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Published in final edited form as: Cell Microbiol. 2013 May 13;15(8):1313–1322. doi: 10.1111/cmi.12152

Caenorhabditis elegans as a model for intracellular pathogen infection

Keir M Balla 1, Emily R Troemel 1,*
PMCID: PMC3894601  NIHMSID: NIHMS545482  PMID: 23617769

Summary

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.

Introduction

An intracellular lifestyle offers benefits as well as challenges for microbial pathogens (Casadevall, 2008). By invading and replicating inside of host cells, intracellular pathogens can bypass antimicrobial defences in the extracellular environment, and gain access to intracellular resources. However, these pathogens must traverse cell membranes to enter and exit the cell, navigate the host cytoskeleton and also evade intracellular defences. Despite these challenges, many pathogens have adopted an intracellular lifestyle. Intracellular pathogens are classified as either facultative intracellular pathogens, which are microbes that can replicate both intracellularly and extracellularly, or obligate intracellular pathogens, which are microbes that replicate exclusively inside of host cells. Obligate intracellular pathogens are widespread throughout nature and cause significant disease, but can be challenging to study in the laboratory because they often are not culturable outside their hosts.

Epithelial cells provide major routes of entry for intracellular pathogens, such as epithelial cells of the skin, and epithelial cells of the intestine (Gallo and Hooper, 2012). Visualizing and studying infections in these cells in vivo is important for understanding the mechanisms of disease pathogenesis and transmission. In this review we will describe the use of the nematode Caenorhabditis elegans as a simple transparent host for studies of intestinal infection by intracellular pathogens in vivo. In particular, we will focus on the recent identification of two natural, obligate intracellular pathogens of the nematode that infect the intestine.

The C. elegans body plan, defence system and ecology

Caenorhabditis elegans is an important genetic model system that has been the platform for many discoveries in biology, including key findings in the fields of neurobiology, development and small RNAs (Fire et al., 1998; The C. elegans Sequencing Consortium, 1998; Marx, 2002). Most of these studies have been performed with the standard laboratory strain of C. elegans called N2, which was isolated from Bristol, England decades ago (Brenner, 1974). In the last few years, the ecology of C. elegans in the wild has gained more attention due to the increased isolation of wild-caught nematodes and their associated microbes (Félix and Duveau, 2012). C. elegans is commonly found in rotting fruits and stems, and appears to feed on a variety of different microbes, which can be nutritious or pathogenic. These studies have provided a global collection of wild-caught nematodes, and have identified some of the natural pathogens of C. elegans (see below).

Caenorhabditis elegans has a simple transparent body plan, which has facilitated many studies in the worm. For example, this transparency allowed C. elegans development to be described in great detail, with every cell division from a fertilized egg to a 1000-cell adult placed in a cell lineage map that illustrates the formation of all tissues in the animal (Sulston et al., 1983). The C. elegans tissues that are in regular contact with microbes are the hypodermis and the intestine. The hypodermis is a single layer epithelium that surrounds the animal and is a multi-nucleate syncytium that secretes collagen to form a tough outer cuticle (Chisholm and Hardin, 2005). C. elegans feeds on microbes that are ingested by the pharynx, which helps degrade food sources before they reach the intestine (Fig. 1A, B). The intestine is comprised of 20 epithelial cells that are mostly in pairs of cells that form a tube that runs the length of the animal (McGhee, 2007). C. elegans intestinal cells share many morphological similarities with human intestinal epithelial cells, including actin-rich microvilli on the apical side of cells that absorb nutrients from the lumen (Fig. 1C, D). These microvilli are anchored into a cytoskeletal structure called the terminal web, which spans the cell and connects into apical cell–cell junctions. Endocytosis and exocytosis in the worm intestine operate through conserved cytoskeletal and trafficking proteins, such as tubulin and small GTPases. Polarized vesicular trafficking occurs in this tissue with distinct sorting to apical and basolateral sides, similar to trafficking in epithelial cells of humans. The ability to visualize these structures and processes in a live animal provides a powerful system in which to examine pathogen infection of the intestine.

Fig. 1.

Fig. 1

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Features of the C. elegans intestine.

A. Simplified body plan of C. elegans. Microbes are ingested by the pharynx (yellow), which helps degrade food before it reaches the intestine (purple).

B. Close-up model of the field highlighted by the dotted box in (A). Divisions between intestinal cells are shown, as well as nuclei and the lumen.

C. Cell biology of intestinal cells.

D. Electron micrograph of a C. elegans intestinal cell.

How does C. elegans defend itself against pathogen infection? C. elegans does not appear to have professional immune cells, such as phagocytes (Irazoqui et al., 2010a). Consequently, in terms of cellular defence, C. elegans appears to rely exclusively on epithelial cell immunity, which is described in more detail below. Although C. elegans does not have a canonical adaptive immune system using specialized immune cells, it will avoid pathogens (Pujol et al., 2001). This avoidance is learned over time, constituting a form of adaptive immunity using behaviour instead of specialized immune cells (Zhang et al., 2005). Induction of these aversive responses is presumably driven by signals emanating from the intestine to signal an infected or `sick' state. RNA interference (RNAi) is another adaptive immune mechanism employed by C. elegans in defence, which protects against viral re-infection after exposure during the life of the animal and can even be passed to its progeny (Rechavi et al., 2011). Thus, with a combination of epithelial immune defences and aversive learning, C. elegans has been able to thrive globally despite widespread microbial challenges (Félix and Braendle, 2010; Félix and Duveau, 2012). Some of the mechanisms employed by C. elegans during pathogenic encounters are described below.

