Chang-Shi Chen et al. (2007) International Worm Meeting "Systematic analysis of the mode of action of Bt crystal toxin Cry5B in Caenorhabditis elegans."

Raffi V. Aroian, Chang-Shi Chen, Kevin Day, Cheng-Yuan Kao

[International Worm Meeting, 2007]

Crystal (Cry) proteins produced by the Gram-positive soil bacterium Bacillus thuringiensis (Bt) have been used extensively in the control of mosquito and black fly vectors that transmit human diseases and in the control of insects that damage crops. Our laboratory is the first to show that individual Cry proteins, in particular Cry5B and Cry21A, can intoxicate the model laboratory nematode Caenorhabditis elegans and other phylogenetically diverse group of nematodes. We have also successfully demonstrated that purified recombinant Cry5B can be a good oral active anthelminthic for the human and animal hookworm parasite Ancylostoma ceylanicum both in vitro and in vivo. The current knowledge about the steps for Cry proteins intoxication are 1) solubilization of Bt crystals 2) proteolytic activation of the protoxin and 3) biding of activated Cry proteins to invertebrate-specific receptors on the midgut, leading to pore-formation. Despite this knowledge of Cry proteins, however, how the Cry5B kills, what parts/domains of the toxin are important for function, and especially what proteases in the intestine of nematode are involved in its activation process, remain unclear. We are applying two systematic approaches to inquire these questions. Firstly, we are carrying out random mutagenesis in Cry5B by error-prone PCR method and performed a high-throughput bioassay in C. elegans to screen hypertoxic Cry5B variants. Several hypertoxic variants that adversely affect worm health more than wild type Cry5B have been identified. Secondly, we are screening all the protease genes found in the Ahringer library to identify by RNAi the proteases involved in Cry5B digestion and activation. Characterization of the Cry5B variants and proteases in C. elegans that enhance toxicity of Cry5B will lead to the elucidation of the mode of action of Cry toxin and shed the light onto the therapeutic potential of Cry5B against human parasitic nematodes.

Chang-Shi Chen et al. (2010) East Asia Worm Meeting "DAF-2 Insulin-like signaling network is involved in Pore-Forming Toxin Cellular Defenses in C. elegans"

Huan-Da Chen, Audrey Bellier, Ferdinand C. O. Los, Chang-Shi Chen, Raffi V. Aroian, Ya-Luen Yang, Cheng-Yuan Kao

[East Asia Worm Meeting, 2010]

Cytolytic pore-forming toxins (PFTs) are a class of potent virulence factor of many disease-causing bacteria that convert from a soluble form to a membrane-integrated pore to intoxicate host cells. Although PFTs play important roles in the development of bacterial disease, we have enormous gaps in our knowledge and are only beginning to understand what these toxins do to the target cells and especially to the whole organism. The DAF-2 insulin/insulin-like growth factor-1 signaling pathway, which regulates lifespan and stress resistance in C. elegans, is known to mutate to resistance to pathogenic bacteria. Here we reveal that reduction of the DAF-2 insulin-like pathway signal cascade also confers the resistance of C. elegans to Crystal toxins, which are PFTs produced by Bacillus thuringiensis. Moreover, this resistance signal diverges at 3-phosphoinositide-dependent kinase 1 (PDK-1) and feeds into a novel signal arm defined by the WW domain Protein 1 (WWP-1). Taken together, our data suggest that WWP-1 and DAF-16 function in parallel within the fundamental DAF-2 signaling network to regulate fundamental cellular responses in C. elegans.

Chang-Shi Chen et al. (2008) C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting "Mutations in DAF-2 Insulin-like Signaling Pathway Contribute to the Bt Crystal Toxin Cry5B Resistance in Caenorhabditis elegans"

Audrey Bellier, Chang-Shi Chen, Raffi Aroian

[C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting, 2008]

Crystal (Cry) proteins produced by the Gram-positive bacterium Bacillus thuringiensis (Bt) are pore-forming toxins (PFTs) that are able to intoxicate and kill C. elegans. This interaction between Cry proteins and C. elegans opens up the first, and to date only, genetic model for studying pore-forming toxins in vivo. PFTs comprise the single most prevalent, and arguably most important, class of bacterial virulence factor. One quarter of all bacterial virulence factors are PFTs, and they are the used by many pathogenic bacteria, such as Staphylococcus aureus and Streptococci. Although we have been focusing on innate defenses in C. elegans, more recently we have been studying intoxication processesthat is, what pathways in the cells contrive with the pore-forming toxins to intoxicate and kill. Virtually nothing is known about this field. The C. elegans DAF-2 insulin-like signaling pathway, which regulates lifespan and stress resistance, has been implicated in resistance to bacterial pathogens. Loss-of-function daf-2 pathway mutants have increased life spans and are also resistant to a variety of bacterial pathogens. This raises the possibility that the increased pathogen resistance of insulin-like signaling pathway mutants might also resistant to Cry5B killing in C. elegans, suggesting that the daf-2 pathway is involved in intoxication. We found that worms mutated in the daf-2 pathways are more resistant to Cry5B killing than wild-type worms. We find, however, that this resistance does not expend to all the parts of the known daf-2 pathway but rather forks off the main pathway at a point not seen before. Our current hypothesis is that the daf-2 pathway has split off a separate arm involved in intoxication against PFTs, as well as perhaps some yet unknown responses. We will present our characterization and models of the daf-2 pathway in response to PFTs.

