ubiquitin-ligase adaptors and *pals* genes

(Thomas, 2006; van Sluijs et al., 2022).

Variation in susceptibility among *C. elegans* isolates was studied for both OrV and microsporidia. First, concerning OrV, the viral load varied among 97 *C. elegans* wild isolates over five orders of magnitude after their infection in the laboratory (Ashe et al., 2013). The examination of natural variation in such a trait can provide insights into the genetic basis of the trait, either using genome-wide association (GWAS) or laboratory crosses (Evans et al., 2021; Andersen and Rockman, 2022). Concerning viral resistance, a GWAS approach first helped to identify the function of DRH-1 in the antiviral response (Ashe et al., 2013). A laboratory cross between two isolates pointed to other QTL on chromosome V associated with OrV susceptibility (Sterken et al., 2021). The transcriptional response to the virus also varies depending on the wild isolate (Sarkies et al., 2013; van Sluijs et al., 2022), as also found for microsporidia (Mok et al., 2023).

Concerning microsporidia, both survival rate and pathogen load vary across *C. elegans* isolates upon infection by *N. parisii* (Balla et al., 2015; Mok et al., 2023). So far loci underlying this variation have been genetically mapped but not identified at the gene level. However, LGG-2/LC3 was found to regulate *Nematocida* intracellular colonization in the CB4856 Hawaiian isolate but not in reference N2 animals. The effect of *C. elegans* natural variation can also depend on the pathogen species: L1 larvae of the CB4856 strain clear *N. ironsii* more efficiently than *N. ausubeli* or *N. parisii* (Balla et al., 2019). Such studies may lead to finding pathogen-specific defense factors.

Studying wild isolates can also provide insight into the evolutionary constraints on the immune response, whose genes may be under balancing selection (van Sluijs et al., 2022). Accordingly, the genotypes differing in their susceptibility to intracellular pathogens can coexist at a local level. For example, microsporidia-susceptible strains with a higher competitive fitness in a pathogen-free environment are outcompeted by the resistant ones in the presence of microsporidia (Richaud et al., 2018). In addition, microsporidia, although clearly detrimental in some conditions, can actually save some *C. elegans* isolates from temperature-depedent multigenerational sterility (Frézal et al., 2023).

# 4. Evolutionary conserved components of responses to infection

In the *Caenorhabditis* genus, *C. briggsae* and *C. elegans* (with genomes as distant as human and mouse; Kiontke et al., 2004) display similar responses when challenged by intracellular pathogens. A comparison of the transcriptional response of *C. elegans* to OrV and *C. briggsae* to Santeuil virus showed that expression of 58 one-to-one orthologs is altered in both species upon infection (Chen et al., 2017a). While the specific genes induced by a microsporidia infection are not all direct orthologs between the two species, the response to microsporidia involves similar gene families (Wan et al., 2022). For example, other *Caenorhabditis* species do not seem to possess one-to-one orthologs of *pals-22* or *pals-25*, given the high rate of gene birth and death in this family (Leyva-Díaz et al., 2017). However, some elements of *C. elegans'* response may be species-specific.

At a larger evolutionary scale, many *C. elegans* immune responses to intracellular pathogens are conserved in vertebrates. This is the case for the RIG-I family members, which are conserved pattern recognition receptors between mammals and *C. elegans* (DRH-1, see above). However, *C. elegans* does not have clear orthologs of interferon and signaling factors activated downstream of RIG-I-like receptors in mammals (Ashe et al., 2013; Sowa et al., 2020). There are certain similarities in the induction of the *C. elegans* IPR and the vertebrate type-I interferon response (Lažetić et al., 2023b).

Mutants in core programmed cell death genes (*ced-3*, *ced-4*, *ced-9*, and *egl-1*) reduce VV replication in *C. elegans* (Liu et al., 2006) and their mammalian counterparts are also involved in viral immunity. Indeed, *ced-3* is an ortholog of human caspase genes which, among other processes, regulate antiviral responses (Chen et al., 2017b). *ced-4* is an ortholog of the human *APAF1* gene (apoptotic peptidase activating
factor 1) and *ced-9* of the *BCL-2* human gene family; the mammalian orthologs are involved in virus-host interactions (Halder et al., 2011; Kvansakul et al., 2017).

Additionally, the genes in the kinase signaling pathway (*nck-1*, *viro-2*, and *sid-3*) required for early stages of OrV infection (Jiang et al., 2017) have mammalian homologs (TNK, XASP, NCK1), which are required for the successful infection of mammalian viruses, such as IAV and Japanese encephalitis virus (Ye et al., 2016; Yángüez et al., 2018; Jiang et al., 2019). The other two genes essential at an OrV pre-replication stage are also conserved in mammals: *drl-1* has a partial similarity with the human MEKK3, which has not been described as involved in virus infections (Sandoval et al., 2019). *hipr-1* has two human orthologs (HIP1 and HIP1R) which are required in human cells for a successful infection by Coxsackie B3 virus (Jiang et al., 2020). The STAT signaling pathway positively regulates a transcriptional immune response in a wide range of animals (Darnell et al., 1994; Paradkar et al., 2012; Villarino et al., 2017; Wang et al., 2022). Yet surprisingly, *C. elegans* lacking the STAT homolog STA-1 constitutively upregulate antiviral response genes and are more resistant to viral infection. The SID-3 tyrosine kinase acts upstream of STA-1 and enables STA-1 to repress the expression of antiviral response genes (Jiang et al., 2017; Tanguy et al., 2017) (Fig. 2).

