El-Houjeiri L et al. (2019) Cell Rep "The Transcription Factors TFEB and TFE3 Link the FLCN-AMPK Signaling Axis to Innate Immune Response and Pathogen Resistance."

Paquette M, Jones R, Puertollano R, Pause A, Vijayaraghavan T, Martina JA, Ma EH, Blanchette P, El-Houjeiri L, Possik E, Kazan JM

[Cell Rep, 2019]

TFEB and TFE3 are transcriptional regulators of the innate immune response, but the mechanisms regulating their activation upon pathogen infection are poorly elucidated. Using C.elegans and mammalian models, we report that the master metabolic modulator 5'-AMP-activated protein kinase (AMPK) and its negative regulator Folliculin (FLCN) act upstream of TFEB/TFE3 in the innate immune response, independently of the mTORC1 signaling pathway. In nematodes, loss of FLCN or overexpression of AMPK confers pathogen resistance via activation ofTFEB/TFE3-dependent antimicrobial genes, whereas ablation of total AMPK activity abolishes this phenotype. Similarly, in mammalian cells, loss of FLCN or pharmacological activation of AMPK induces TFEB/TFE3-dependent pro-inflammatory cytokine expression. Importantly, a rapid reduction in cellular ATP levels in murine macrophages is observed upon lipopolysaccharide (LPS) treatment accompanied by an acute AMPK activation and TFEB nuclear localization. These results uncover an ancient, highly conserved, and pharmacologically actionable mechanism coupling energy status with innate immunity.

El Mouridi, Sonia et al. (2021) MicroPubl Biol

AlHarbi, Sarah, El Mouridi, Sonia, Frkjr-Jensen, Christian

[MicroPubl Biol, 2021]

For El Mouridi, S; AlHarbi, S; Frkjr-Jensen, C (2021). A histamine-gated channel is an efficient negative selection marker for C. elegans transgenesis. microPublication Biology. 10.17912/micropub.biology.000349.

Po MD et al. (2012) Neuron "Releasing the inner inhibition for axon regeneration."

Po MD, Calarco JA, Zhen M

[Neuron, 2012]

The adult mammalian central nervous system exhibits restricted regenerative potential. Chen etal. (2011) and El Bejjani and Hammarlund (2012) used Caenorhabditis elegans to uncover intrinsic factors that inhibit regeneration of axotomized mature neurons, opening avenues for potential therapeutics.

Shivakumar S et al. (1991) International C. elegans Meeting "CHARACTERIZING A C. ELEGANS WNT GENE"

Shackleford G, Shiue L, Kenyon CJ, Varmus HE, Shivakumar S

[International C. elegans Meeting, 1991]

Members of the Wnt gene family encode cysteine-rich secretory glycoproteins that have been postulated to play a role in embryonic pattern formation in flies, frogs and mice. We are interested in studying the role of a C. elegans wnt gene in the development of the worm. A portion of a C. elegans wnt gene (Ce-wnt-l ) was previously cloned by PCR, using degenerate oligonucleotide primers corresponding to the conserved protein sequence motifs present in the Wnt family (Kamb et al., P.N.A.S., 86: 4372,1989). One genomic clone (2.7 kb) and two cDNA clones (0.56 kb and 1.3 kb) have subsequently been isolated and sequenced (G. Shackleford, L. Shiue, J. Mason and A. Kamb, unpublished results). Comparison of the genomic and cDNA sequences reveal marked differences in exon/intron organization between the Ce-wnt-l gene and other Wnt genes. The Ce-wnt-l gene was kindly mapped by John Sulston and Alan Coulson to a contig that has not yet been mapped to a chromosome. As with all other known members of the Wnt family, the predicted Ce- wnt-l protein is cysteine-rich and 21 of the 23 cysteines are conserved in other Wnt proteins. The degree of identity between the predicted Ce-wntI protein and other Wnt proteins ranges between 34% and 40%. The longer (1.3 kb) cDNA contains a putative translation start site, a polyadenylation signal and hybridizes to a single 1.5 kb mRNA on Northern blots containing C. elegans RNA. Preliminary studies suggest the the Ce-wnt-l RNA is expressed predominantly in embryos. Primer extension analysis predicts a transcription start site 98 bp upstream of the translation start site. We are currently attempting to isolate mutants with deletions in the Ce-wnt-l gene. After EMS mutagenesis, F2 animals are screened by PCR for loss of restriction sites in the Ce-wnt-l gene. PCR products are then analyzed by Southern hybridization. We have used the unc-54(el 90) mutant which has a 412 bp deletion in reconstruction experiments to test this method. We have been able to detect and isolate one unc-54( el 90) mutant in a background of 40,000 wild-type worms.

Hunter CP et al. (1991) International C. elegans Meeting "NON-AUTONOMY OF HER-1 FUNCTION IMPLICATES CELL INTERACTIONS IN C. ELEGANS SEX DETERMINATION."

