Microbial pathogenesis and host defense in the nematode C. elegans

Abstract

Epithelial cells line the surfaces of the body, and are on the front lines of defense against microbial infection. Like many other metazoans, the nematode C. elegans lacks known professional immune cells and relies heavily on defense mediated by epithelial cells. New results indicate that epithelial defense in C. elegans can be triggered through detection of pathogen-induced perturbation of core physiology within host cells and through autophagic defense against intracellular and extracellular pathogens. Recent studies have also illuminated a diverse array of pathogenic attack strategies used against C. elegans. These findings are providing insight into the underpinnings of host/pathogen interactions in a simple animal host that can inform studies of infectious diseases in humans.

Introduction

Epithelial cells cover the internal and external body surfaces and thus are often the first responders to pathogenic attack by microbes [1,2]. The significance of epithelial cells for immunity in humans is increasingly appreciated, and epithelial cells are now realized to be key players in defense against infection of the lung, skin and intestine. Furthermore, inappropriate activation of epithelial cell immune pathways can lead to inflammatory diseases, and thus understanding the immune pathways in these cells is of critical importance for human health. Epithelial cells, especially those of the intestine, are in regular contact with a wide array of microbes that include pathogenic microbes that cause disease, as well as innocuous or even beneficial microbial species. Therefore, epithelial cells have the challenge not just of how to discriminate self from non-self (a classic question in immunology), but also how to discriminate pathogen from non-pathogen. Canonical innate immune pathways triggered by pattern-recognition receptors (PRRs) are not sufficient to provide this distinction [3]. PRRs detect molecules associated with broad classes of microbes – these molecules were originally called pathogen-associated molecular patterns, or PAMPs, but they are now commonly called microbe-associated molecular patterns, or MAMPs, in keeping with the idea that they can be found in both pathogenic and non-pathogenic microbes [4,5] (Figure 1). Understanding how hosts distinguish pathogens from other microbes is a rapidly developing field within the area of infection and innate immunity, and is particularly relevant for epithelial cell defense [5,6].

Figure 1. Model for diverse triggers of defense gene expression in response to infection.

C. elegans uses effector-triggered immunity, as well as DAMP-triggered immunity, to upregulate defense gene expression in response to infection. MAMP-triggered immunity is well-described for other hosts, but has not yet been described for C. elegans.

Background on C. elegans infections and host defense

Many animal species lack professional immune cells, and yet thrive in a diverse microbial world by relying on defense by epithelial cells. The nematode Caenorhabditis elegans is one such species, and indeed nematodes are among the most numerous animals on the planet [7]. Many different microbial pathogens have been shown to attack and induce a defense response in the epithelial cells of C. elegans [8-16]. Pseudomonas aeruginosa is an opportunistic bacterial pathogen of humans and the most commonly studied pathogen in C. elegans, where it causes a lethal infection of intestinal epithelial cells [17]. In addition, several other bacterial, fungal and viral pathogens can infect the C. elegans intestine, and penetrating fungal species can infect epithelial cells of the epidermis. C. elegans has no known dedicated, migratory immune cells like macrophages to aid in defense against infection of the intestine or epidermis, and does not appear to have canonical cytokine and chemokine signaling pathways used to recruit those cells. However, C. elegans does use system-wide signaling to respond to stress and infection by upregulating defense pathways in epithelial cells, which is a topic that has been covered in other reviews [9,12,18-21].