Other proteins with conserved evolutionary function are the argo-
naute ALG-1 and AIN-1, whose ALG-1 binds in the RISC complex and are
essential for Orsay virus replication (Cubillas et al., 2023). ALG-1
orthologs in humans and fruit flies have been described to have both
pro- and anti-viral functions. However, for these orthologs the slicing
activity is important and, in the nematodes, ALG1 the slicing is not
required to promote OrV replication, suggesting that the mechanism
may be distinct (Cubillas et al., 2023). Remarkably, some *C. elegans*
genes have human orthologs that could have immune functions but have
not yet been studied. For example, the *pals* genes have a mammal
ortholog that encodes a single, non-essential, member of this family,
FLACC1 (Chotiner et al., 2020). The study of immune response genes
across species has revealed both evolutionary conserved defense
mechanisms and unique adaptations.

**5. Future directions**

The availability of intracellular pathogens infecting *C. elegans* make the nematode an ideal model to study microbial pathogenesis and host responses to intracellular infection (Balla and Troemel, 2013). The future of this research field is bright given the many open questions, the collaborative spirit of the "worm community," and the growing number of researchers working on *C. elegans* intracellular pathogens.

Of note, we still have few obligate intracellular pathogens that infect *C. elegans* and among them, a single virus species. Note however that three related viruses were found to infect *Caenorhabditis briggsae* (Franz et al., 2014; Frézal et al., 2019). In addition, some RNA molecules encoding viral-like RNA-dependent RNA polymerases were found in the germline of other *Caenorhabditis* species (Richaud et al., 2019). In *C. elegans* some retrotransposons, such as *Cer1*, encode capsids (Dennis et al., 2012; Sun et al., 2023). They are expressed in the germline but may be involved in inter-tissue and potentially inter-generational small RNA transfer (Moore et al., 2021). *Polinton* virus-like transposons likely caused horizontal gene transmission between *Caenorhabditis* species (Widen et al., 2023). The intracellular bacterium *Bordetella atropi* was also recently described in a related nematode species (Tran et al., 2021) and the host immune response against this intracellular bacterium is yet to be studied. The discovery and study of new intracellular pathogens, especially in tissues other than the intestine, would improve our understanding of immunity to intracellular pathogens. Recent work with epidermal microsporidia will also be interesting to follow (Luallen et al., 2016; Mok et al., 2023; Wadi et al., 2023). Studying a variety of pathogens helps to understand the specificity of the interactions. The use of natural selection on resistant genotypes in the laboratory appears

promising for mapping of loci affecting susceptibility. For the diversity
of microsporidia, the analysis of their genomes reveals the conservation
of core cellular processes among these organisms (Wadi et al., 2023).
However, the proteins predicted to interact with hosts significantly
diverge among them (Reinke et al., 2017).

In the past decade, the discovery of natural viruses and microsporidia
in *C. elegans* has facilitated the discovery of new immune mechanisms.
However, we still lack an understanding of certain aspects of the re-
sponses, such as the mode of action of the *pals* gene family, the relevant
substrates of the ubiquitin-mediated pathway in mediating the immune
responses to intracellular pathogens, the STAT pathway or the
communication between tissues. A meta-analysis of genes differentially
expressed upon infection suggests that the immune response to the OrV
involves still unstudied pathways (Mishra et al., 2019). Beyong genetic
approaches, chemical screens may also be an entrypoint, including for
anti-microsporidia drug development (Murareanu et al., 2022). The
interaction of immune responses and their variation among natural
isolates, their degree of pathogen-specificity, and how the responses
vary under different environments are also important research lines for
the future.

With so much potential yet to be explored, many exciting discoveries
in the field are sure to follow.

CRediT authorship contribution statement

**Rubén González:** Conceptualization, Funding acquisition, Visualization, Writing – original draft, Writing – review & editing.
**Marie-Anne Félix:** Conceptualization, Funding acquisition, Writing – original draft, Writing – review & editing.

Data availability

No data was used for the research described in the article.

**Acknowledgements**

Research on Félix laboratory on viruses and microsporidia is sup-
ported by the CNRS and EMBO (ALTF 311–2021) and previously sup-
ported by the Fondation pour la Recherche Médicale DEQ20150331704.
We are grateful to the Troemel, Wang and Luallen labs for many
discussions.

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