C. elegans infection by facultative intracellular pathogens and host defence

Most infection studies in C. elegans thus far have focused on clinically relevant pathogens, which have provided a strong framework for studying infections in this host (Irazoqui et al., 2010a; Pukkila-Worley and Ausubel, 2012). Feeding-based infections of C. elegans are relatively straightforward to perform, which aids studies of intestinal infections in this host. The primary food source in a laboratory setting is the E. coli strain OP50, but other bacteria and fungi also support growth and reproduction (Brenner, 1974; Mylonakis et al., 2002). Several feeding-based infection models have been developed, which involve simply substituting a microbial pathogen in place of the normal food source of E. coli. The lifespan of C. elegans on E. coli is only about 2 weeks, which makes it easy to assess whether pathogens cause a shortening of lifespan. The wide variety of microbial pathogens that have been shown to infect and kill C. elegans have been described in other reviews (e.g. see table 1 in Sifri et al. 2005), so we will only briefly point out here the infections with facultative intracellular pathogens of humans, and then highlight some features of C. elegans immunity.

There are several facultative intracellular pathogens of clinical importance that have been shown to infect C. elegans (Table S1). These microbes include the human fungal pathogen Cryptococcus neoformans, and the human bacterial pathogens Shigella spp., Salmonella enterica and Listeria spp. (Mylonakis et al., 2002; Burton et al., 2006; Kesika and Balamurugan, 2012). Unlike in mammals, these pathogens remain extracellular throughout infection of C. elegans, with the exception of Salmonella enterica Serovar Typhimurium. Early reports of S. typhimurium infection indicated that it remained extra-cellular (Aballay et al., 2000; Labrousse et al., 2000), but a more recent report has indicated that S. typhimurium can be found intracellularly and kills more efficiently when autophagy genes are disrupted (Jia et al., 2009). Autophagy may therefore constitute one of the conserved mechanisms employed by C. elegans to defend against intracellular pathogens. Listeria species have been studied in C. elegans models of infection, but there are conflicting reports of their pathogenicity and no intestinal invasion has been observed (Thomsen et al., 2006; Forrester et al., 2007; Guha et al., 2013). Part of the difficulty in modelling intracellular infections in C. elegans with human pathogens may be due to intrinsic differences in tissue architecture. For instance, Listeria monocytogenes invades mammalian intestinal cells specifically at junctions during cell extrusion or through the lumenal side of goblet cells, neither of which are features of the C. elegans intestine (Pentecost et al., 2006; Nikitas et al., 2011). In order to use C. elegans as a host to model intracellular intestinal infection by human pathogens, it may be necessary to use pathogens that invade the lumenal side of epithelial cells.

Caenorhabditis elegans responds to bacterial and fungal pathogens by robust transcriptional upregulation of many effector genes (Shivers et al., 2008; Irazoqui et al., 2010b). Effectors include those that appear to provide intracellular defence, such as UDP-glucoronysyl transferases and cytochrome P450s that could inactivate intracellular toxins, and P-glycoprotein pumps that could excrete toxins out of the cell. C. elegans also has a vast repertoire of genes that could provide extracellular defence. Indeed about 17% of the genes in the C. elegans genome are predicted to encode secreted proteins, and a third of those are upregulated by exposure to pathogens (Suh and Hutter, 2012). These factors often are part of expanded gene families and include some proteins conserved with mammals, such as secreted C-type lectins, which have anti-microbial activity, but most of these secreted factors are nematode-specific. Two notable examples include the C. elegans nlps or neuro-peptide-like proteins and caenacins, both of which are secreted peptides that have anti-fungal activity.

Effector gene expression is controlled by several distinct signalling pathways in C. elegans. For example, the conserved PMK-1 p38 MAP kinase pathway controls induction of nlps and other subsets of effectors, and provides defence against bacterial and fungal infection. What induces activation of this and other defence pathways? Classical pattern recognition receptors have not yet been described in C. elegans. Instead, recent studies indicate that defensive responses can be induced by pathogen-directed perturbation of core processes, so-called effector-triggered immunity (ETI). ETI was originally described in plants, and is increasingly appreciated to be important in animal immunity as well (Stuart et al., 2013). For example, Pseudomonas aeruginosa-directed block of host translation was shown to induce expression of some PMK-1 dependent genes, and also activate the ZIP-2 bZIP transcription factor defence pathway in C. elegans (Dunbar et al., 2012; McEwan et al., 2012). Interestingly, some of the other pathogen-induced transcriptional responses in C. elegans appear to be controlled non-cell-autonomously by the nervous system. For example, fungal infection induces expression of a TGF-beta ligand in neurons, which then activates a hypodermal pathway to induce expression of antimicrobial caenacins (Zugasti and Ewbank, 2009). This interplay of tissues has parallels with mammalian immunity, for which the nervous system has increasingly been recognized as playing a regulatory role (Chiu et al., 2012). Altogether, these findings provide insights into how C. elegans can integrate signals throughout the body to sense and respond to microbial infection, and provide a foundation for making progress with the recent discoveries of C. elegans infection by natural, obligate intracellular pathogens, which are the subject for the rest of the review.

C. elegans as a host for intracellular infection by viruses

Natural viral infection models, as well as models using non-natural viruses of C. elegans, have been established. Viral infections of C. elegans can be followed from the stages of invasion through to transmission. Furthermore, C. elegans shares a conserved antiviral RNA interference (RNAi) defence system with other animals. These advantages make C. elegans a flexible platform for studying host–virus interactions.