C. elegans provides a powerful model system to address questions about innate immune pathways that are independent of classic PRR/MAMP signaling: C. elegans lacks components of some of the PRR pathways used by other metazoans, and it has yet to be shown to respond to MAMPs. In particular, C. elegans does not have an obvious NFκB ortholog, nor does it have Nod-like receptors (NLRs), and its single Toll-like receptor (TLR) does not play a substantial role in defense [14,22]. Interestingly, these signaling components are found in cnidaria, a clade that includes coral, jellyfish and hydra. Like C. elegans, these animals have outer and inner epithelial layers without known dedicated immune cells [23], so the presence of these immune signaling components does not correlate with the presence of a professional immune system. Given the evolutionary relationships among these animals, C. elegans most likely lost these genes during evolution, and presumably other pathways have been able to compensate for their role. Importantly, C. elegans does have a robust inducible defense system. In response to both intestinal and epidermal infection, C. elegans epithelial cells upregulate secreted antimicrobial peptides, detoxifying enzymes and efflux pumps, with distinct responses to distinct pathogens [24]. While some of this transcriptional response might be due to MAMP detection in C. elegans [25-27], it is clear that other signals from pathogens trigger a substantial part of the C. elegans transcriptional response to infection [28-30]. Previous studies of the inducible transcriptional response to infection have indicated that several signaling pathways control these responses, but one central pathway is a p38 MAP kinase (MAPK) pathway that includes a p38 MAPK called PMK-1 [31]. The PMK-1 p38 kinase cascade is an evolutionarily conserved pathway and is important for defense against microbial attack of both the C. elegans intestine and the epidermis. Several transcription factors have been shown to act downstream of PMK-1 in different contexts to control inducible defenses upon infection [32]. Other defense pathways operate in parallel to the p38 kinase cascade, including one regulated by the bZIP transcription factor ZIP-2 [8]. The upstream activators of these pathways, both pathogen-derived and host-derived, are just now being elucidated as described below.

Mechanisms of microbial pathogenesis and host defense in C. elegans have been reviewed previously [8-16]. Here we describe major developments from the last two years with a focus on bacterial infections, but also mention infections by other microbes when relevant. An emerging body of data suggests that nematodes monitor disruptions in cellular homeostasis as a means to detect pathogen infection and mount protective host responses. New data implicate these signals in the activation of conserved immune pathways, including the p38 pathway. In addition, several studies have implicated a conserved role for epithelial autophagy in C. elegans host defense against a broad array of pathogens. Finally, studies of bacterial pathogens have yielded insights both into the strategies employed by microbes to establish infection and the pathogen-encoded factors that lead to C. elegans immune pathway activation.

Surveillance or "effector-triggered" immunity induces host defense by monitoring core processes perturbed by pathogens

One feature that distinguishes pathogens from other microbes is their delivery of toxins and other effector molecules into host cells to disable core processes and pathways that might otherwise aid in defense. The immune responses to these attacks have been termed “effector-triggered” immunity or surveillance immunity, which is a concept that has been pioneered in plant immunity and more recently been appreciated in animal hosts, including C. elegans [4,5,33] (Figure 1).

A common mode of bacterial attack is to disable the process of host mRNA translation, which can prevent production of anti-microbial molecules and therefore improve bacterial survival. An effector-triggered immune pathway activated by inhibition of translation elongation was discovered in C. elegans through analysis of the transcriptional response to P. aeruginosa infection. Previous studies indicated that P. aeruginosa pathogenicity induced protective transcriptional responses mediated by the p38 pathway as well as by the ZIP-2 transcription factor [34]. More recently, it has been shown that a trigger for these pathways is inhibition of translation by the P. aeruginosa secreted Exotoxin A [35]. Studies by McEwan et al demonstrated that C. elegans detects the translation-blocking effects of this toxin, instead of directly detecting Exotoxin A molecular structure [36]. Furthermore, they showed that ZIP-2, as well as the p38 pathway and the G-protein-coupled receptor FSHR-1 provide defense against its toxic effects. In a parallel study, Dunbar et al used an RNAi screen and found that translational inhibition as well as perturbation of several other core processes, such as mitochondrial pathways and transcription-related pathways, could trigger ZIP-2-mediated defense gene expression [37]. They found that host endocytosis of Exotoxin A appeared to block translation specifically in the intestine, which then led to a paradoxical increase in levels of ZIP-2 protein via an upstream open reading frame that controls ZIP-2 expression. In mammals and flies, perturbation of translation has been shown to upregulate cytokine expression, demonstrating a broadly conserved link between surveillance of translation and immune responses [38,39]. In a related study, Melo and Ruvkun knocked down genes in core processes in C. elegans and observed induction of infection response genes and avoidance of normally attractant food, which is a characteristic behavioral response to infection that allows animals to avoid pathogenic microbes [40]. They found that inhibition of many core processes, such as translation, ATP synthesis, proteasome function, in the hypodermis and/or the intestine (common sites of infection) was sufficient to induce avoidance, and expression of defense genes. Altogether, these three studies indicated that C. elegans can sense perturbations of core processes as a method to detect pathogenic attack, and provided insight to a specific mechanism by which blockade of mRNA translation by the pathogen P. aeruginosa can induce host defense gene expression (Table 1, Figure 1).

