Both the Caspase CSP-1 and a Caspase-Independent Pathway Promote Programmed Cell Death in Parallel to the Canonical Pathway for Apoptosis in Caenorhabditis elegans

Caspases are cysteine proteases that can drive apoptosis in metazoans and have critical functions in the elimination of cells during development, the maintenance of tissue homeostasis, and responses to cellular damage. Although a growing body of research suggests that programmed cell death can occur in the absence of caspases, mammalian studies of caspase-independent apoptosis are confounded by the existence of at least seven caspase homologs that can function redundantly to promote cell death. Caspase-independent programmed cell death is also thought to occur in the invertebrate nematode Caenorhabditis elegans. The C. elegans genome contains four caspase genes (ced-3, csp-1, csp-2, and csp-3), of which only ced-3 has been demonstrated to promote apoptosis. Here, we show that CSP-1 is a pro-apoptotic caspase that promotes programmed cell death in a subset of cells fated to die during C. elegans embryogenesis. csp-1 is expressed robustly in late pachytene nuclei of the germline and is required maternally for its role in embryonic programmed cell deaths. Unlike CED-3, CSP-1 is not regulated by the APAF-1 homolog CED-4 or the BCL-2 homolog CED-9, revealing that csp-1 functions independently of the canonical genetic pathway for apoptosis. Previously we demonstrated that embryos lacking all four caspases can eliminate cells through an extrusion mechanism and that these cells are apoptotic. Extruded cells differ from cells that normally undergo programmed cell death not only by being extruded but also by not being engulfed by neighboring cells. In this study, we identify in csp-3; csp-1; csp-2 ced-3 quadruple mutants apoptotic cell corpses that fully resemble wild-type cell corpses: these caspase-deficient cell corpses are morphologically apoptotic, are not extruded, and are internalized by engulfing cells. We conclude that both caspase-dependent and caspase-independent pathways promote apoptotic programmed cell death and the phagocytosis of cell corpses in parallel to the canonical apoptosis pathway involving CED-3 activation.     

Autophagy activity contributes to programmed cell death in Caenorhabditis elegans

The physiological relationship between autophagy and programmed cell death during C. elegans development is poorly understood. In C. elegans, 131 somatic cells and a large number of germline cells undergo programmed cell death. Autophagy genes function in the removal of somatic cell corpses during embryogenesis. Here we demonstrated that autophagy activity participates in germ-cell death induced by genotoxic stress. Upon γ ray treatment, fewer germline cells execute the death program in autophagy mutants. Autophagy also contributes to physiological germ-cell death and post-embryonic cell death in ventral cord neurons when ced-3 caspase activity is partially compromised. Our study reveals that autophagy activity contributes to programmed cell death during C. elegans development.     

Caenorhabditis elegans Myotubularin MTM-1 Negatively Regulates the Engulfment of Apoptotic Cells

During programmed cell death, apoptotic cells are recognized and rapidly engulfed by phagocytes. Although a number of genes have been identified that promote cell corpse engulfment, it is not well understood how phagocytosis of apoptotic cells is negatively regulated. Here we have identified Caenorhabditis elegans myotubularin MTM-1 as a negative regulator of cell corpse engulfment. Myotubularins (MTMs) constitute a large, highly conserved family of lipid phosphatases. MTM gene mutations are associated with various human diseases, but the cellular functions of MTM proteins are not clearly defined. We found that inactivation of MTM-1 caused significant reduction in cell corpses in strong loss-of-function mutants of ced-1, ced-6, ced-7, and ced-2, but not in animals deficient in the ced-5, ced-12, or ced-10 genes. In contrast, overexpression of MTM-1 resulted in accumulation of cell corpses. This effect is dependent on the lipid phosphatase activity of MTM-1. We show that loss of mtm-1 function accelerates the clearance of cell corpses by promoting their internalization. Importantly, the reduction of cell corpses caused by mtm-1 RNAi not only requires the activities of CED-5, CED-12, and CED-10, but also needs the functions of the phosphatidylinositol 3-kinases (PI3Ks) VPS-34 and PIKI-1. We found that MTM-1 localizes to the plasma membrane in several known engulfing cell types and may modulate the level of phosphatidylinositol 3-phosphate (PtdIns(3)P) in vivo. We propose that MTM-1 negatively regulates cell corpse engulfment through the CED-5/CED-12/CED-10 module by dephosphorylating PtdIns(3)P on the plasma membrane.     

