Kuo, Cheng-Ju et al. (2017) International Worm Meeting "Natural variation in C. elegans resistance and xenophagy against a natural intracellular parasite."

Kuo, Cheng-Ju, Balla, Keir, Troemel, Emily

[International Worm Meeting, 2017]

Microsporidia are obligate intracellular parasites that infect animals ranging from single-celled ciliates to humans. Nematocida ironsii (ERTm5), is a natural microsporidian parasite of Caenorhabditis elegans isolated from Hawaii (Balla et al PLoS Pathogns 2015), and we previously found that the Hawaiian C. elegans strain CB4856 can clear infection by N. ironsii, in contrast to the standard N2 laboratory strain from England that always succumbs to infection. Quantitative trait locus mapping indicated this enhanced immunity in CB4856 is a complex trait, and maps to four separate loci in the genome. We constructed and tested near isogenic lines to confirm that two of these loci (on chromosomes II and V) confer resistance against infection. Interestingly these two loci contain some of the most rapidly evolving genes in the C. elegans genome (Thomas, Genome Research 2006), including F-box-containing genes, which are predicted to be substrate-binding subunits in multi-subunit ubiquitin ligase complexes. These findings led to the hypothesis that the CB4856 genome encodes for ubiquitin ligase adaptors that are able to target microsporidia parasite cells with ubiquitin, in order to recruit the autophagy machinery and facilitate so-called 'xenophagy' to clear these pathogens from intestinal cells. Here we present unpublished data that support our hypothesis that CB4856 animals are better able to target the natural parasite N. ironsii for xenophagic clearance. We found that when we compared ubiquitin localization to N. ironsii parasite cells in N2 and CB4856 animals, the increased resistance to N. ironsii infection by CB4856 animals correlates with increased localization of ubiquitin to parasite cells in these animals. Surprisingly, when we found that the frequency of localization of the key autophagy marker LGG-1 (GABARAP orthologs) to parasite cells was NOT higher in CB4856 animals compared to N2 animals. Instead, we found that the less well-studied autophagy protein LGG-2* (LC3 ortholog), like ubiquitin, was localized to a higher percentage of parasite cells in CB4856 animals compared to N2 animals. We are currently investigating a functional role for LGG-2 in the ability of CB4856 Hawaiian animals to clear this Hawaiian Nematocida species. Our findings may shed new light onto the mechanisms underlying xenophagy and intracellular parasite clearance, as well as natural host/pathogen co-evolution. * Thanks to Renaud Legouis' lab for GFP::LGG-2 plasmid used to generate these strains.

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.

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.

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.

Yang, Ya-Luen et al. (2013) International Worm Meeting "Sestrin confers the regulations of muscle aging and lifespan in Caenorhabditis elegans."

Yang, Ya-Luen, Chu, I-Hua, Liou, Bang-Yu, Loh, Kah-Sin, Chen, Huan-Da, Chen, Chang-Shi, Kuo, Cheng-Ju

[International Worm Meeting, 2013]

Aging is associated with the progressive decline in body function and physiology that is shared by all multicellular organisms. The free radical theory argues that the accumulation of damage caused by reactive oxygen species (ROS) over the time is the cause of aging. Sestrins are evolutionarily conserved in metazoans and are required for ROS clearance. However, whether sestrins per se regulate longevity in multicellular organisms is still unclear. Here, we report that SESN-1, the only Sestrin ortholog in Caenorhabditis elegans, is a positive regulator of lifespan. C. elegans sesn-1 mutants exhibit shorter lifespans, hypersensitivity to oxidative stress, and premature aging in muscle. When sesn-1 gene was overexpressed and rescued, the lifespan of sesn-1 mutant worms could be extended. These findings imply that SESN-1 is required to protect against general life stressors, is important in regulation of lifespan and healthspan late in life, and might play a key role in muscle integrity.

Chen, Huan-Da et al. (2017) International Worm Meeting "HLH-30 regulates epithelium intrinsic cellular defenses in intestinal cells against bacterial pore-forming toxin in C. elegans."

Kao, Cheng-Yuan, Chen, Chang-Shi, Chen, Yu-Hung, Kuo, Cheng-Ju, Chen, Huan-Da, Liu, Bang-Yu, Huang, Shin-Whei, Wang, Horng-Dar, Huang, Cheng-Rung, Ruan, Jhen-Wei, Aroian, Raffi, Lin, Yen-Hung

[International Worm Meeting, 2017]

Autophagy is an evolutionarily conserved self-clearance system that maintains cellular homeostasis by degrading and recycling various superfluous or damaged cellular components. The transcription factor HLH-30 (TFEB in mammals) has been reported to modulate bacterial infection by regulating autophagy, but less is known about the bona fide bacterial effector that activates HLH-30 and autophagy. Here, we unveal that bacterial membrane pore-forming toxin (PFT) induces cellular autophagy via an HLH-30-dependent manner in Caenorhabditis elegans. The PFT-activated autophagy controls the tolerance of host to PFT intoxication through xenophagic degradation of PFT and repair of membrane-pore cell-autonomously within the PFT-targeted intestinal cells. All together, our data demonstrate a new connection between HLH-30 and epithelium intrinsic cellular defense (INCED) against the single most common mode of bacterial attack in vivo.

G Kao et al. (1999) International C. elegans Meeting "Characterization of Laminin beta and gamma Genes."