Table 1.

Recent discoveries about microbial pathogens, their virulence factors, and the immune pathways they activate in C. elegans.

Infection	Virulence Factor /Attack	Immunity Triggers	C. elegans innate immune pathways	References
P. aeruginosa (Slow killing model)	Exotoxin A	Translational inhibition	ZIP-2, PMK-1, FSHR-1 dependent pathways	[36,37,40]
	unknown	Mitochondrial stress	ATFS-1, ZIP-2, and ceramide/mevalonate pathways	[37,40-42]
P. aeruginosa (Liquid killing model)	Pyoverdin (iron scavenging)	Hypoxia	HIF-1 associated defense response	[62]
P. aeruginosa (Fast killing model)	Phenazine-1- carboxylic acid	Oxidative stress	AGE-1 PI3K pathway	[60,61]
N. parisii	unknown	Proteasomal inhibition	Ubiquitylation-related pathways	[44]
D. coniospora	Wounding of the cuticle	HPLA	DCAR-1 receptor activation of PMK-1 pathway	[47]
S. aureus, P. aeruginosa (Slow killing model), S. enterica, N. parisii	unknown	unknown	Autophagy-mediated defense response (TFEB-1 mediated for S. aureus)	[44,56,57]

Mitochondrial function is essential for the organismal health, and there are many genera of bacteria found in the C. elegans natural habitat that induce mitochondria stress upon infection. As mentioned above, perturbation of mitochondrial pathways was able to induce aversive behavioral responses, as well as infection response gene expression [37,40]. Two new studies provide mechanistic insight about how mitochondrial surveillance pathways trigger this defense gene expression [41,42]. Pellegrino et al found an immune role for the bZIP transcription factor ATFS-1, as well as ZIP-2, in mitochondrial stress [42]. Previously, these authors had shown that during mitochondrial stress, ATFS-1 is prevented from trafficking to mitochondria, where it normally localizes, and instead moves to the nucleus where it induces the mitochondrial unfolded protein response [43]. More recently, these authors have shown that this ATFS-1 nuclear localization also occurs upon P. aeruginosa infection, and ATFS-1 can induce expression of anti-microbial peptides and secreted lysozyme as part of the innate immune response, acting together with the ZIP-2 transcription factor [42]. Liu et al showed that ceramide and mevalonate biosynthetic pathways are involved in mitochondrial surveillance responses, and ceramide appears to act upstream of ATFS-1. In addition, they demonstrated that certain bacterial species block the induction of defense gene expression triggered by mitochondrial surveillance. Thus, C. elegans uses ATFS-1, ZIP-2, ceramide and mevalonate surveillance pathways to detect disruption of mitochondrial function and induce immune responses.

Another core host system recently shown to be involved in the inducible response to infection in C. elegans is the ubiquitin proteasome system, which targets proteins for degradation. Two intracellular pathogens of C. elegans shown to infect worms in the natural environment are the Orsay virus and the fungal-like microsporidian pathogen Nematocida parisii. Surprisingly, these very distinct pathogens were found to induce very similar transcriptional responses in C. elegans, which were distinct from responses to other pathogens and were characterized by an upregulation of ubiquitin ligase components [44,45]. These ubiquitin ligase components provided defense against infection, and interestingly, their expression could also be induced by inhibition of proteasome function, suggesting that detection of these intracellular pathogens is related to surveillance of the core cellular process of proteasomal degradation [40,44].

Upstream activators of the p38 MAPK immune pathway are diverse and damage-associated