Evolutionary conservation of a genetic pathway of programmed cell death

Genetic analysis of programmed cell death in Caenorhabditis elegans has led to the identification of 13 genes that constitute a developmental pathway of programmed cell death. Two of the three key genes in this pathway, ced-9, a cell death suppressor, and ced-3, a cell death inducer, were found to encode proteins that share structural and functional similarities with the mammalian proto-oncogene product Bcl-2 and interleukin-1β converting enzyme, respectively. These results suggest that the genetic pathway of programmed cell death may be evolutionarily conserved from worms to mammals. © 1996 Wiley-Liss, Inc.     

Lipid Droplet Protein LID-1 Mediates ATGL-1-Dependent Lipolysis during Fasting in Caenorhabditis elegans

Lipolysis is a delicate process involving complex signaling cascades and sequential enzymatic activations. In Caenorhabditis elegans, fasting induces various physiological changes, including a dramatic decrease in lipid contents through lipolysis. Interestingly, C. elegans lacks perilipin family genes which play a crucial role in the regulation of lipid homeostasis in other species. Here, we demonstrate that in the intestinal cells of C. elegans, a newly identified protein, lipid droplet protein 1 (C25A1.12; LID-1), modulates lipolysis by binding to adipose triglyceride lipase 1 (C05D11.7; ATGL-1) during nutritional deprivation. In fasted worms, lipid droplets were decreased in intestinal cells, whereas suppression of ATGL-1 via RNA interference (RNAi) resulted in retention of stored lipid droplets. Overexpression of ATGL-1 markedly decreased lipid droplets, whereas depletion of LID-1 via RNAi prevented the effect of overexpressed ATGL-1 on lipolysis. In adult worms, short-term fasting increased cyclic AMP (cAMP) levels, which activated protein kinase A (PKA) to stimulate lipolysis via ATGL-1 and LID-1. Moreover, ATGL-1 protein stability and LID-1 binding were augmented by PKA activation, eventually leading to increased lipolysis. These data suggest the importance of the concerted action of lipase and lipid droplet protein in the response to fasting signals via PKA to maintain lipid homeostasis.     

Genes Required for the Engulfment of Cell Corpses during Programmed Cell Death in Caenorhabditis Elegans

After programmed cell death, a cell corpse is engulfed and quickly degraded by a neighboring cell. For degradation to occur, engulfing cells must recognize, phagocytose and digest the corpses of dying cells. Previously, three genes were known to be involved in eliminating cell corpses in the nematode Caenorhabditis elegans: ced-1, ced-2 and nuc-1. We have identified five new genes that play a role in this process: ced-5, ced-6, ced-7, ced-8 and ced-10. Electron microscopic studies reveal that mutations in each of these genes prevent engulfment, indicating that these genes are needed either for the recognition of corpses by other cells or for the initiation of phagocytosis. Based upon our study of double mutants, these genes can be divided into two sets. Animals with mutations in only one of these sets of genes have relatively few unengulfed cell corpses. By contrast, animals with mutations in both sets of genes have many unengulfed corpses. These observations suggest that these two sets of genes are involved in distinct and partially redundant processes that act in the engulfment of cell corpses.     

DISSECT Method Using PNA-LNA Clamp Improves Detection of EGFR T790m Mutation

Non-small cell lung cancer (NSCLC) patients treated with small molecule EGFR inhibitors, such as gefitinib, frequently develop drug resistance due to the presence of secondary mutations like the T790M mutation on EGFR exon 20. These mutations may originate from small subclonal populations in the primary tumor that become dominant later on during treatment. In order to detect these low-level DNA variations in the primary tumor or to monitor their progress in plasma, it is important to apply reliable and sensitive mutation detection methods. Here, we combine two recently developed methodologies, Differential Strand Separation at Critical Temperature (DISSECT), with peptide nucleic acid-locked nucleic acid (PNA-LNA) polymerase chain reaction (PCR) for the detection of T790M EGFR mutation. DISSECT pre-enriches low-abundance T790M EGFR mutations from target DNA prior to implementing PNA-LNA PCR, a method that can detect 1 mutant allele in a background of 100–1000 wild type alleles. The combination of DISSECT and PNA-LNA PCR enables the detection of 1 mutant allele in a background of 10,000 wild type alleles. The combined DISSECT-PNA-LNA PCR methodology is amenable to adaptation for the sensitive detection of additional emerging resistance mutations in cancer.     