Bellier, Audrey et al. (2009) International Worm Meeting "Pore-forming toxins, stress and the hypoxia pathway in C. elegans."

Aroian, Raffi, Chen, Chang-Shi, Bellier, Audrey

[International Worm Meeting, 2009]

Pore-forming toxins (PFTs) represent the largest class of bacterial protein toxins and often play an important role in the bacterial pathogenesis. The pores formed by these proteins in the membrane of target cells lead to ion imbalances and signal transduction activation that represent a tremendous stress on cell. We are studying pore-forming Crystal (Cry) proteins made by the bacterium Bacillus thuringiensis (Bt) to understand how cellular factors and pathways respond to PFTs. In particular, our lab is interested in understanding what genes and pathways act in concert with the pore to undermine cellular health and integrity. The prediction is elimination of such gene and pathways will lead to animals resistant to the PFTs. A previous genetic screen in the laboratory for C. elegans resistance mutants to the PFT Cry5B led to the identification of glycolipid carbohydrates as key receptors for Cry5B toxin. These results, although important, did not actually point to broadly applicable mechanisms. Therefore, to try to get to these intoxication pathways, we screened for mutants resistant to a different PFT, Cry21A. These results will be presented here. Despite extensive screening, only one robust mutant resistant to Cry21A was found. Surprisingly this mutant presents an early stop codon in egl-9, which encodes for a key regulator of the hypoxia pathway. In an egl-9 mutant, the hypoxia-inducible factor (HIF-1) is stable and activates transcription of genes involved in cell survival under hypoxia. We found that egl-9 mutants are also resistant to various other Cry PFTs. These data suggest that repression of the hypoxia pathway by egl-9 makes animals more susceptible to PFTs. Loss-of-function mutation in egl-9 has already been reported to confer resistance to pathogenic attack (Darby et al., 1999), therefore we tested egl-9 to different stresses. We found that egl-9 mutants are also resistant to heat shock, oxidative stress, pathogenic bacteria and have an extended lifespan. However, the dependence of these stress responses on HIF-1 can be diametrically opposite, suggesting that both stabilization and loss of function of HIF-1 lead to stress resistance, depending on the stress. We believe we have also identified a factor downstream of HIF-1 required for a normal response to PFTs. Our data show that HIF-1 levels play an important role on cellular stress resistance and longevity and that maintaining a proper and dynamic balance of HIF-1 activity is crucial for proper survival of the animal under varying conditions. Lethal paralysis of Caenorhabditis elegans by Pseudomonas aeruginosa. 1999. Darby C, Cosma CL, Thomas JH, Manoil C. Proc Natl Acad Sci U S A. 96:15202-7.

Kuo, Cheng-Ju et al. (2015) International Worm Meeting "A forward genetic screen for host factors involved in enterohemorrhagic E. coli colonization in Caenorhabditis elegans."

Kuo, Cheng-Ju, Chen, Chang-Shi, Yang, Fang-Jung

[International Worm Meeting, 2015]

Enterohemorrhagic E. coli (EHEC) causes hemorrhagic diarrhea and hemolytic uremic syndrome worldwide. In our previous study, we have established an EHEC-C. elegans infection model to study the bacterial pathogenesis in vivo. We found that EHEC clinical isolated strains can colonize and replicate in the intestine of C. elegans. Several published literatures have identified the key bacterial factors required for EHEC colonization, however the host factors involved in EHEC colonization in vivo remain ambiguous. Here, we applied the EMS-mediated forward genetic screen for identifying the host factors involved in EHEC colonization in C. elegans. We isolated 32 EMS-induced mutants conferred the INC (enhanced intestinal colonization) phenotype. We then examined whether these alleles exhibit a different host susceptibility to EHEC infection. Intriguingly, four independent inc mutants also exhibited a significant EHEC resistant phenotype compared to the wild-type N2 animals. Given that the mutations in these four alleles enhance the EHEC intestinal colonization yet confer resistance to EHEC infection in C. elegans, the phenomenon is contradictory to our current knowledge about bacterial infection. We therefore turned our attentions to clone these four alleles. We believe through our study on the function of the host factors required for EHEC colonization may shed light on the novel aspects of immunity in EHEC infection.

