A UPR-independent infection-specific role for a BiP/GRP78 protein in the control of antimicrobial peptide expression in C. elegans epidermis

The nematode C. elegans responds to infection by the fungus Drechmeria coniospora with a rapid increase in the expression of antimicrobial peptide genes. To investigate further the molecular basis of this innate immune response, we took a two-dimensional difference in-gel electrophoresis (2D-DIGE) approach to characterize the changes in host protein that accompany infection. We identified a total of 68 proteins from differentially represented spots and their corresponding genes. Through class testing, we identified functional categories that were enriched in our proteomic data set. One of these was “protein processing in endoplasmic reticulum,” pointing to a potential link between innate immunity and endoplasmic reticulum function. This class included HSP-3, a chaperone of the BiP/GRP78 family known to act coordinately in the endoplasmic reticulum with its paralog HSP-4 to regulate the unfolded protein response (UPR). Other studies have shown that infection of C. elegans can provoke a UPR. We observed, however, that in adult C. elegans infection with D. coniospora did not induce a UPR, and conversely, triggering a UPR did not lead to an increase in expression of the well-characterized antimicrobial peptide gene nlp-29. On the other hand, we demonstrated a specific role for hsp-3 in the regulation of nlp-29 after infection that is not shared with hsp-4. Epistasis analysis allowed us to place hsp-3 genetically between the Tribbles-like kinase gene nipi-3 and the protein kinase C delta gene tpa-1. The precise function of hsp-3 has yet to be determined, but these results uncover a hitherto unsuspected link between a BiP/GRP78 family protein and innate immune signaling.


Introduction
The nematophagous fungus Drechmeria coniospora infects various species of nematodes. Its spores adhere to the surface of a worm, germinate and perforate the cuticle. The worm's body is then totally invaded by the fungus, rapidly causing death (reviewed by Engelmann and Pujol 1 ). When D. coniospora infects Caenorhabditis elegans this triggers the expression of a large number of genes including those encoding antimicrobial peptides (AMPs) of the NLP family. [2][3][4] The induction of several nlp genes is dependent upon a protein kinase C delta (PKC∂)/p38 MAPK pathway that can be activated in the epidermis either by infection or by sterile wounding. 5 In both cases, signaling passes via TPA-1, a PKC∂ that acts upstream of TIR-1, the nematode ortholog of SARM, and a MAPK cassette constituted of a MAP3K (NSY-1), MAP2K (SEK-1) and the p38 MAPK PMK-1. This then acts upstream of the STAT-like transcription factor STA-2 to regulate nlp gene expression. 6 The elements that contribute to signaling upstream of TPA-1 have only been partially characterized. Wounding and infection require G-protein signaling upstream of TPA-1, while infection specifically involves the Tribbles-like kinase NIPI-3. 5,7 Part of the innate defenses against intestinal pathogens and toxins are also mediated by a p38 MAPK cascade that shares many but not all of the elements that act in the epidermis; 8-14 reviewed by Partridge et al. 15 and Coleman and Mylonakis. 16 Intestinal infection or exposure to bacterial toxins can also induce an unfolded protein response (UPR); this too is linked to the p38 pathway. 17,18 The UPR in C. elegans is divided into constitutive and inducible pathways, the former being essential during development. 19,20 Part of the UPR involves activation of the endoribonuclease IRE-1 that leads to the production of an alternatively spliced isoform of the mRNA of the transcription factor XBP-1. 21 Compounds such as thapsigargin, dithiothreitol and tunicamycin that perturb endoplasmic reticulum (ER) homeostasis trigger a UPR and lead to IRE-1 activation. The subsequent production of the specific form of XBP-1 then leads to the expression throughout the organism of a large number of genes, many involved in metabolism, or the secretory pathway, including chaperones. [19][20][21] Feeding worms the bacterial pore-forming toxin Cry5B also activates IRE-1 and upregulates chaperone expression specifically in the intestine. This requires the p38 MAPK signaling cassette. 17 The Gram-negative bacterial pathogen Pseudomonas aeruginosa also induces IRE-1-mediated splicing of xbp-1 mRNA in larvae and consequent chaperone gene expression, in a p38 MAPKdependent manner. 18 As the UPR-deficient xbp-1 mutants arrest as larvae when cultured on P. aeruginosa, 18 in this case, it was suggested that the ER cannot cope with the combined developmental and defense demands placed upon it (reviewed in Ewbank and Pujol 22 ).