Identification of a Peptide Inhibitor of the RPM-1·FSN-1 Ubiquitin Ligase Complex

The Pam/Highwire/RPM-1 (PHR) proteins include: Caenorhabditis elegans RPM-1 (Regulator of Presynaptic Morphology 1), Drosophila Highwire, and murine Phr1. These important regulators of neuronal development function in synapse formation, axon guidance, and axon termination. In mature neurons the PHR proteins also regulate axon degeneration and regeneration. PHR proteins function, in part, through an ubiquitin ligase complex that includes the F-box protein FSN-1 in C. elegans and Fbxo45 in mammals. At present, the structure-function relationships that govern formation of this complex are poorly understood. We cloned 9 individual domains that compose the entire RPM-1 protein sequence and found a single domain centrally located in RPM-1 that is sufficient for binding to FSN-1. Deletion analysis further refined FSN-1 binding to a conserved 97-amino acid region of RPM-1. Mutagenesis identified several conserved motifs and individual amino acids that mediate this interaction. Transgenic overexpression of this recombinant peptide, which we refer to as the RPM-1·FSN-1 complex inhibitory peptide (RIP), yields similar phenotypes and enhancer effects to loss of function in fsn-1. Defects caused by transgenic RIP were suppressed by loss of function in the dlk-1 MAP3K and were alleviated by point mutations that reduce binding to FSN-1. These findings suggest that RIP specifically inhibits the interaction between RPM-1 and FSN-1 in vivo, thereby blocking formation of a functional ubiquitin ligase complex. Our results are consistent with the FSN-1 binding domain of RPM-1 recruiting FSN-1 and a target protein, such as DLK-1, whereas the RING-H2 domain of RPM-1 ubiquitinates the target.     