G Kao, C Huang, WG Wadsworth

[International C. elegans Meeting, 1999]

We have characterized laminin b (lam-1) and laminin g (lam-2) genes. The sequencing project has uncovered genes for one isoform each of the two subunits and two isoforms of laminin a , epi-1 and lam-3. (See abstract by Cheng et al.). This indicates that two types of trimers likely are form in basement membranes : a A bg and a B bg . A hypomorphic mutation (rh219) exists in lam-1, whose phenotypes we have described in previous meetings (IWM 1997). Both lam-1(RNAi) and lam-2(RNAi) animals arrest development during morphogenesis. Significantly, this phenotype is identical to the arrest seen in double knockout of epi-1 and lam-3, and is more severe than either knockout alone. We examined the expression patterns of lam-1 and lam-2 using promoter:GFP fusions. Both genes are expressed in body wall muscles, pharynx, vulval muscles, the rectal muscle group and the distal tip cells. The expression patterns of b and g subunit genes overlap with and are more widespread than either epi-1 or lam-3 alone. Taken together these results suggest that lam-1 and lam-2 are found in all basement membranes and indicate that trimers a A bg and a B bg are formed.

Cho, Jihye et al. (2021) International Worm Meeting "Identify the function of mechanosensitive channel PEZO-1 in C. elegans males"

Cho, Jihye, Kim, Kyuhyung

[International Worm Meeting, 2021]

The conversion of mechanical force to biological signals is crucial for the survival of animals. Mechanosensitive ion channels play a direct role in sensing physical stimuli and are evolutionarily conserved from bacteria to mammals. In mammals, PIEZO channels have been identified as ion channels that directly detect mechanical stimuli (Coste et al., 2010, Gnanasambandam et al., 2015, Syeda et al., 2015). C. elegans genome has a single PIEZO gene, pezo-1, which encodes 14 isoforms (Bai X et al., 2020). However, the function of PEZO-1 in C. elegans has not been fully understood yet. To investigate its function, we first grouped 14 isoforms into short or long isoforms depending on the mRNA length and observed their expression patterns. We found that the promoter region of short isoforms is expressed in several tail neurons, including the 6th ray neurons of C. elegans males. Nine pairs of male ray neurons appear to have functional specialization and have been implicated to be involved in mating behavior (Zhang H, Yue X, Cheng H, et al., 2018). Interestingly, sensory endings of ray 6 neurons, but not other ray neurons, are not exposed to the external environment (Sulston and Horvitz, 1977), suggesting a distinct role in mechanotransduction during C. elegans mating. We are currently examining the contribution of PEZO-1 to mating behavior in males.

Ravichandiran Kumarasamy et al. (2002) European Worm Meeting "Dna polymorphism in Pristionchus pacificus"

Ralf J Sommer, Ravichandiran Kumarasamy

[European Worm Meeting, 2002]

To understand the evolution of developmental process at the microevolutionary level, it is important to study the biogeography and the evolutionary alterations of the developmental processes in detail. To acuminate this, we are working on the satellite model Pristionchus pacificus. The genus Pristionchus has a worldwide distribution and different strains of Pristionchus pacificus exist from Washington (PS 1843), Hawaii (JU 138), Ontario (AF 8130), Poland (RS 106) and the wild type strain from California (PS 312). Previous AFLP studies showed a high degree of polymorphisms between the strains from Washington, Hawaii and California. BAC end sequencing and SSCP analysis of BAC end fragments between the strains from California and Washington confirmed these original observations. To obtain a better picture of the distribution of polymorphisms between the strains of P. pacificus, we have chosen a random 5 Kb region on Chromosome III that contains no open reading frame. Our results showed a high degree of polymorphism (3.46%) in both Washington and Hawaii when compared to the laboratory strain California. There were ~ 0.7% transitions, ~ 0.5% transversions, ~ 0.3% insertions and ~ 0.1% deletions. Most of the polymorphisms observed were single base pair events except a few which were up to 5 bp . Further studies are underway to extend this study, comparing the ceh-20 and ceh-40 genes in these strains and to eventually include the different species' of Pristionchus namely P. maupasi and P. lheritieri.

Riga, Amalia et al. (2019) International Worm Meeting "The role of basolateral polarity regulators in epithelial tissue homeostasis."

Boxem, Mike, Pires, Helena, Riga, Amalia

[International Worm Meeting, 2019]

Polarization of epithelial cells into apical and basolateral domains is essential for the functioning of epithelia as selectively permeable barriers. The basolateral Scribble group proteins (Scrib, Lgl, Dlg) were originally identified as tumor suppressors in D. melanogaster and were later shown to play key roles in the establishment and maintenance of polarity in a broad range of cell types. In addition to promoting basolateral identity, Scribble group proteins regulate diverse processes including tissue growth, differentiation and directed cell migration, and therefore are major regulators of tissue development and homeostasis. In C. elegans, loss of LET-413/Scrib and DLG-1/Dlg results in defects in epithelial polarization and junction formation during embryonic development, resulting in embryonic lethality. However, their importance in larval epithelia is less well understood. To study the role of LET-413 in larval epithelia, we generated an allele that allows inducible protein degradation using the auxin-inducible system recently developed for C. elegans (1). We find that ubiquitous degradation of LET-413 in larval tissues results in a developmental arrest. Tissue-specific degradation of LET-413 in the larval intestine does not cause defects in development. In contrast, degradation of LET-413 in the seam cells and hypodermis mimics the developmental arrest seen upon ubiquitous LET-413 degradation. Live-cell imaging of seam cells shows a severe effect on outgrowth and reattachment following the L1 asymmetric division. In addition, we observed that loss of LET-413 results in punctate and discontinuous localization pattern of DLG-1, similar to previous observations in C. elegans embryos. We are currently following the effects of LET-413 inactivation on cell recognition and migration pathways to understand the outgrowth defects of the seam cells. (1) Zhang L, Ward JD, Cheng Z, Dernburg AF. Development. 2015 Dec 15;142(24):4374-84.