In addition to the UPR-mediated changes in protein maturation, turnover and trafficking, the innate immune response may also affect the activity, post-translational modification and subcellular localization of signal transduction proteins. These can be analyzed at a global level through proteomic approaches. Indeed, there have already been a number of informative studies addressing the changes in the proteome that accompany infection of C. elegans by several different bacterial pathogens. [23][24][25] To extend our characterization of the response of C. elegans to D. coniospora we have now compared the proteomes of infected and control worms using two-dimensional difference gel electrophoresis (2D-DIGE). This fluorescence-based method allows two different protein samples tagged with two distinct fluorescent dyes to be run on the same gel, thereby improving comparative quantitation. We decided to focus on a single time point, early in the infection, with the hope of detecting changes in proteins involved in signal transduction, rather than finding proteins altered by the pathophysiological consequences of infection. We found that few changes were detected in whole extracts, but after fractionation we detected changes in many proteins. For one of these candidates, a C. elegans BiP/GRP78 homolog, we defined a novel role in the regulation of AMP gene expression.

Results
Protein fractionation reveals changes in the proteome induced by fungal infection. The infection of C. elegans by D. coniospora induces significant changes in gene expression within a matter of hours 26 (and unpublished data). We used a standard 2D-DIGE approach to identify alterations at the protein level in C. elegans after 5 h of infection with D. coniospora. As with any 2D gel approach, with DIGE it is appropriate to refer to changes in representation, rather than stating that a protein is more or less abundant, unless all protein spots are identified and quantified. With whole animal extracts (FT), when we used a narrow-range pH gradient for isoelectric focusing, although 890 protein spots were detected, we observed no differences between extracts of infected and control worms. With a broad-range pH gradient that allowed 1,478 spots to be resolved, just three differentially represented proteins were detected. Only one of these was present in sufficient quantities to allow identification; it corresponded to the galectin LEC-6 (see Materials and Methods for access to data). We therefore adopted a more laborious approach, separating the extracts into four fractions (F1, F2, F3 and FNS), and performing DIGE as above for each one. The fractionation allowed many more spots to be detected (9,246 in total), and revealed differences in intensity for 67 and 103 spots in the narrow-and broad-range pH gradient gels, respectively. All these spots were excised and analyzed by mass-spectrometry, leading to an identification of a protein from 98 spots ( Tables 1 and 2). This clearly illustrates the interest of combining DIGE with a prior protein fractionation approach.
Classification of differentially represented proteins. In some cases, the same protein was identified from more than one spot, either within the same fraction on the same gel, or from different fractions and/or gels. As a consequence, the 98 characterized spots corresponded to 67 individual proteins that were differentiallyrepresented between infected and control worms ( Table 3). We used WormMart 27 (WS220) to match each of the 68 identified proteins (LEC-6 and the 67 others) with its corresponding C. elegans gene ( Table 3). They fall into many different structural and functional classes (Table S1). We therefore performed two complementary bioinformatic analyses to find common themes. We first used the KEGG database 28 to determine whether there was an over-representation of higher-level systemic functions within the list of 68 genes. The most populated categories ( Table 3) were "implicated in a metabolic pathway" (13 genes), and "protein processing in endoplasmic reticulum" (8 genes). We then used EASE, 29 with our extensive in-house annotations culled from the C. elegans literature and referenced to WS220. 4 There were 24 functional classes identified as significantly enriched (p , 0.001, Fisher exact test; see Materials and Methods). Among these classes, 6 were related to the response of C. elegans to infection, with a further 10 linked to aging and stress-resistance, including to the insulin/DAF-2 pathway (Tables 4 and S2). Given the intimate connection between stress-resistance and susceptibility to infection, a part of the observed protein changes could thus be directly or indirectly associated with an innate immune response. The EASE analysis also revealed a potential connection with protein processing in the endoplasmic reticulum, as had been seen with KEGG.