The engulfment receptor Draper is required for autophagy during cell death

Autophagy is a process to degrade and recycle cytoplasmic contents. Autophagy is required for survival in response to starvation, but has also been associated with cell death. How autophagy functions during cell survival in some contexts and cell death in others is unknown. Drosophila larval salivary glands undergo programmed cell death requiring autophagy genes, and are cleared in the absence of known phagocytosis. Recently, we demonstrated that Draper (Drpr), the Drosophila homolog of C. elegans engulfment receptor CED-1, is required for autophagy induction during cell death, but not during cell survival. drpr mutants fail to clear salivary glands. drpr knockdown in salivary glands prevents the induction of autophagy, and Atg1 misexpression in drpr null mutants suppresses salivary gland persistence. Surprisingly, drpr knockdown cell-autonomously prevents autophagy induction in dying salivary gland cells, but not in larval fat body cells following starvation. This is the first engulfment factor shown to function in cellular self-clearance, and the first report of a cell-death-specific autophagy regulator.Key words: autophagy, Draper, programmed cell death, engulfment, developmentProgrammed cell death is required for animal development and tissue homeostasis. Improper cell death leads to pathologies including autoimmunity and cancer. Several morphological forms of cell death occur during animal development, including apoptosis and autophagic cell death. Autophagic cell death is characterized by the presence of autophagosomes in dying cells that are not known to be engulfed by phagocytes. Autophagic cell death is observed during several types of mammalian developmental cell death, including regression of the corpus luteum and involution of mammary and prostate glands.During macroautophagy (autophagy), cytoplasmic components are sequestered by autophagosomes and delivered to the lysosome for degradation. Autophagy is a cellular response to stress required for survival in response to starvation. Whereas autophagy has been associated with cell death, it is unknown how autophagy is distinguished during cell death and cell survival. Autophagy is induced in Drosophila in response to starvation in the fat body where it promotes cell survival, while autophagy is induced by the steroid hormone ecdysone in salivary glands where it promotes cell death. This allows studies of autophagy in different cell types and in response to different stimuli.Drosophila larval salivary glands die with autophagic cell death morphology and autophagy is required for their degradation. Expression of the caspase inhibitor p35 enhances salivary gland persistence in Atg mutants, suggesting that caspases and autophagy function in parallel during salivary gland degradation. Either activation of caspases or Atg1 misexpression is sufficient to induce ectopic salivary gland clearance. We queried genome-wide microarray data from purified dying salivary glands and noted the induction of engulfment genes, those required for a phagocyte to consume and degrade a dying cell. We also noted few detectable changes in engulfment genes in Drosophila larvae during starvation.We found that Drpr, the Drosophila orthologue of C. elegans engulfment receptor CED-1, is enriched in dying salivary glands, and drpr null mutants have persistent salivary glands. Interestingly, whereas knockdown of drpr in phagocytic blood cells fails to influence salivary gland clearance, expression of drpr-RNAi in salivary glands prevents gland clearance. Drosophila drpr is alternatively spliced to produce three isoforms. We found that drpr-I-specific knockdown prevents salivary gland degradation and Drpr-I expression in salivary glands of drpr null mutants rescues salivary gland persistence. Therefore, drpr is autonomously required for salivary gland clearance. However, how Drpr is induced or activated during hormone-regulated cell death remains to be determined.drpr knockdown fails to influence caspase activation, and caspase inhibitor p35 expression in drpr null mutants enhances salivary gland persistence, suggesting that Drpr functions downstream or parallel to caspases in dying salivary glands. Interestingly, we found that drpr knockdown in salivary glands prevents the formation of GFP-LC3 puncta. Further, Atg1 misexpression in salivary glands of drpr null mutants suppresses salivary gland persistence. drpr is therefore required for autophagy induction in salivary glands, and Atg1 functions downstream of Drpr in this tissue. We found that several other engulfment genes are required for salivary gland degradation. However, the Drpr signaling mechanism leading to autophagy induction in salivary glands remains to be elucidated.We tested whether drpr is a general regulator of autophagy. The Drosophila fat body is a nutrient storage and mobilization organ akin to the mammalian liver, and is a well-established model to study starvation-induced autophagy. We found that drpr-RNAi expression in fat body clone cells fails to prevent GFP-Atg8 puncta formation in response to starvation. Similarly, drpr null fat body clone cells form Cherry-Atg8 puncta after starvation. Strikingly, drpr-RNAi expression in salivary gland clone cells inhibits the formation of GFP-Atg8 puncta. Therefore, drpr is cell-autonomously required for autophagy induction in dying salivary gland cells, but not for autophagy induction in fat body cells after starvation. These findings suggest that distinct signaling mechanisms regulate autophagy in response to nutrient deprivation compared to steroid hormone induction. Little is known about what distinguishes autophagy function in cell survival versus death. It is possible that varying levels of autophagy are induced during specific cell contexts and that high levels of autophagy could overwhelm a cell—leading to cell death. Autophagic degradation of specific cargo, such as cell death inhibitors, could also contribute to cell death.Given recent interest in manipulation of autophagy for therapies, it is possible that factors such as Drpr could be used as biomarkers to distinguish autophagy leading to cell death versus cell survival. While it is generally accepted that augmentation of protein clearance by autophagy during neurodegeneration would be beneficial, the role of autophagy in tumor progression is less clear. For example, monoallelic loss of the human Atg6 homolog beclin 1 is prevalent in human cancers, suggesting that autophagy is a tumorsuppressive mechanism. Thus, autophagy enhancers have been proposed for cancer prevention. However, autophagy occurs in tumor cells as a survival mechanism, and autophagy inhibitors have been proposed for anti-cancer therapies. Understanding how autophagy is regulated in different contexts is critical for appropriate therapeutic strategies.     

A bacterial cytotoxin identifies the RhoA exchange factor Net1 as a key effector in the response to DNA damage

Background

Exposure of adherent cells to DNA damaging agents, such as the bacterial cytolethal distending toxin (CDT) or ionizing radiations (IR), activates the small GTPase RhoA, which promotes the formation of actin stress fibers and delays cell death. The signalling intermediates that regulate RhoA activation and promote cell survival are unknown.

Principal Findings

We demonstrate that the nuclear RhoA-specific Guanine nucleotide Exchange Factor (GEF) Net1 becomes dephosphorylated at a critical inhibitory site in cells exposed to CDT or IR. Expression of a dominant negative Net1 or Net1 knock down by iRNA prevented RhoA activation, inhibited the formation of stress fibers, and enhanced cell death, indicating that Net1 activation is required for this RhoA-mediated responses to genotoxic stress. The Net1 and RhoA-dependent signals involved activation of the Mitogen-Activated Protein Kinase p38 and its downstream target MAPK-activated protein kinase 2.

Significance

Our data highlight the importance of Net1 in controlling RhoA and p38 MAPK mediated cell survival in cells exposed to DNA damaging agents and illustrate a molecular pathway whereby chronic exposure to a bacterial toxin may promote genomic instability.     