Non-natural virus models

Models of Flock House virus (FHV), vesicular stomatitis virus (VSV) and vaccinia virus (VV) infections have been established in C. elegans (Lu et al., 2005; Schott et al., 2005; Wilkins et al., 2005; Liu et al., 2006). FHV has been studied in C. elegans by introducing a genetically encoded viral replicon in transgenic animals, while VSV infections have been modelled by adding recombinant virus particles to primary embryonic C. elegans cell cultures. Experiments with these models identified a prominent role for RNAi in defence against viruses, which is in agreement with data from host–virus interactions modelled in plants and insects. C. elegans mutants defective for RNAi had higher levels of viral infection, while mutants with hyperactive RNAi responses had lower levels of viral infection. Thus, FHV and VSV subvert the wild-type C. elegans antiviral responses enough to replicate, but can replicate more successfully when host defences are defective. Further work in the FHV model identified host factors that are conserved with mammalian antiviral proteins (Lu et al., 2009). Among the genes that were involved in viral interactions were Dicer-related helicases. Homologous proteins in mammals have been shown to both positively and negatively regulate immune responses to viruses. Likewise, drh-1 was found to positively regulate immunity to FHV in C. elegans, while drh-2 exhibits negative regulation. These conserved RNAi responses are hypothesized to have evolved in part to limit viruses that have double-stranded RNA genomes or that generate double-stranded RNA intermediates during replication within host cells. VV is a double stranded DNA virus that may generate double stranded RNA, but mutants in the RNAi pathway did not affect the outcome of infection in C. elegans. Instead, core members of the programmed cell death pathway appear to limit VV replication, as mutant animals have higher pathogen load. Interestingly, mutants in the programmed cell death pathway were not more susceptible to killing, suggesting that alternative mechanisms of resistance likely exist.

The experimental viral models discussed above have provided valuable tools, and further work with these models will likely reveal additional insight in to the mechanisms of antiviral defence pathways. And with the recent discovery of natural viral infections of C. elegans described below, researchers should also be able to address other key aspects of host-viral interactions, such as transmission among live animals and the identification of genetic substrates participating in co-evolutionary processes in the wild.

Natural virus models

Natural viral infections in C. elegans have recently been identified, providing powerful models for investigating co-evolution of intracellular interactions. Field surveys carried out by Félix and colleagues led to the identification of two infected Caenorhabditis isolates, a C. elegans strain called JU1580 isolated from a rotting apple from Orsay, and a Caenorhabditis briggsae strain called JU1264 isolated from a snail found on a rotting grape from Santeuil (Félix et al., 2011). Both strains exhibited a range of intestinal pathologies observable by microscopy, which overall appear to compromise the integrity of intestinal cells (Fig. 2A). These phenotypes were not observed in the progeny of bleached parents, arguing against the possibility that the viruses were vertically transmitted. Furthermore, the phenotypes could be reinitiated in bleached progeny by adding a 0.2 μm filtrate from affected animals, suggesting viral infection. Electron micrographs of affected animals revealed the ultrastructural details of these cell pathologies, and detected electron dense particles that were reminiscent of virions. Sequencing of affected animals identified two novel viruses most closely related to the Nodaviridae family as the causes of infection. And now a third virus infecting a wild-caught C. briggsae from Le Blanc, France, has recently been reported, which is also related to the Nodaviridae family (Franz et al., 2012). Altogether, these are the first models of natural host–virus infections in Caenorhabditis.

Fig. 2.

Fig. 2

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Intracellular pathogens studied in C. elegans and the cell biology of natural intracellular infections in C. elegans intestinal cells.

A. Model of the predicted Orsay virus lifecycle and intestinal phenotypes associated with infection. Virions are drawn as purple hexagons, viral RNA is drawn as purple curved lines for positive strand transcripts and green for negative strand transcripts. Shorter lines represent small RNAs. Actin is drawn as vertical orange lines, terminal web as blue lines, and apical junctions as black ovals.

B. Model of the N. parisii lifecycle and intestinal phenotypes associated with microsporidia infection. Spores are drawn as green ovals, polar tube is green line from invading spore, replicating meronts are irregular green shapes. Actin is drawn as vertical orange lines, terminal web as blue lines, and apical junctions as black ovals.

Nodaviruses are well studied due to their small genome sizes and ability to induce and suppress host RNAi defences. The Orsay and Santeuil viruses were identified as positive-sense RNA viruses with small bi-partite genomes. Phylogenetic analyses positioned the Orsay and Santeuil viruses as distantly related to all other characterized nodaviruses, and most similar to each other. A predicted open reading frame unique to the Orsay and Santeuil viruses was identified, but no functional role has been attributed. No obvious homologue to the RNAi suppression protein found in FHV was located in the Orsay or Santeuil genomes, but several experiments suggest that an antiviral RNAi response is initiated during infection. Small RNAs from the Orsay genome were identified in infected JU1580 animals. These small RNAs had range of nucleotide lengths for the sense strand RNAs, which is suggestive of non-specific Dicer products. Antisense RNAs showed length and nucleotide biases, favouring 22 nucleotide transcripts beginning with a guanidine residue. The significance of this pattern is not fully understood, but is generally indicative of a regulated antiviral RNAi response. Additional support for the antiviral response mounted during Orsay virus infection is the observation that the N2 strain, while normally 100-fold more resistant to infection (i.e. has 100-fold less viral transcript) than JU1580, becomes just as susceptible when the RNAi pathway is disrupted, such as in rde-1 Argonaute mutants. Thus, like nodaviruses, the Orsay virus of C. elegans is genomically small and provokes RNAi defences.