Fungal infection in adults does not provoke the UPR. The proteins linked to protein processing in the endoplasmic reticulum included the calreticulin CRT-1, the protein disulphide  isomerase PDI-2 and HSP-3. HSP-3 and the closely related HSP-4 represent the worm's BiP/GRP78 homologs. All these proteins function in the ER to ensure the correct folding of nascent polypeptides and are important components of the UPR. Given the reported link between the UPR and resistance to bacterial toxins and infection, 17,18,22,30,31 we decided to investigate whether the UPR is involved in the host response to D. coniospora infection.
A direct measure of the activation of the UPR is provided by the detection of a specific UPR-associated alternatively spliced isoform of the transcription factor XBP-1. In contrast to the splicing of xbp-1 observed when young adult worms were treated with the UPR-inducing drug tunicamycin, the alternatively spliced isoform of xbp-1 was not detected following D. coniospora infection (Fig. 1A). Another indicator of the UPR is an increased expression of hsp-3 and hsp-4. In C. elegans, the UPR is often monitored in vivo using a phsp-4::GFP transgene reporter, which has a lower constitutive expression and higher level of induction during a UPR than phsp-3::GFP. 32 In contrast to tunicamycintreated worms, there was neither induction of an phsp-4::GFP transgene reporter after infection (Fig. 1B), nor increase of the hsp-4 transcript as measured by qRT-PCR (data not shown). This is consistent with previous genome-wide transcriptome studies that found that the expression of hsp-4 (and hsp-3) was not significantly altered following infection with D. coniospora. 3,4 When worms carrying a pnlp-29::GFP transgene reporter were exposed to tunicamycin, strong GFP expression was observed in young larvae. This is consistent with a previous microarray study that reported the induction of a number of epidermal AMP genes, including nlp-29, in L2 larvae treated with tunicamycin. 20 A marked increase in reporter gene expression was also seen in young larvae carrying a pnlp-30::GFP reporter transgene. On the other hand, no induction of either of these reporters was seen in L4 or adult worms ( Fig. 1B and data not shown). Similar results were obtained using the UPR-inducing agents dithiothreitol and thapsigargin (data not shown).
We also tested whether direct activation of effector genes in the epidermis would trigger a UPR. PMA activates the PKCd TPA-1 that controls multiple AMP genes, including nlp-29. 7 It provokes very high levels of AMP gene expression within 4 h (unpublished results). Treating worms with PMA for 5 h did not lead to splicing of xbp-1 nor to induction of hsp-4 or the phsp-4::GFP transgene reporter (Fig. 1A, data not shown). The expression of many epidermal genes, including some AMPs, is strongly upregulated by osmotic stress. 3,33 Although exposure to high salt did induce a pnlp-29::GFP transgene reporter as expected, it did not cause a measurable increase in phsp-4::GFP expression (Fig. 1B). Thus, neither fungal infection nor the strong induction of gene expression by PMA or salt provokes a UPR, and conversely a UPR does not trigger the expression of anti-fungal immune effectors in adult C. elegans. hsp-3 regulates nlp-29 AMP gene expression. While the results described above suggested that the UPR did not play a direct role in the antifungal innate immune response, the representation of a number of ER-resident proteins is modulated by infection. This led us to assay directly the role of the corresponding genes in the regulation of nlp-29 by RNAi. While several of the tested genes had an effect (results not shown), hsp-3 stood out for its strong effect, essentially totally blocking the induction of pnlp-29::GFP normally observed upon infection in adult worms ( Fig. 2A). A similar abrogation of reporter gene expression was seen in an atf-6 mutant, but not in a pek-1 mutant background (Fig. S1).