Peng, Hsiao-Fang et al. (2017) International Worm Meeting "Male-specific proteins SMZ-1 and SMZ-2 are required for normal sperm generation in C. elegans."

Peng, Hsiao-Fang, Chen, Chang-Shi, Hsu, Ya-Chu, Wu, Jui-Ching

[International Worm Meeting, 2017]

In sexually reproducing animals, sperm are generated in highly-specialized male germline. Therefore, male-specific regulators should be participated in functional sperm production. SMZ-1 and SMZ-2, two highly identical PDZ domain proteins, were found abundant in spermatogenic chromatin through proteomic study in C. elegans. While PDZ domain-containing proteins are known for acting as scaffolds for cell signal transduction, little is known about the roles of PDZ domain-containing proteins in sperm generation. To investigate the roles of SMZ-1 and SMZ-2 in fertility, we first obtained two smz-2 deletion mutants using CRISPR/Cas9 techniques, and generated smz-1;smz-2 double mutant worms. We compared total brood size and hatch rate of single as well as double mutant hermaphrodites and found that worms bearing either smz-1 or smz-2 single deletion can normally generate offspring. On the other hand, smz-1; smz-2 hermaphrodites do not have any countable progeny. The results show that SMZ-1 and SMZ-2 are functionally redundant in fertility. Examination of hermaphrodite uterus revealed smz-1; smz-2 uterus filled with unfertilized oocytes. This suggests failure of fertilization, which can result from defects in sperm or oocyte function. To distinguish where the defects take place, we examined SMZ-1 and SMZ-2 protein expression in worms generating either sperm or oocytes. Immunoblotting analyses showed that SMZ-1 and SMZ-2 are highly expressed in sperm but not in oocytes or somatic cells. In agreement to this, the infertility in smz-1; smz-2 double mutant hermaphrodites can be rescued by mating with wild type males. These results indicate SMZ-1 and SMZ-2 only function in sperm. In order to define the origin defects of smz-1; smz-2 in sperm generation, we examined fixed smz-1; smz-2 male gonad. We found smz-1; smz-2 male germline failed to generate mature sperm comparing with wild type, suggesting there are meiotic division defects. Time-lapse recording of smz-1; smz-2 spermatocytes showed that chromosomes failed to progress into metaphase I. Taken together, SMZ-1/2 are male specific fertility factors required for initiation of meiotic divisions in spermatocytes. We are currently investigating the cellular factors that are regulated by SMZ-1/2.

Chen, Huan-Da et al. (2011) International Worm Meeting "SIR-2.3 is involved in Cry5B pore-forming toxin defense in C. elegans."

Kao, Cheng-Yuan, Aroian, Raffi V., Chen, Huan-Da, Chen, Chang-Shi

[International Worm Meeting, 2011]

Pore-Forming toxins(PFTs) are bacterial toxins that perforate holes at the plasma membrane of host cells and play essential roles in the pathogenesis of many human pathogens. Crystal(Cry) toxins are PFTs produced by Bacillus thuringiensis(Bt), and have been widely used as natural insecticides in agriculture for many decades. C. elegans has been developed as an in vivo model host to study the epithelial defenses against Cry toxins, including Cry5B in this study, and has proven instrumental in the identification of several molecular PFT-defense pathways. Here we showed that the transcription of the sir-2.3 gene was significantly upregulated by Cry5B and sir-2.3 was required for Cry5B defense in C. elegans. SIR-2.3 is a member of the SIR2 family proteins with NAD-dependent histone deacetylase activity. Our previous study has demonstrated that DAF-2 insulin-like signaling pathways were important for the intrinsic epithelial defense against PFTs. Interestingly, it has been reported that the mammalian SIR-2.3 homolog, SIRT4, can inhibit the insulin secretion in pancreatic b cells. Our current genetic epistasis data suggested that sir-2.3 may act upstream of daf-2 and function in the same signal pathway in response to Cry5B intoxication in C. elegans.

Chou, Ting-Chen et al. (2011) International Worm Meeting "Characterization of the pathogenicity of Enterohaemorrhagic E. coli in C. elegans."