In the Félix et al. study, the authors also provide evidence for natural variation among strains in their ability to resist viral infection. They tested a small collection of genetically diverse C. elegans strains for responses to exogenously added double-stranded RNA and the Orsay virus, and found variation in the efficiency of endogenous and antiviral RNA responses. A loose trend emerged, suggesting that strains with compromised endogenous RNAi pathways showed more symptoms of viral infection. However, this hypothesis could not explain the data from all the strains, pointing towards the likelihood that independent mechanisms can regulate antiviral defences. The phenotypes scored for antiviral responses were the intestinal pathologies observed by microscopy. This method could allow for detection of variation in the cell biology of viral responses among strains. Expanding these studies with additional strains is expected to permit genetic mapping of loci that have evolved in C. elegans to cope with natural viruses.

C. elegans as a host for intracellular infection by microsporidia

The first natural intracellular pathogen identified in C. elegans defined a new genus and species of microsporidia (Troemel et al., 2008). It was named Nematocida parisii (strain ERTm1), or nematode-killer from Paris, because it was found in wild-caught C. elegans from a compost pit near Paris, and causes a lethal intestinal infection in its host. Microsporidia comprise a phylum of obligate intracellular parasites with over 1200 species, which are most closely related to fungi (Texier et al., 2010). They parasitize all animal phyla, infecting single-celled ciliates to mammals, with some microsporidian species able to infect only one host, and other species able to infect a broad range of hosts. Microsporidia infections in humans and agriculturally important animals can be lethal, but the mechanisms underlying these interactions are not well understood (Didier and Weiss, 2011; Troemel, 2011).

The N. parisii life cycle inside the C. elegans host shares overall similarities with other microsporidia infections (Fig. 2B). First, an ingested spore encounters a host environment that triggers firing of an infection apparatus called a polar tube, which serves to inject a nucleus and sporoplasm into an intestinal cell. This invasion leads to formation of a membrane-bound intracellular stage called a meront that often replicates as a multi-nucleate meront. These meronts eventually differentiate into spores, which exit the cell and propagate infection. The details of these processes may differ in other host/microsporidia pairs, but the overall developmental structure appears to be well conserved. As with many other intestinal pathogens, N. parisii is transmitted via a faecal–oral route, whereby spores are defecated out by the animal and subsequently consumed by new hosts to continue the life cycle.

Little is known about the genetic and molecular details of these steps in the microsporidia lifecycle in any system, because it is not possible to manipulate microsporidia genetically, and they can only grow inside of host cells. The discovery of natural pathogens of C. elegans has provided a tractable model to study microsporidia infection. In addition to N. parisii (ERTm1), microsporidia have been isolated from nematodes around the world, indicating that these pathogens are a common cause of infection for nematodes in the wild (Troemel et al., 2008; M.-A. Félix, unpubl. data).

Nematocida parisii infection of C. elegans has recently been used to illuminate key features of the exit strategy of this species of microsporidia, which can be divided into two main phases (Fig. 2B) (Troemel et al., 2008; Estes et al., 2011). The first phase involves a restructuring of the terminal web, which is a conserved structure of intestinal cells. Intestinal actin is normally restricted to the apical side of intestinal cells in the microvilli and terminal web. However, as meronts develop, actin is ectopically redistributed to the basolateral side of the cell where it forms filaments. Soon after this relocalization, there are gaps that appear in intermediate filaments at the terminal web. These gaps may be triggered by actin redistribution away from the apical side, as reducing levels of actin caused gap formation in the absence of infection. Other pathogens of C. elegans do not appear to cause gaps to form in the terminal web, indicating that gaps are due to a specific perturbation caused by N. parisii infection. This terminal web restructuring may serve to remove a barrier to exit, so that spores can exit out the apical side of cells back into the intestinal lumen.

In the second phase of exit spores are released from the cell and defecated by the animal, with thousands of spores being shed per hour. Spores begin to form shortly after the appearance of holes in the terminal web, and then exit the cell in a non-lytic manner. No host membrane is visible around spores that have exited to the intestinal lumen, suggesting that non-lytic exit does not occur via budding. Spore exit appears to be highly directional – spores only exit from the intestine on the apical side of cells and not on the basolateral side. Thus, spores likely use a directional cue from the host in order to escape into the lumen, which is required for its faecal–oral life cycle. The precise molecular mechanisms of this directional escape by N. parisii remain to be determined.

To learn more about the pathogenic mechanisms of N. parisii, as well as other microsporidian species, the Microsporidian Genomes Consortium was recently formed with the goal of sequencing several species of microsporidia. The genomes of three Nematocida strains were recently sequenced as part of this consortium, including N. parisii (ERTm1), a related N. parisii strain (ERTm3) isolated from wild-caught C. elegans in Santeuil, France, and a divergent strain called Nematocida sp1 (ERTm2) isolated from a C. briggsae host in India (Cuomo et al., 2012). Microsporidia have the smallest known eukaryotic genomes (Keeling and Corradi, 2011), and in keeping with that, Nematocida genomes are extremely reduced. Genome sizes and sequence were highly similar between N. parisii ERTm1 and ERTm3, sharing approximately 4.1 Mb of sequence that is 99.8% identical. Nematocida sp1 ERTm2 was slightly larger at 4.6 Mb and shared only 68.3% average identity with N. parisii. The high divergence between ERTm1 and ERTm2 does not appear to be due solely to differential host specification, as both are infective to C. elegans and C. briggsae. Over 70% of the N. parisii genome is predicted to be coding and intronless, with non-coding intergenic regions that are short. Thus, Nematocida genomes are compact like some other microsporidia genomes. This reduction is thought to reflect a dependency on host factors, which can be redirected to facilitate parasitic growth.