The mRNA sequence of the second BiP/GRP78 gene in C. elegans, hsp-4, is highly similar to that of hsp-3 (1472/1873 nucleotides identical, including several contiguous stretches of more than 21 nucleotides) and would thus be predicted to be targeted by the hsp-3 RNAi construct. At the same time, there is a reciprocal control of hsp-3 and hsp-4, such that a decrease in hsp-3 expression normally leads to an increase in the level of hsp-4, and vice versa. 34 As RNAi with hsp-4 did block pnlp-29::GFP induction upon infection in adult worms (data not shown), we sought to discriminate between the two genes using available null mutants. We observed a strong reduction in pnlp-29::GFP expression only in an hsp-3 mutant, not in an hsp-4 mutant background (Fig. 2B). Attempts to establish a hsp-3;hsp-4 strain were confounded by the fact that homozygous double mutants were sterile. When we inactivated hsp-3 by RNAi in the hsp-4 mutant background, the adult worms were sterile, and the induction of pnlp-29::GFP expression upon infection was blocked (data not shown).
In C. elegans, fertility and pathogen resistance are interlinked, via the FOXO transcription factor DAF-16, 35,36 which also plays a role in the UPR. 37 We therefore assayed the effect of hsp-3 RNAi on pnlp-29::GFP expression in a daf-16 mutant background. Loss of daf-16 had no effect on the abrogation of pnlp-29::GFP expression provoked by hsp-3 RNAi, or by RNAi with the STATlike transcription factor sta-2, previously characterized for its role in nlp-29 expression 6 ( Fig. 2A), indicating that the effect of hsp-3, and of sta-2, is independent of daf-16.
We then determined the specificity of the effect of hsp-3 on reporter gene expression. In clear contrast to the near-complete block of pnlp-29::GFP expression after infection, in an hsp-3 mutant the induction of the reporter gene was at least as strong as in the wild-type background when triggered by PMA, salt or wounding. In the hsp-4 mutant, however, no effect was seen under any of the experimental conditions (Fig. 2B). These results underline the specific role hsp-3 plays in regulating pnlp-29::GFP only after infection, and place hsp-3 genetically upstream of, or parallel to, the PKCd TPA-1.
hsp-3 acts downstream of nipi-3 to regulate nlp-29 AMP gene expression. The only previously known component of the innate immune signaling pathways that regulates nlp-29 expression specifically upon infection is the Tribbles-like kinase nipi-3. Overexpression of nipi-3 leads to an induction of pnlp-29::GFP. 5 This induction was blocked in the hsp-3 mutant background, placing hsp-3 genetically downstream of nipi-3 (Fig. 3A). Consistent with this result, hsp-3 did not block the increased expression of pnlp-29::GFP provoked by an activated form of GPA-12 that triggers TPA-1 independently of NIPI-3 7 (Fig. 3B). Together these results indicate that hsp-3 acts between nipi-3 and tpa-1 to control the expression of nlp-29 upon fungal infection (Fig. 4).

Discussion
Much of our previous characterization of the innate immune response of C. elegans to D. coniospora has been focused on the host transcriptional changes that accompany infection. In Drosophila, the expression of many components of immune signaling pathways are themselves highly regulated upon infection. 38,39 In contrast, none of 18 genes known to influence nlp-29 expression, including the p38 MAPK cascade components nsy-1, sek-1 and pmk-1, show a marked change in their expression level after infection. 4 In a previous DIGE-based pilot study, we identified RACK-1 as a factor involved in the regulation of anti-fungal defenses. 7 In an attempt to identify additional candidates, we extended the approach and undertook a comprehensive proteomic study of the changes that accompany fungal infection.
A number of other comparative gel-based proteomic studies have been performed using C. elegans 40,41 including two looking at protein changes upon bacterial infection of the intestine. 23,24 It is striking that certain proteins, such as ACT-4, SODH-1, VHA-13 and PDI-2, appear in almost every published list. This may reflect an intrinsic bias in the approach, since the measured expression level 4 for the genes corresponding to the proteins that we identified as differentially represented was very significantly higher than that of genes in general, (71.7% . 5 dcpm vs. 6.6% for all transcripts; p , 0.001 binomial test). It may also result from the fact that all these analyses used whole-animal extracts, potentially masking tissue-specific biologically relevant variations in protein abundance, and underlines the interest for developing efficient and simple methods to allow protein extraction from a specific C. elegans tissue.
