Danielle L. Huffman et al. (2006) European Worm Meeting "PMK-1 MAP Kinase Pathway in Response and Defense to Pore-Forming Toxins"

Mindy Hsieh, Raffi V. Aroian, Danielle L. Huffman

[European Worm Meeting, 2006]

Danielle L. Huffman, Mindy Hsieh and Raffi V. Aroian Pathogenic bacteria can produce a wide range of gene products that contribute to their virulence. The most common virulence factors produced by pathogenic bacteria are pore-forming toxins (PFTs). Bacillus thuringiensis is a soil pathogen that produces a family of PFTs called Cry toxins. Cry toxins work by binding to receptors on the surface of the hosts intestine and inserting into the bilayer to form a pore (~2nm) in the host cell membrane. Our lab has shown that the nematicidal Cry toxin Cry5B is toxic to C. elegans in a dose-dependent manner. In an effort to learn how animal cells deal with and protect themselves against PFTs, we are using Cry5B and C. elegans to identify and characterize host genes and pathways that protect the animal against PFTs.. From our studies, we have discovered that the p38 MAP kinase cascade (NSY-1/SEK-1/PMK-1) helps protect C. elegans from Cry5B toxin. We also found that the protective function of this pathway is conserved in a mammalian cell line exposed to a PFT from a bacterium that causes human disease. We further investigated the role of this pathway in Cry5B defense using a combination of microarray analysis and reverse genetics to find the transcriptional targets of the pathway that are important to toxin defense. This analysis led to the identification of the ttm gene class (toxin-regulated target of MAP kinase). We have recently found that Cry5B exposure induces phosphorylation of the C. elegans PMK-1 MAP kinase, indicating that the host responds to toxin by activating this protective pathway. Furthermore, we have determined that the NSY-1 MAP3K is not absolutely required for activation of PMK-1 in Cry5B treated animals, indicating an alternative upstream activator for this pathway that has yet to be discovered. In an attempt to identify some of the genes responsible for toxin-responsive activation of PMK-1, we have begun a genome-wide RNAi screen for animals that are hypersensitive to Cry5B and unable to activate PMK-1 in response to Cry5B exposure. This screen has thus far identified over 70 new genes involved in toxin defense, including one that is important for activation of PMK-1 by Cry5B.

Cheng-Yuan Kao et al. (2007) International Worm Meeting "Discovery of a p38-centered gene network involved in Caenorhabditis elegans host defense against the bacterial pore-forming toxin."

Chih Hsu, Cheng-Yuan Kao, Danielle Huffman, Raffi V. Aroian, Mindy Hsieh, Li Chen

[International Worm Meeting, 2007]

Pore-forming toxins (PFTs), the largest single class of bacterial virulence factors, are important virulence factors made by many pathogenic bacteria such as Streptococcus pneumoniae and Staphylococcus aureus. These toxins form holes in cell plasma membranes and disrupt normal cell functions. Our laboratory has developed a powerful system for studying the effects of pore-forming toxins, the intoxication of C. elegans by the bacterial PFT Cry5B. Using this system, we have demonstrated that cells can use a signal transduction pathway, namely the p38 mitogen-activated protein kinase (MAPK) pathway, to protect against PFTs (Huffman et al., 2004). To follow up on this work, we have carried out a genome-wide RNAi screening to globally determine which genes in the C. elegans genome are important for defense against the PFT Cry5B (i.e. genes that when reduced by RNAi give rise to a hypersensitive to pore-forming toxin or Hpo phenotype). We have screened through all C. elegans chromosomes using the Ahringer library and have identified 264 hpo genes. In order to fully elucidate whether these hpo genes contribute to p38 host defense pathway, we performed network analysis based on RNAi results. A network analysis will allows us to predict PFT defense networks by the guilt-by-association principle whereas the RNAi data will allow us to determine the functionality of these networks. By integrating our RNAi data with the C. elegans Interactome, we have assembled functional Cry5B PFT defense networks. The biggest sub-network contains 5 RNAi target genes and 21 nodes. Most interestingly, two of the HPOs in this sub-network are bridged by PMK-1/p38 MAPK, which indicates their physical interaction with p38 MAPK, and the upstream MAPKKK NSY-1 is also found in this sub-network. Since this sub-network was the only one found to contain PMK-1 and NSY-1, we therefore hypothesize that, this sub-network is a central p38-mediated PFT defense network. All candidates in this p38 sub-network are currently being validated with genetic mutants or by a two-step RNAi protocol in liquid toxicity assay. By dissecting the role of the p38 MAPK signaling network in response to PFTs, it will bring important and groundbreaking insights into the field of bacterial pathogenesis and host innate immunity against this arguably most important class of bacterial virulence factor.