How does a genomically reduced eukaryotic pathogen succeed in a complex host environment? Phylogenomic characterization of Nematocida, together with several other microsporidia species, uncovered some of the potential evolutionary strategies used by these pathogens for obligate intracellular growth (Cuomo et al., 2012). RNA-seq data of N. parisii expression over the course of ERTm1 infection in C. elegans demonstrated a dramatic takeover of intestinal cells, growing within infected cells at an estimated doubling time of approximately three hours. This rate is similar to yeast growing in rich media, indicating that N. parisii is well-adapted to grow in this intracellular environment. Common features identified in microsporidian genomes that may explain their rapid growth include the loss of the retinoblastoma (Rb) cell cycle inhibitor, which is lost in most human cancers. Because Rb serves as quality control in DNA replication, loss of Rb may lead to a higher mutation rate and explain the high sequence divergence among microsporidian genomes. Another interesting observation that came out of this study with potential parallels with cancer is that microsporidia appear to secrete the metabolic enzyme hexokinase into host cells early during infection. It is unclear if this secreted hexokinase would benefit pathogen or host, but one possibility is that it could benefit the pathogen by driving the host to upregulate glycolysis and the pentose phosphate pathway to provide more building blocks for growth like lipids, nucleotides and amino acids, similar to the Warburg effect in cancer. Genome studies have also revealed that microsporidia have horizontally acquired nucleoside transporters (Katinka et al., 2001; Cuomo et al., 2012), which presumably could import these host-derived building blocks to favour pathogen growth. Finally, genetic diversity in Nematocida and other microsporidia may be increased through mating and recombination, both of which were hypothesized from Nematocida genome data. Together with the diversity driven by rapid and mistake-prone growth, mating and recombination may allow Nematocida to keep up with the ongoing host–pathogen evolutionary arms race likely taking place between hosts and their microsporidian parasites.

Summary and future directions

Caenorhabditis elegans is an especially convenient whole-animal model for obligate intracellular pathogen infection, made possible by the recent identification of natural virus and microsporidian pathogens that infect its intestine. Although these pathogens infect internal tissues, the transparency of C. elegans makes it possible to visualize and study pathogenic mechanisms of entry, replication and exit, as well as host defence mechanisms within whole animals. The progression of infection can be observed in real-time by light microscopy; a technique that has been utilized to describe some of the basic cell biology phenotypes associated with viral and microsporidian infections (Fig. 2). Powerful new optogenetic techniques may facilitate further dissection of these infections. For example, specific protein–protein interactions between host and pathogen could be observed and manipulated by tagging with a genetically encoded mini singlet oxygen generator, allowing for visualization by fluorescence microscopy, specific and localized protein destruction by over-production of reactive oxygen species, and high-resolution imaging by electron microscopy (Tour et al., 2003; Shu et al., 2011). These kinds of experiments are ideally suited to the small, transparent, genetically tractable C. elegans model.

Intracellular infections of C. elegans by natural pathogens are simple to propagate in the lab, providing a convenient system in which to study the mechanisms of intracellular pathogen transmission. Horizontal transmission is commonly observed among intracellular pathogens, and was observed for the Orsay and Santeuil viruses in Caenorhabditis. Similarly, only horizontal transmission was observed during Nematocida infections, and recent studies indicate that Nematocida exit occurs in a directional manner into the intestinal lumen. This polarized exit is crucial for all pathogens transmitted via a faecal–oral route, but the underlying mechanisms are unknown. Neither the virus nor microsporidia infections of C. elegans characterized so far appear to be transmitted vertically. The mechanisms that prevent this kind of transmission are not well understood. Studying transmission strategies in C. elegans may clarify how and why one strategy may be favoured over another. Additionally, transmission of the Orsay virus was only successful in strains of C. elegans, while the Santeuil virus could only be propagated in the C. briggsae isolate JU1264. In contrast, all three Nematocida strains could infect both C. elegans and C. briggsae. These models may therefore be informative for understanding the factors that specify the transmission strategies and host range of intracellular pathogens.

Defences against intracellular pathogens seem to be distinct from those defined for extracellular pathogens. The RNAi pathway is a well-characterized regulator of antiviral immunity in both C. elegans and other organisms (Ding, 2010). Data from the first natural viral infections of C. elegans indicate that other pathways will be involved in regulating viral pathology. These may be elucidated in both natural and artificial models of viral infection. Resistance mechanisms that limit microsporidia infections are not well understood for any host. In mammals, resistance is known to involve T cells of adaptive immunity, but the molecular mechanisms are unknown (Didier and Weiss, 2011). In C. elegans, the major immune defence pathways regulated by p38 MAP kinase and insulin signalling pathways do not appear to be involved in defence against N. parisii infection (Troemel et al., 2008), indicating that new pathways will be found to regulate defence against this intracellular pathogen.