If a particular spot on a gel increases or decreases in intensity, one cannot always infer that the total level of the corresponding protein was changed. For example, post-translational modifications may render a protein more difficult to extract, so that spot intensity does not reflect protein abundance. Similarly, posttranslational modifications may also lead to an alteration of the sub-cellular localization of a protein, which may cause a protein to be found in different extraction fractions, and thereby affect spot intensity. As many proteins give rise to multiple spots, generally because of post-translational modifications, only if all the spots for a given protein were identified and quantified from each fraction would one be able to quantitate protein abundance. The development of alternative methods, such as metabolic labeling coupled to mass spectrometry holds considerable promise for profiling changes during pathogenic challenge in C. elegans. 25 In the meantime, caution needs to be exercised when analyzing gel-based proteomic results. For this reason, until we have further functional evidence for a role in innate immunity for the various candidate proteins we identified, any discussion of a putative role would be premature.
The exception is HSP-3, which clearly has a specific function in regulating AMP gene expression. The hsp-3 gene is expressed at a high-level and is unchanged by infection, 4 so the total level of HSP-3 may not change upon infection. We have not established the change in HSP-3 (e.g., degradation, phosphorylation, etc.) that leads to a change in intensity of the corresponding spot. It is noteworthy that in an atf-6 mutant background there is a marked reduction of pnlp-29::GFP expression after infection. This may reflect a role for ATF-6 in regulating HSP-3 levels.
During development, hsp-3 has an unambiguous role in the UPR. 19,42 The data presented here indicate that the immune function of hsp-3 is independent of its function in the UPR. There is, however, evidence for a link between the UPR and AMP gene regulation in larvae. As mentioned above, a number of genes, including cnc-4, fipr-26, nlp-28 and nlp-29 are induced in L2 larvae after treatment with tunicamycin, apparently independently of xbp-1. 20 But although tunicamycin does provoke upregulation of pnlp-29::GFP in larvae, D. coniospora infection does not induce phsp-4::GFP expression either in larvae or adults. Further, this UPR-induced expression of pnlp-29::GFP is independent of the p38 MAPK pathway, as it is observed in pmk-1 mutant background, as well as in a tpa-1 and nipi-3 mutant backgrounds (data not shown), and overall, there is only a minimal overlap between the genes upregulated by tunicamycin and D. coniospora infection. 3,20 So the relationship between anti-fungal innate immunity and the UPR is not straightforward.
It is interesting, nonetheless, to speculate on how HSP-3 might exert its influence on AMP expression. There are several plausible models that are based on the idea that although genetically hsp-3 is positioned between nipi-3 and tpa-1, it seems unlikely that it plays a direct role in signal transduction. HSP-3 might be needed to ensure the correct intracellular localization of NIPI-3, itself, or of a protein that acts downstream of NIPI-3 and upstream of TPA-1. It may therefore be worthwhile to look at NIPI-3 localization in wild-type and hsp-3 mutant worms. The presence of two almost identical BiP/GRP78 proteins in the nematode is intriguing, as mammals, for example, only have one. It is conceivable that HSP-3 has a UPR-independent function outside the ER. Interestingly, one of the areas of sequence divergence between the two proteins is at the C-terminus; where HSP-3 has the ER retention signal KDEL sequence, HSP-4 has HDEL.
There are many examples of heat shock proteins playing a more or less direct role in innate immune responses. 43 For example, they can function as endogenous danger signals to indicate cell stress and tissue damage to the immune system. As another example, the conserved SGT1/HSP90 complex binds NLR proteins, and modulates innate immune signaling in plants and animals, 44 although it should be noted that there are no obvious NLR proteins in C. elegans. A study of the intracellular localization of HSP-3, and of the consequences of artificially expressing it in the cytoplasm could be merited. It is interesting to note that in an hsp-3 mutant there is some residual induction of the nlp-29 reporter gene, but this is fully abolished if the mutants are subject to RNAi against hsp-4 (C.C., unpublished observations). On the other hand, we have shown that loss of hsp-4 function alone has no effect on nlp-29 reporter gene expression. This suggests that hsp-4 can partially compensate for the absence of hsp-3.