Mutations in Grxcr1 Are The Basis for Inner Ear Dysfunction in the Pirouette Mouse

Recessive mutations at the mouse pirouette (pi) locus result in hearing loss and vestibular dysfunction due to neuroepithelial defects in the inner ear. Using a positional cloning strategy, we have identified mutations in the gene Grxcr1 (glutaredoxin cysteine-rich 1) in five independent allelic strains of pirouette mice. We also provide sequence data of GRXCR1 from humans with profound hearing loss suggesting that pirouette is a model for studying the mechanism of nonsyndromic deafness DFNB25. Grxcr1 encodes a 290 amino acid protein that contains a region of similarity to glutaredoxin proteins and a cysteine-rich region at its C terminus. Grxcr1 is expressed in sensory epithelia of the inner ear, and its encoded protein is localized along the length of stereocilia, the actin-filament-rich mechanosensory structures at the apical surface of auditory and vestibular hair cells. The precise architecture of hair cell stereocilia is essential for normal hearing. Loss of function of Grxcr1 in homozygous pirouette mice results in abnormally thin and slightly shortened stereocilia. When overexpressed in transfected cells, GRXCR1 localizes along the length of actin-filament-rich structures at the dorsal-apical surface and induces structures with greater actin filament content and/or increased lengths in a subset of cells. Our results suggest that deafness in pirouette mutants is associated with loss of GRXCR1 function in modulating actin cytoskeletal architecture in the developing stereocilia of sensory hair cells.     

The C. elegans Opa1 homologue EAT-3 is essential for resistance to free radicals

The C. elegans eat-3 gene encodes a mitochondrial dynamin family member homologous to Opa1 in humans and Mgm1 in yeast. We find that mutations in the C. elegans eat-3 locus cause mitochondria to fragment in agreement with the mutant phenotypes observed in yeast and mammalian cells. Electron microscopy shows that the matrices of fragmented mitochondria in eat-3 mutants are divided by inner membrane septae, suggestive of a specific defect in fusion of the mitochondrial inner membrane. In addition, we find that C. elegans eat-3 mutant animals are smaller, grow slower, and have smaller broodsizes than C. elegans mutants with defects in other mitochondrial fission and fusion proteins. Although mammalian Opa1 is antiapoptotic, mutations in the canonical C. elegans cell death genes ced-3 and ced-4 do not suppress the slow growth and small broodsize phenotypes of eat-3 mutants. Instead, the phenotypes of eat-3 mutants are consistent with defects in oxidative phosphorylation. Moreover, eat-3 mutants are hypersensitive to paraquat, which promotes damage by free radicals, and they are sensitive to loss of the mitochondrial superoxide dismutase sod-2. We conclude that free radicals contribute to the pathology of C. elegans eat-3 mutants.     

SDA,a DNA Aptamer Inhibiting E- and P-Selectin Mediated Adhesion of Cancer and Leukemia Cells,the First and Pivotal Step in Transendothelial Migration during Metastasis Formation

Endothelial (E-) and platelet (P-) selectin mediated adhesion of tumor cells to vascular endothelium is a pivotal step of hematogenous metastasis formation. Recent studies have demonstrated that selectin deficiency significantly reduces metastasis formation in vivo. We selected an E- and P-Selectin specific DNA Aptamer (SDA) via SELEX (Systematic Evolution of Ligands by EXponential enrichment) with a K _d value of approximately 100 nM and the capability of inhibiting the interaction between selectin and its ligands. Employing human colorectal cancer (HT29) and leukemia (EOL-1) cell lines we could demonstrate an anti-adhesive effect for SDA in vitro. Under physiological shear stress conditions in a laminar flow adhesion assay, SDA inhibited dynamic tumor cell adhesion to immobilized E- or P-selectin. The stability of SDA for more than two hours allowed its application in cell-cell adhesion assays in cell culture medium. When adhesion of HT29 cells to TNFα-stimulated E-selectin presenting human pulmonary microvascular endothelial cells was analyzed, inhibition via SDA could be demonstrated as well. In conclusion, SDA is a potential new therapeutic agent that antagonizes selectin-mediated adhesion during metastasis formation in human malignancies.     