Chiu, Hao-Chieh, Chou, Ting-Chen, Chen, Chang-Shi, Wu, Ching-Ming, Kuo, Cheng-Ju

[International Worm Meeting, 2011]

Strains of Enterohaemorrhagic Escherichia coli (EHEC) that produce verocytotoxin, such as E. coli O157:H7, form an important group of zoonotic pathogens. E. coli O157:H7 has been implicated in large outbreaks as well as in sporadic cases of haemorrhagic colitis and the sometimes fatal haemolytic uremic syndrome. Here we applied the genetic tractable animal, C. elegans, as a model host to study the virulence of EHEC as well as the host innate immunity to this pathogen. Our preliminary data showed that EHEC intoxicated, induced severe Bag (bag-of-worms adults) phenotype, and eventually killed the worms. The transmission electron microscopy results indicated that EHEC induced attaching and effacing (A/E) lesions in the intestine. By further genetic analysis, we found that some important virulence factors of EHEC were required for its toxicity in C. elegans, and some conserved innate immune signal pathways were mediated in host susceptibility. Our results suggest that this EHEC-C. elegans model is suitable for future comprehensive genetic screens in both bacterial and host factors involved in the pathogenesis of EHEC.

Hu, Yan et al. (2009) International Worm Meeting "HAT's off: a novel and powerful class of nematicides for soil-transmitted nematode infections."

Chen, Chang-Shi, Georghiou, Sophia, Platzer, Edward G., Kao, Cheng-Yuan, Kelleher, Alan, Aroian, Raffi V., Hu, Yan

[International Worm Meeting, 2009]

Soil-transmitted nematodes, hookworm, Ascaris, and Trichuris (HAT), are amongst the most prevalent human parasites, infecting more than 1 billion of the poorest peoples around the world. We have limited number drugs for treating these parasites, and, for practical reasons, only one (albendazole) is commonly used in mass drug administration (MDA). All our current drugs have limitations as used in MDAs, including moderate efficacy against hookworms, low efficacy against Trichuris, and threat of drug resistance. Therefore there is need for new human anthelmintics (anti-worm drugs) that are safe, effective and broad spectrum in scope. Our laboratory is working to develop Crystal (Cry) proteins produced by soil bacteria Bacillus thuringiensis (Bt) as a new class of anthelmintics. Bt Cry proteins are commonly used as insecticides in agriculture, are safe to humans, and have a mechanism of action (form pores in invertebrate intestine) different from nematicides currently use. Several Cry proteins have been identified that target nematodes, including C. elegans and plant-parasites. We are now using the mouse intestinal parasitic nematode, Heligmosomoides polygyrus, as a model for human disease. Here we will present our latest data showing the efficacy of Cry proteins against this rodent parasitic nematode, as well as our system to improve the anthelmintic activity of Cry proteins by looking for point mutant Cry variants that are hyperactive against C. elegans.

Kuo, Cheng-Ju et al. (2013) International Worm Meeting "Shiga-like toxin 1 confers the full pathogenicity of Enterohaemorrhagic Escherichia coli and activation of the p38/MAPK pathway in Caenorhabditis elegans."

Chiu, Wen-Tai, Chen, Chang-Shi, Chiu, Hao-Chieh, Kuo, Cheng-Ju, Syu, Wan-Jr, Chou, Ting-Chen

[International Worm Meeting, 2013]

Enterohaemorrhagic Escherichia coli (EHEC), a major foodborne pathogen, is responsible for life-threatening diseases in humans as a consequence of the production of Shiga-like toxins. Currently, there is no specific treatment for EHEC infection and the use of antibiotics is contraindicated. Moreover, lack of a good animal model system hinders the study of EHEC virulence with systematic methods in vivo. To these regards, we applied the genetic tractable animal model, Caenorhabditis elegans, as a surrogate host to study the virulence of EHEC as well as the host innate immunity to this human pathogen. Our results show that E. coli O157:H7, a serotype of EHEC, infects and kills C. elegans. We also demonstrate that E. coli O157:H7 colonizes and replicates in C. elegans, stimulates the ectopic expression of microvillar actin, and induces the characteristic attaching and effacing (A/E) lesions in the intact intestinal epithelium in vivo. Moreover, our genetic analyses corroborate that the Shiga-like toxin 1 (Stx1) of E. coli O157:H7 is required partly for its toxicity. The C. elegans p38 MAP kinase signaling pathways, an evolutionally conserved innate immune signaling pathways, are activated and mediated in the regulation of host susceptibility to EHEC infection in a Stx1-dependent manner. Given that the bacterivore C. elegans and bacterium, in this case E. coli, may have generated a diverse arsenal for both parties to defend each other during the long-term evolution of this predator-prey relationship, our results suggest that this EHEC-C. elegans model is suitable for future comprehensive genetic screens for both novel bacterial and host factors involved in the pathogenesis of EHEC infection.