In plant defense, effector-triggered immunity can detect pathogen-induced changes to host cells before there is overt damage, and the same may hold true in animal defense (Figure 1). In addition, prolonged or extensive damage can also trigger defense against infection, through so-called "damage-associated molecular patterns" or DAMPs [46]. DAMPs in mammalian immunity include a diverse range of molecules, including ATP, interleukin 1α, uric acid, S100 cytoplasmic proteins, the nuclear protein HMGB1 and the extracellular matrix molecule hyaluronan. Key insights into DAMP-mediated activation of p38 signaling in C. elegans have come from recent studies of fungal infection, which previously had been shown to trigger a G-protein signaling cascade upstream of p38 in epidermal cells [32,47]. These signaling events can be activated by infection with the fungus Drechmeria coniospora, which penetrates the C. elegans cuticle outside the epidermis, and by sterile wounding of the epidermis, indicating that damage to the epidermis elicits an immune response. Zugasti et al used RNAi screening to identify a G-protein coupled receptor called DCAR-1 to be responsible for activating p38 signaling in both cases [47]. Furthermore, they identified an endogenous ligand called HPLA, which is a tyrosine derivative generated by infection, and likely also by wounding. Thus, HPLA can be considered a DAMP, and together with DCAR-1 provide the first-described ligand/receptor pair that induce immune responses in C. elegans. Future studies will likely investigate the mechanism by which HPLA is induced, and whether a similar signaling pathway exists in mammals.

Although less is known about the upstream activators of p38 in response to bacterial infection in C. elegans, a recent study indicated that this pathway can be activated by an exogenous compound to promote resistance against infection [48]. An earlier high-throughput screen for anti-infectives yielded several compounds that appeared to act on the host, instead of the pathogen [49]. Pukkila-Worley et al characterized one of these compounds and demonstrated that it provided protection against killing by P. aeruginosa infection. Although its exact molecular target is unknown, they demonstrated that this small molecule could activate genes that are also induced by P. aeruginosa infection and the p38 pathway, and that its effects on pathogen resistance were partially dependent on this pathway. Further studies by these authors identified the conserved Mediator subunit MDT-15/ MED15 to play a role in compound-mediated resistance through the p38 pathway [50]. Given that this compound is toxic to worms, these studies suggest bacterial activation of the intestinal p38 pathway may be due to pathogen-induced damage or perturbation of core physiology. Indeed, a wide variety of bacterial-associated stimuli appear to induce the p38 pathway. Recent discoveries indicate that the translation-blocking Shiga toxin from pathogenic E. coli (which has the same mechanism of action as Exotoxin A from P. aeruginosa) can induce the p38 pathway [51]. Furthermore, non-pathogenic soil-associated bacteria have been shown to induce immune responses via p38 signaling [52]. It is still unclear in these contexts the exact pathogenic triggers and how they are detected by C. elegans.

Autophagy mediates defense against a broad range of pathogenic microbes

Another form of immunity that can be triggered by damage is the cellular process of autophagy, or self-eating, and several recent studies in C. elegans have demonstrated a role for autophagy in defense against infection. Autophagy is the process of de novo membrane formation to engulf large, damaged cellular components for digestion and recycling. This process has gained attention for having a key role in defense against intracellular pathogens in professional immune cells as well as in epithelial cells [53]. Targeting intracellular pathogens for degradation via the autophagy pathway has been deemed 'xenophagy', and can be triggered by damage to intracellular membranes. In C. elegans, autophagy was first shown to have a role in defense against infection by the human bacterial pathogen Salmonella enterica, which is a facultative intracellular pathogen [54]. In these studies, Salmonella was only found intracellular in the C. elegans intestine if autophagy was compromised, indicating that C. elegans autophagy prevents invasion or replication within intestinal cells. However, a subsequent study did not confirm that inhibition of autophagy leads to intracellular Salmonella in the intestine, although it did show that autophagy was required in the intestine to promote survival upon infection [55]. Direct localization of C. elegans autophagy machinery was recently shown for the intracellular pathogen N. parisii, and autophagy played a role in controlling levels of this pathogen [44].

In addition to promoting defense against intracellular microbes, autophagy has recently been shown to promote defense against bacteria that are predominantly extracellular. In particular, Visvikis et al demonstrated that the conserved autophagy transcription factor TFEB-1 activated cytoprotective and anti-microbial gene expression in response to infection with the Gram-positive pathogen Staphylococcus aureus and promoted survival [56]. These authors found a similar role for TFEB-1 in response to S. aureus infections in mammalian cells. An unusual role for autophagy in defense was shown by Zou et al, who demonstrated that autophagy activation prevents necrosis, and provides defense against P. aeruginosa infection [57]. Interestingly, autophagy did not reduce pathogen load (i.e. increase resistance) in S. aureus or P. aeruginosa infections, but rather improved tolerance of pathogen infection [58]. Thus, these new findings indicate that autophagy promotes tolerance against extracellular pathogens by inducing defense gene expression and preventing infection-induced damage, in addition to promoting resistance by directly targeting intracellular pathogens for destruction (xenophagy) and lowering pathogen load.