Effect of the mitochondrial unfolded protein response on hypoxic death and mitochondrial protein aggregation

Mitochondria are the main oxygen consumers in cells and as such are the primary organelle affected by hypoxia. All hypoxia pathology presumably derives from the initial mitochondrial dysfunction. An early event in hypoxic pathology in C. elegans is disruption of mitochondrial proteostasis with induction of the mitochondrial unfolded protein response (UPR^mt) and mitochondrial protein aggregation. Here in C. elegans, we screen through RNAis and mutants that confer either strong resistance to hypoxic cell death or strong induction of the UPR^mt to determine the relationship between hypoxic cell death, UPR^mt activation, and hypoxia-induced mitochondrial protein aggregation (HIMPA). We find that resistance to hypoxic cell death invariantly mitigated HIMPA. We also find that UPR^mt activation invariantly mitigated HIMPA. However, UPR^mt activation was neither necessary nor sufficient for resistance to hypoxic death and vice versa. We conclude that UPR^mt is not necessarily hypoxia protective against cell death but does protect from mitochondrial protein aggregation, one of the early hypoxic pathologies in C. elegans.Subject terms: Necroptosis, Energy metabolism     

The Putative Thiosulfate Sulfurtransferases PspE and GlpE Contribute to Virulence of Salmonella Typhimurium in the Mouse Model of Systemic Disease

The phage-shock protein PspE and GlpE of the glycerol 3-phosphate regulon of Salmonella enterica serovar Typhimurium are predicted to belong to the class of thiosulfate sulfurtransferases, enzymes that traffic sulfur between molecules. In the present study we demonstrated that the two genes contribute to S. Typhimurium virulence, as a glpE and pspE double deletion strain showed significantly decreased virulence in a mouse model of systemic infection. However, challenge of cultured epithelial cells and macrophages did not reveal any virulence-associated phenotypes. We hypothesized that their contribution to virulence could be in sulfur metabolism or by contributing to resistance to nitric oxide, oxidative stress, or cyanide detoxification. In vitro studies demonstrated that glpE but not pspE was important for resistance to H₂O₂. Since the double mutant, which was the one affected in virulence, was not affected in this assay, we concluded that resistance to oxidative stress and the virulence phenotype was most likely not linked. The two genes did not contribute to nitric oxid stress, to synthesis of essential sulfur containing amino acids, nor to detoxification of cyanide. Currently, the precise mechanism by which they contribute to virulence remains elusive.     

The Caenorhabditis elegans genes ced-3 and ced-4 act cell autonomously to cause programmed cell death

Mutations in the genes ced-3 and ced-4 prevent almost all of the programmed cell deaths that occur during Caenorhabditis elegans development. To determine the sites of action of these two genes, we performed genetic mosaic analyses. We generated C. elegans animals that carried a free chromosomal duplication bearing either ced-3(+) or ced-4(+) in an otherwise homozygous ced-3 or ced-4 genetic background. We used other genes on the duplication as markers to identify genetic mosaic animals in which the duplication was present in some but not all cells. The patterns of cell death survivors in these mosaic animals indicated that the products of both ced-3 and ced-4 function within dying cells to cause cell death.     

Cranberry Extract Standardized for Proanthocyanidins Promotes the Immune Response of Caenorhabditis elegans to Vibrio cholerae through the p38 MAPK Pathway and HSF-1

Botanicals are rich in bioactive compounds, and some offer numerous beneficial effects to animal and human health when consumed. It is well known that phytochemicals in cranberries have anti-oxidative and antimicrobial activities. Recently, an increasing body of evidence has demonstrated that cranberry phytochemicals may have potential benefits that promote healthy aging. Here, we use Caenorhabditis elegans as a model to show that water-soluble cranberry extract standardized to 4.0% proanthocyanidins (WCESP), a major component of cranberries, can enhance host innate immunity to resist against Vibrio cholerae (V. cholerae; wild type C6706 (O1 El Tor biotype)) infection. Supplementation of WCESP did not significantly alter the intestinal colonization of V. cholerae, but upregulated the expression of C. elegans innate immune genes, such as clec-46, clec-71, fmo-2, pqn-5 and C23G10.1. Additionally, WCESP treatment did not affect the growth of V. cholerae and expression of the major bacterial virulence genes, and only slightly reduced bacterial colonization within C. elegans intestine. These findings indicate that the major components of WCESP, including proanthocyanidins (PACs), may play an important role in enhancing the host innate immunity. Moreover, we engaged C. elegans mutants and identified that the p38 MAPK signaling, insulin/IGF-1 signaling (IIS), and HSF-1 play pivotal roles in the WCESP-mediated host immune response. Considering the level of conservation between the innate immune pathways of C. elegans and humans, the results of this study suggest that WCESP may also play an immunity-promoting role in higher order organisms.