Other promising future directions for these models will exploit the fact that the host and pathogens have presumably co-evolved. In other systems, studies of natural host/pathogen pairs have provided insights into the multilayered interactive networks that can occur through coevolving bouts of adaptation and counter adaptation (Woolhouse et al., 2002). One outcome of these interactions can be mimicry by both host and pathogen of each other (Elde and Malik, 2009). Efforts towards understanding the selective pressures that have shaped immunity and pathogenesis will be greatly bolstered by the ongoing progress in identifying the ecology and global diversity of C. elegans and its pathogens (Félix and Braendle, 2010; Andersen et al., 2012; Cuomo et al., 2012; Félix and Duveau, 2012). Recent work in C. elegans has provided experimental support for pathogens being a driving force behind sexual reproduction in their hosts, and has demonstrated that co-evolutionary dynamics generate genetic changes in host and pathogen that affect fitness within a relatively brief time frame (Morran et al., 2011). Combining the increasing genetic data from wild isolates with experimental co-evolution approaches should facilitate the discovery of the specific molecular interactions that determine the evolutionary trajectory of C. elegans and its pathogens. While still quite new, the discovery of two natural intracellular infections in C. elegans is expected to provide new insights into the logic underlying the structure of the C. elegans immune response and more generally, the functional interactions between hosts and their intra-cellular pathogens.

Supplementary Material

Supp Table 1

Pathogens tested for intracellular infection in C. elegans and features of immune response.

Acknowledgements

We thank David Wang, Marie-Anne Félix, Suzy Szumowski and Michael Botts for helpful comments on the manuscript. Supported by NSF GRFP predoctoral fellowship to K.M.B., and NIAID R01 AI087528, the Searle Scholars Program, Ray Thomas Edwards Foundation, David & Lucile Packard Foundation fellowship to E.R.T.

Footnotes

Supporting information Additional Supporting Information may be found in the online version of this article at the publisher's web-site:

References

  1. Aballay A, Yorgey P, Ausubel FM. Salmonella typhimurium proliferates and establishes a persistent infection in the intestine of Caenorhabditis elegans. Curr Biol. 2000;10:1539–1542. doi: 10.1016/s0960-9822(00)00830-7. [DOI] [PubMed] [Google Scholar]
  2. Andersen EC, Gerke JP, Shapiro JA, Crissman JR, Ghosh R, Bloom JS, et al. Chromosome-scale selective sweeps shape Caenorhabditis elegans genomic diversity. Nat Genet. 2012;44:285–290. doi: 10.1038/ng.1050. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brenner S. The genetics of Caenorhabditis elegans. Genetics. 1974;77:71–94. doi: 10.1093/genetics/77.1.71. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Burton EA, Pendergast AM, Aballay A. The Caenorhabditis elegans ABL-1 tyrosine kinase is required for Shigella flexneri pathogenesis. Appl Environ Microbiol. 2006;72:5043–5051. doi: 10.1128/AEM.00558-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Casadevall A. Evolution of intracellular pathogens. Annu Rev Microbiol. 2008;62:19–33. doi: 10.1146/annurev.micro.61.080706.093305. [DOI] [PubMed] [Google Scholar]
  6. Chisholm AD, Hardin J. Epidermal morphogenesis. WormBook. 2005;1 December:1–22. doi: 10.1895/wormbook.1.35.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chiu IM, von Hehn CA, Woolf CJ. Neurogenic inflammation and the peripheral nervous system in host defense and immunopathology. Nat Neurosci. 2012;15:1063–1067. doi: 10.1038/nn.3144. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cuomo CA, Desjardins CA, Bakowski MA, Goldberg J, Ma AT, Becnel JJ, et al. Microsporidian genome analysis reveals evolutionary strategies for obligate intracellular growth. Genome Res. 2012;22:2478–2488. doi: 10.1101/gr.142802.112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Didier ES, Weiss LM. Microsporidiosis: not just in AIDS patients. Curr Opin Infect Dis. 2011;24:490–495. doi: 10.1097/QCO.0b013e32834aa152. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Ding SW. RNA-based antiviral immunity. Nat Rev Immunol. 2010;10:632–644. doi: 10.1038/nri2824. [DOI] [PubMed] [Google Scholar]
  11. Dunbar TL, Yan Z, Balla KM, Smelkinson MG, Troemel ER. C. elegans detects pathogen-induced translational inhibition to activate immune signaling. Cell Host Microbe. 2012;11:375–386. doi: 10.1016/j.chom.2012.02.008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Elde NC, Malik HS. The evolutionary conundrum of pathogen mimicry. Nat Rev Microbiol. 2009;7:787–797. doi: 10.1038/nrmicro2222. [DOI] [PubMed] [Google Scholar]
  13. Estes KA, Szumowski SC, Troemel ER. Nonlytic, actin-based exit of intracellular parasites from C. elegans intestinal cells. PLoS Pathog. 2011;7:e1002227. doi: 10.1371/journal.ppat.1002227. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Félix MA, Braendle C. The natural history of Caenorhabditis elegans. Curr Biol. 2010;20:R965–R969. doi: 10.1016/j.cub.2010.09.050. [DOI] [PubMed] [Google Scholar]
  15. Félix MA, Duveau F. Population dynamics and habitat sharing of natural populations of Caenorhabditis elegans and C. briggsae. BMC Biol. 2012;10:59. doi: 10.1186/1741-7007-10-59. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Félix MA, Ashe A, Piffaretti J, Wu G, Nuez I, Belicard T, et al. Natural and experimental infection of Caenorhabditis nematodes by novel viruses related to nodaviruses. PLoS Biol. 2011;9:e1000586. doi: 10.1371/journal.pbio.1000586. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature. 1998;391:806–811. doi: 10.1038/35888. [DOI] [PubMed] [Google Scholar]
  18. Forrester S, Milillo SR, Hoose WA, Wiedmann M, Schwab U. Evaluation of the pathogenicity of Listeria spp. in Caenorhabditis elegans. Foodborne Pathog Dis. 2007;4:67–73. doi: 10.1089/fpd.2006.64. [DOI] [PubMed] [Google Scholar]
  19. Franz CJ, Zhao G, Félix MA, Wang D. Complete genome sequence of Le Blanc virus, a third Caenorhabditis nematode-infecting virus. J Virol. 2012;86:11940. doi: 10.1128/JVI.02025-12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Gallo RL, Hooper LV. Epithelial antimicrobial defence of the skin and intestine. Nat Rev Immunol. 2012;12:503–516. doi: 10.1038/nri3228. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Guha S, Klees M, Wang X, Li J, Dong Y, Cao M. Influence of planktonic and sessile Listeria monocytogenes on Caenorhabditis elegans. Arch Microbiol. 2013;195:19–26. doi: 10.1007/s00203-012-0841-y. [DOI] [PubMed] [Google Scholar]
  22. Irazoqui JE, Urbach JM, Ausubel FM. Evolution of host innate defence: insights from Caenorhabditis elegans and primitive invertebrates. Nat Rev. 2010a;10:47–58. doi: 10.1038/nri2689. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Irazoqui JE, Troemel ER, Feinbaum RL, Luhachack LG, Cezairliyan BO, Ausubel FM. Distinct pathogenesis and host responses during infection of C. elegans by P. aeruginosa and S. aureus. PLoS Pathog. 2010b;6:e1000982. doi: 10.1371/journal.ppat.1000982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Jia K, Thomas C, Akbar M, Sun Q, Adams-Huet B, Gilpin C, Levine B. Autophagy genes protect against Salmonella typhimurium infection and mediate insulin signaling-regulated pathogen resistance. Proc Natl Acad Sci USA. 2009;106:14564–14569. doi: 10.1073/pnas.0813319106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Katinka MD, Duprat S, Cornillot E, Metenier G, Thomarat F, Prensier G, et al. Genome sequence and gene compaction of the eukaryote parasite Encephalitozoon cuniculi. Nature. 2001;414:450–453. doi: 10.1038/35106579. [DOI] [PubMed] [Google Scholar]
  26. Keeling PJ, Corradi N. Shrink it or lose it: balancing loss of function with shrinking genomes in the microsporidia. Virulence. 2011;2:67–70. doi: 10.4161/viru.2.1.14606. [DOI] [PubMed] [Google Scholar]
  27. Kesika P, Balamurugan K. Studies on Shigella boydii infection in Caenorhabditis elegans and bioinformatics analysis of immune regulatory protein interactions. Biochim Biophys Acta. 2012;1824:1449–1456. doi: 10.1016/j.bbapap.2012.07.008. [DOI] [PubMed] [Google Scholar]
  28. Labrousse A, Chauvet S, Couillault C, Kurz CL, Ewbank JJ. Caenorhabditis elegans is a model host for Salmonella typhimurium. Curr Biol. 2000;10:1543–1545. doi: 10.1016/s0960-9822(00)00833-2. [DOI] [PubMed] [Google Scholar]