New insights into strategies of pathogenic attack in C. elegans infections

Pathogens often rely on an extensive arsenal of virulence factors to attack their hosts, and they can deploy different factors under different conditions. One pathogen in particular that has a large number of virulence factors is P. aeruginosa, which can cause a range of different infections in humans. To explore the variety of virulence strategies used by P. aeruginosa, several pathogenesis models have been developed in C. elegans. All the studies mentioned above used the most common pathogenesis model for P. aeruginosa, which is the slow-killing model, where bacteria are grown on minimal media plates and fed to C. elegans. Under these conditions, killing of C. elegans requires the bacteria to be alive and involves accumulation of bacteria in the intestinal lumen of C. elegans. A recently described genome-wide screen with this model identified 170 genes required for virulence, although it did not identify secretion systems or obvious individual effector molecules [59]. This study together with others mentioned above indicate that individual virulence factors such as Exotoxin A are not required for gene induction or for killing, but are sufficient to induce gene expression and cause damage, implying there is extensive redundancy in the toxins deployed by P. aeruginosa under these slow-killing conditions [36,37].

Two other models for P. aeruginosa pathogenesis have been developed in C. elegans. One model is the "fast-killing" model, where growth of P. aeruginosa on nutrient-rich, high osmolarity plates leads to rapid killing of C. elegans. This killing does not require P. aeruginosa to be alive during exposure, does not involve accumulation of P. aeruginosa cells within the intestine, and appears to be caused by diffusible toxins. Previous work had implicated host oxidative stress responses and bacterial-derived phenazines as key factors in this infection model [60], and Cezairliyan et al used a combination of mutant, biochemical and metabolomic studies to demonstrate that the predominant toxin in this model is phenazine-1-carboxylic acid [61]. A new model for P. aeruginosa pathogenicity in C. elegans is the "liquid-killing" format, which identified a separate set of factors important for pathogenicity [62]. Kirienko et al showed that iron-scavenging by P. aeruginosa-derived pyoverdin is a virulence strategy, and can lead to a hypoxic crisis in C. elegans. The C. elegans ortholog of hypoxia-inducible factor (HIF-1) promoted survival upon infection in this model; HIF-1 has also been shown to regulate host resistance in other infection models [63,64]. Given that pyoverdin and phenazines have been implicated in P. aeruginosa killing in mammals, further analysis of these pathogenesis strategies in medically relevant infections is warranted.

In addition to toxin-based killing, a visually dramatic form of pathogenic attack was described by Hodgkin et al, who showed how Leucobacter bacteria attack the epidermal surface of C. elegans and use a trapping strategy to create "worm-stars", which are structures of dozens of worms captured by their tails radiating outward, preventing their mobility and leading to their early demise [65]. These studies focused on natural epidermal pathogens and found that C. elegans resistance to one strain caused by changes in surface glycosylation led to susceptibility to a second strain, and a sub-lethal infection with the second strain induced resistance to the first strain. Thus, they demonstrated the trade-offs that are often found in host defense against microbial infection, and also identified a novel mechanism by which bacterial pathogens can attack their hosts.

Conclusion

Discoveries in recent years have shown how C. elegans can detect pathogens not simply through the identification of their physical presence, but through detecting the common perturbations they cause in core processes to upregulate defense. Studies have also indicated that host autophagy serves to increase pathogen tolerance against extracellular pathogens, as well as pathogen resistance against intracellular pathogens. Recent findings have also highlighted the diversity of pathogenic strategies that bacteria use to attack C. elegans. This animal host provides a powerful model for further investigating pathogen attack and damage-triggered or effector-triggered immunity in epithelial cells, because it appears to be the foundation of defense for C. elegans.

Highlights.

• C. elegans detects infection with surveillance or "effector-triggered" immunity

• Multiple upstream inputs activate the p38 MAPK pathway

• Autophagy provides defense against intracellular and extracellular pathogens

• Pathogen attack involves toxins, as well as physical trapping of the host