Leyva-Diaz, Eduardo et al. (2017) International Worm Meeting "pals-22, a member of an expanded C. elegans gene family, is involved in repetitive transgene silencing."

Leyva-Diaz, Eduardo, Hobert, Oliver, Stefanakis, Nikolaos, Carrera, Ines

[International Worm Meeting, 2017]

Repetitive DNA sequences are subject to chromatin-based gene silencing in various animal species. Under specific circumstances repetitive DNA sequences can escape such silencing. For example, while exogenously added, extrachromosomal DNA sequences that are stably inherited in multicopy, repetitive arrays in the nematode C. elegans are often silenced in the germline, such silencing often does not occur in the soma. This indicates that somatic cells may utilize factors that prevent repetitive DNA silencing. Such "anti-silencing" factors have been revealed through genetic screens that identified mutant loci in which repetitive transgenic arrays are aberrantly silenced in the soma (Fischer et al. 2008, Grishok et al. 2005, Hsieh et al. 1999, Knight and Bass 2002). We describe here a novel mutant locus, pals-22 (for protein containing ALS2CR12 domain) with a transgene silencing phenotype. We find that pals-22 is a member of a large family of divergent genes, defined by the presence of an ALS2CR12 domain (39 members, including 2 pseudogenes). While family members are highly divergent they show striking patterns of genomic clustering. The family expansion appears C. elegans specific and has not occurred to the same extent in other nematode species (e.g. C. briggsae only codes for 6 pals genes). A PALS-22::GFP fusion protein that is fully functional in rescue assays displays a cytoplasmic subcellular localization, which rule out any direct chromatin-mediated silencing mechanism. This fusion protein is expressed across different tissues: the nervous system (pan-neuronally), muscle (pharyngeal, vulval, anal and body wall muscle), gut and seam cells. Lastly, the transgene silencing phenotype of pals-22 depends on the biogenesis of small RNAs, since silencing is abolished in the RNAi defective mutant rde-4, suggesting that pals-22 might regulate RNAi dependent silencing in the cytoplasm of neurons and other tissues.

Rong-Jeng Tseng et al. (2005) International Worm Meeting "A novel gene lex-1 affects lin-48 expression in the C. elegans hindgut"

Helen M Chamberlin, Rong-Jeng Tseng

[International Worm Meeting, 2005]

lin-48 encodes an Ovo-like zinc finger protein and is required for specification of specific cell types in C. elegans. Previous studies in our lab have shown that lin-48 is important for normal development of the hindgut, the excretory duct, and the male spicule. GFP tagged LIN-48 shows the expression in a subset of hindgut cells, the excretory duct cell, and some neuronal support cells in head and tail. To understand how lin-48 transcription is regulated, we carried out a genetic screen for mutants with altered lin-48::gfp expression. This screen identified mutations in three genes important to promote lin-48 expression in hindgut. One gene is egl-38, shown previously to encode a PAX transcription factor necessary for lin-48 expression1. In addition, multiple mutations were recovered in the gene tam-12, and a new gene, lex-1 (lin-48 expression abnormal).lex-1 encodes a protein containing a bromodomain and an ATPase domain. lex-1 Mutant alleles gu24 and gu48 have a point mutation in the ATPase domain and the bromodomain, respectively. Interestingly, gu24 and gu48 can complement each other while both fail to complement a third allele, gu47, which causes frameshift of the last exon. This suggests the LEX-1 protein may function as a multimer. In addition to the preferential reduction of lin-48::gfp expression in the hindgut, lex-1 mutants also affects endogenous lin-48 expression in the hindgut as lex-1 mutants enhance male spicule abnormality in lin-48 heterozygous animals. Moreover, lex-1 mutants show different levels of egg-laying defect, abnormal male tail and male mating deficiency. tam-1 has been characterized previously to play a role in gene regulation and functions as a class B synMuv gene2. Since tam-1 and lex-1 mutants exhibit a similar mutant phenotype of lin-48 expression and the gu48 allele of lex-1 shows allele-specific gene interference with tam-1 mutant alleles, we tested whether lex-1 is also a synMuv gene. Indeed, lex-1 double mutants with a class A synMuv gene exhibit a multivulva phenotype. Given the fact that 1) a number of class B synMuv genes encode components of histone deacetylase complex3; 2) bromodomains are found in a variety of chromatin-associated proteins; 3) bromodomains can interact with acetylated lysine4, we hypothesize that LEX-1 plays a role in assembly or activity of a large protein complex that modulates gene transcription.1. Johnson et al. (2001) Development 128, 2857-28652. Hsieh et al. (1999) Genes Dev. 13, 2958-29703. Ceol et al. (2001) Mol. Cell 7, 461-4734.Jeanmougin et al. (1997) Trends Biochem. Sci. 22, 151-153