  29. Liu WH, Lin YL, Wang JP, Liou W, Hou RF, Wu YC, Liao CL. Restriction of vaccinia virus replication by a ced-3 and ced-4-dependent pathway in Caenorhabditis elegans. Proc Natl Acad Sci USA. 2006;103:4174–4179. doi: 10.1073/pnas.0506442103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Lu R, Maduro M, Li F, Li HW, Broitman-Maduro G, Li WX, Ding SW. Animal virus replication and RNAi-mediated antiviral silencing in Caenorhabditis elegans. Nature. 2005;436:1040–1043. doi: 10.1038/nature03870. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Lu R, Yigit E, Li WX, Ding SW. An RIG-I-Like RNA helicase mediates antiviral RNAi downstream of viral siRNA biogenesis in Caenorhabditis elegans. PLoS Pathog. 2009;5:e1000286. doi: 10.1371/journal.ppat.1000286. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. McEwan DL, Kirienko NV, Ausubel FM. Host translational inhibition by Pseudomonas aeruginosa Exotoxin A Triggers an immune response in Caenorhabditis elegans. Cell Host Microbe. 2012;11:364–374. doi: 10.1016/j.chom.2012.02.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. McGhee JD. The C. elegans intestine. WormBook. 2007;27 March:1–36. doi: 10.1895/wormbook.1.133.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Marx J. Nobel prize in physiology or medicine. Tiny worm takes a star turn. Science. 2002;298:526. doi: 10.1126/science.298.5593.526. [DOI] [PubMed] [Google Scholar]
  35. Morran LT, Schmidt OG, Gelarden IA, Parrish RC, 2nd, Lively CM. Running with the Red Queen: host-parasite coevolution selects for biparental sex. Science. 2011;333:216–218. doi: 10.1126/science.1206360. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Mylonakis E, Ausubel FM, Perfect JR, Heitman J, Calderwood SB. Killing of Caenorhabditis elegans by Cryptococcus neoformans as a model of yeast pathogenesis. Proc Natl Acad Sci USA. 2002;99:15675–15680. doi: 10.1073/pnas.232568599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Nikitas G, Deschamps C, Disson O, Niault T, Cossart P, Lecuit M. Transcytosis of Listeria monocytogenes across the intestinal barrier upon specific targeting of goblet cell accessible E-cadherin. J Exp Med. 2011;208:2263–2277. doi: 10.1084/jem.20110560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Pentecost M, Otto G, Theriot JA, Amieva MR. Listeria monocytogenes invades the epithelial junctions at sites of cell extrusion. PLoS Pathog. 2006;2:e3. doi: 10.1371/journal.ppat.0020003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Pujol N, Link EM, Liu LX, Kurz CL, Alloing G, Tan MW, et al. A reverse genetic analysis of components of the Toll signaling pathway in Caenorhabditis elegans. Curr Biol. 2001;11:809–821. doi: 10.1016/s0960-9822(01)00241-x. [DOI] [PubMed] [Google Scholar]
  40. Pukkila-Worley R, Ausubel FM. Immune defense mechanisms in the Caenorhabditis elegans intestinal epithelium. Curr Opin Immunol. 2012;24:3–9. doi: 10.1016/j.coi.2011.10.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Rechavi O, Minevich G, Hobert O. Transgenerational inheritance of an acquired small RNA-based anti-viral response in C. elegans. Cell. 2011;147:1248–1256. doi: 10.1016/j.cell.2011.10.042. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Schott DH, Cureton DK, Whelan SP, Hunter CP. An antiviral role for the RNA interference machinery in Caenorhabditis elegans. Proc Natl Acad Sci USA. 2005;102:18420–18424. doi: 10.1073/pnas.0507123102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Shivers RP, Youngman MJ, Kim DH. Transcriptional responses to pathogens in Caenorhabditis elegans. Curr Opin Microbiol. 2008;11:251–256. doi: 10.1016/j.mib.2008.05. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Shu X, Lev-Ram V, Deerinck TJ, Qi Y, Ramko EB, Davidson MW, et al. A genetically encoded tag for correlated light and electron microscopy of intact cells, tissues, and organisms. PLoS Biol. 2011;9:e1001041. doi: 10.1371/journal.pbio.1001041. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Sifri CD, Begun J, Ausubel FM. The worm has turned – microbial virulence modeled in Caenorhabditis elegans. Trends Microbiol. 2005;13:119–127. doi: 10.1016/j.tim.2005.01.003. [DOI] [PubMed] [Google Scholar]
  46. Stuart LM, Paquette N, Boyer L. Effector-triggered versus pattern-triggered immunity: how animals sense pathogens. Nature Reviews. Immunology. 2013;13:199–206. doi: 10.1038/nri3398. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Suh J, Hutter H. A survey of putative secreted and transmembrane proteins encoded in the C. elegans genome. BMC Genomics. 2012;13:333. doi: 10.1186/1471-2164-13-333. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Sulston JE, Schierenberg E, White JG, Thomson JN. The embryonic cell lineage of the nematode Caenorhabditis elegans. Dev Biol. 1983;100:64–119. doi: 10.1016/0012-1606(83)90201-4. [DOI] [PubMed] [Google Scholar]
  49. Texier C, Vidau C, Vigues B, El Alaoui H, Delbac F. Microsporidia: a model for minimal parasite-host interactions. Curr Opin Microbiol. 2010;13:443–449. doi: 10.1016/j.mib.2010.05.005. [DOI] [PubMed] [Google Scholar]
  50. C. elegans Sequencing Consortium Genome sequence of the nematode C. elegans: a platform for investigating biology. Science. 1998;282:2012–2018. doi: 10.1126/science.282.5396.2012. [DOI] [PubMed] [Google Scholar]
  51. Thomsen LE, Slutz SS, Tan MW, Ingmer H. Caenorhabditis elegans is a model host for Listeria monocytogenes. Appl Environ Microbiol. 2006;72:1700–1701. doi: 10.1128/AEM.72.2.1700-1701.2006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Tour O, Meijer RM, Zacharias DA, Adams SR, Tsien RY. Genetically targeted chromophore-assisted light inactivation. Nat Biotechnol. 2003;21:1505–1508. doi: 10.1038/nbt914. [DOI] [PubMed] [Google Scholar]
  53. Troemel ER. New models of microsporidiosis: infections in zebrafish, C. elegans, and honey bee. PLoS Pathog. 2011;7:e1001243. doi: 10.1371/journal.ppat.1001243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Troemel ER, Félix MA, Whiteman NK, Barriere A, Ausubel FM. Microsporidia are natural intracellular parasites of the nematode Caenorhabditis elegans. PLoS Biol. 2008;6:2736–2752. doi: 10.1371/journal.pbio.0060309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Wilkins C, Dishongh R, Moore SC, Whitt MA, Chow M, Machaca K. RNA interference is an antiviral defence mechanism in Caenorhabditis elegans. Nature. 2005;436:1044–1047. doi: 10.1038/nature03957. [DOI] [PubMed] [Google Scholar]
  56. Woolhouse ME, Webster JP, Domingo E, Charlesworth B, Levin BR. Biological and biomedical implications of the co-evolution of pathogens and their hosts. Nat Genet. 2002;32:569–577. doi: 10.1038/ng1202-569. [DOI] [PubMed] [Google Scholar]
  57. Zhang Y, Lu H, Bargmann CI. Pathogenic bacteria induce aversive olfactory learning in Caenorhabditis elegans. Nature. 2005;438:179–184. doi: 10.1038/nature04216. [DOI] [PubMed] [Google Scholar]
  58. Zugasti O, Ewbank JJ. Neuroimmune regulation of antimicrobial peptide expression by a noncanonical TGF-beta signaling pathway in Caenorhabditis elegans epidermis. Nat Immunol. 2009;10:249–256. doi: 10.1038/ni.1700. [DOI] [PubMed] [Google Scholar]

Associated Data

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Supplementary Materials

Supp Table 1

Pathogens tested for intracellular infection in C. elegans and features of immune response.

NIHMS545482-supplement-Supp_Table_1.xlsx (40.3KB, xlsx)

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