www.landesbioscience.com Virulence
It has been shown that during C. elegans development, the activation of a p38 MAPK pathway that follows intestinal infection with the P. aeruginosa strain PA14 causes a UPR. 18 This is believed to be a consequence of the increased expression of innate immune effectors that overload the protein folding machinery in the ER. The results we have presented here show that infection of adult C. elegans by D. coniospora and the resultant induction of a large number of defense proteins in the epidermis does not provoke a UPR. This might reflect a relatively low constitutive activity of the secretory pathway in the epidermis, and therefore a buffering capacity in adult animals to cope with the consequences of infection. It will be interesting to dissect further the complex interplay between developmental, physiological (e.g., production of digestive enzymes in the intestine) and induced processes that put stress on the ER, both in C. elegans and other organisms. Additional study is also required to understand fully the UPR-independent role of BiP/ GRP78 in innate immunity in C. elegans and to determine whether it might play any such role in other organisms.
Splicing of xbp-1. N2 worms were grown and maintained on NGM plates with OP50. When they reached the young adult stage, worms were infected with D. coniospora by transferring them to NGM/OP50 plates previously spread with a dense suspension of spores. These had been freshly harvested in M9 buffer. Otherwise uninfected young adult worms were tranferred onto NGM/OP50 plates containing 10 mg/mL or 20 mg/mL tunicamycin (Sigma) or 1 mg/mL PMA (Sigma). After 5 h worms were harvested and RNA extracted with Trizol as described. 46 Reverse transcription used High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) and PCR analyses were performed as described. 17 Samples were normalized by Q-PCR as described 26 with eft-2 17 as an internal control.
RNAi. All RNAi feeding experiments were performed essentially as described, 47 using clones from the Ahringer library. All RNAi clones were sequence verified before use. The experiments were performed with worms cultured on OP50 until the L2 stage.
Infection, wounding, osmotic stress, PMA stress. Infection, and wounding were performed as described. 5 For exposure to osmotic stress, PMA and tunicamycin, compounds were added to NGM plates to a final concentration of 300 mM for NaCl, 1 mg/mL for PMA and 10 or 20 mg/mL for tunicamycin. Worms were grown and maintained on NGM plates with OP50. When they reached the young adult stage, worms were transferred onto the appropriate modified NGM/OP50 plates. Similar conditions were used to assay the induction of GFP expression in the strains IG274 and IG1320 (shown in Fig. 1B), except images were taken after only 5 h.
Biosorter. The quantification of fluorescent reporter gene (GFP) expression was performed with the COPAS Biosort (Union Biometrica), essentially as described. 26 Generally, animals were analyzed for length (time of flight), optical density (extinction), green fluorescence, and red fluorescence (if appropriate).
Protein extraction. A synchronized population of L4 IG274 worms was infected with D. coniospora. After 5 h, when there was a clear induction of GFP, indicative of the innate immune response to a productive infection, worms were harvested by washing plates with M9 buffer. Worms were pelleted by decantation, washed twice in the M9 buffer, then twice with PBS buffer. Proteins were extracted from a pellet of 500 mL of worms either by sonication in 8 M urea, 2 M thiourea, 4% (w/v) CHAPS, pH 8.5, containing a phosphatase inhibitor cocktail (Roche), or using the 2D fractionation kit (Amersham) according to the manufacturer's instructions.
Protein fractionation, labeling, gel electrophoresis and identification. Full experimental details are publically available at http://miapegeldb.expasy.org/experiment/118. The comprehensive set of analytical data from this study is available at the World-2DPAGE database http://world-2dpage.expasy.org/ repository/0042.

Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.