Hunter CP, Wood WB

[International C. elegans Meeting, 1991]

Activity of the her-l gene is required for normal male development in C. elegans. Loss-of-function her-l mutations cause feminization of XO animals (normally male) to produce fertile hermaphrodites, while gain-of-function her-l mutations cause masculinization of XX animals ( normally hermaphrodite) to produce pseudo-males. Genetic analysis indicated that her-l functions early in the hierarchy of regulatory interactions among the major sex determination genes (1). We have analyzed her-l genetic mosaics to determine which cells must express her-l for wild-type male development. If her-l functions cell- autonomously, then in XO animals her-l (+) cells will follow male fates, and her-1(-) cells will follow hermaphrodite fates. If her-l or a her-l regulated activity functions non-autonomously, then the her-l genotype of a cell may not determine its sexual phenotype. The phenotypes of XO her-l genetic mosaics are variable, but clearly show that both her-1(-) and her-l (+) cells can follow either hermaphrodite or male fates. We conclude that her-l is neither necessary nor sufficient to cell-autonomously determine male sexual phenotypes and, therefore, that her-l or a her-l regulated activity must be able to function non-autonomously. The observed patterns of non-autonomous effects suggest that many or all cells express and respond to her-l activity. These findings implicate cell interactions in C. elegans sex determination. Earlier genetic analysis indicated that her-l might directly control tra-2 activity (1). Molecular characterization of the gene products of her-l and tra-2 (see abstracts by M. Perry et al. and P. Kuwabara et al) is consistent with a model in which a secreted ligand encoded by her-l interacts with a cell-surface receptor encoded by tra-2. We will discuss the possible role of such an interaction in coordinating the sex determination decision.

Forrester S et al. (2007) Foodborne Pathog Dis "Evaluation of the pathogenicity of Listeria spp. in Caenorhabditis elegans."

Schwab U, Wiedmann M, Hoose WA, Milillo SR, Forrester S

[Foodborne Pathog Dis, 2007]

Caenorhabditis has proven to be a useful model for studying host-pathogen interactions as well as the ability of nematodes to serve as vectors for the dispersal of foodborne pathogens. In this study, we evaluated whether C. elegans can serve as a host for Listeria spp. While there was an effect of growth media on C. elegans killing, C. elegans exposed to L. monocytogenes and L. innocua pregrown in Luria-Bertani medium showed reduced survival when compared to nonpathogenic E. coli OP50, while L. seeligeri showed survival similar to E. coli OP50. In a preference assay, C. elegans preferred E. coli over L. monocytogenes and L. innocua, but showed no preference between L. monocytogenes and L. innocua. A gentamicin assay indicated that L. monocytogenes did not persist within the C. elegans intestinal tract. Our findings that L. monocytogenes and L. innocua strains tested have equally deleterious effects on C. elegans and that L. monocytogenes did not establish intestinal infection conflict with other recently published results, which found intestinal infection and killing of C. elegans by L. monocytogenes. Further studies are thus needed to clarify the interactions between L. monocytogenes and C. elegans, including effects of environmental conditions and strain differences on killing and intestinal infection.

Kenyon CJ et al. (1994) Worm Breeder's Gazette "A polyray Suppressor"

Kenyon CJ, Maloof JN

[Worm Breeder's Gazette, 1994]

A polyray-l suppressor Julin Maloof and Cynthia Kenyon, Dept of Biochemistry, U.C.S.F, San Francisco, CA 94143 The polyray-l (pry-l) gene is required to keep homeotic selector (HOM-C) genes repressed where their expression has not been initiated (see accompanying abstract and WBG 12(5):39). pry-l mutant worms have numerous homeotic transformations and are very sick, so we were intrigued by a plate of pry-l mutant worms which appeared much healthier than normal. I outcrossed the strain and found F2s with the full pry-l mutant phenotype, suggesting that the "healthy" strain contained both pry-l and an extragenic suppressor. Does the suppressor have a phenotype on its own? Since pry-l causes homeotic transformations due to ectopic HOM-C expression, it seemed possible that the suppressor would cause transformations in the opposite direction, similar to those seen in HOM-C loss of function mutants. Indeed some F2s from the outcross had descendants of the QL neuroblast in the anterior of the worms, a phenotype also caused by loss-of function mutations in the HOM-C gene mab-5. We are calling this newly identified gene spy-l for suppressor of polyray-l. spy-l seems to suppress all of the pry-l mutant-phenotypes: pry-l; spy-l double mutant worms do not have extra rays, are no longer Muv, are not Unc, and have anteriorly placed QR descendants. spy-l could suppress pry-l either by preventing ectopic HOM-C expression, or by preventing ectopically expressed HOM-C genes from having any effect. mab-5-lacZ expression was examined and found to be normal in the p1y-l; spy-l double, so wild-type spy-l is required for the ectopic HOM-C expression seen in pry-l mutant animals. The fact that there is a QL migration defect in worms mutant for spy-l alone suggests that spy-l may normally be required to turn on mab-5 in QL. We compared expression of mab-5-lacZ in wild- type and spy-l mutant worms at 3.5-5 hours and at 8-10 hours after hatching. At 3.5-5 hours 100% (n=20) of the mutant and 95% (n=20) of the wild-type worms had expression in QL. At 8- 10 hours 0% (n=30) of the mutant worms has expression of mab- 5 in the descendants of QL, but 87% (n=30) of the wild-type worms showed expression. It appears that spy-l is not required to initiate mab-5 expression in QL, but that it is required to maintain expression once it is initiated. It is possible that spy-l is required to maintain the ON state of a number of HOM-C genes, in a manner analogous to the brahma and trithorax genes of Drosophila. spy-l is located on X, in a region well covered by cosmids. Hopefully a molecular analysis of spy-l along with the isolation and study of more spy-l alleles will enable us to determine how similar the C. elegans and Drosophila maintenance systems are, and how spy-l and other maintenance genes are used to maintain and specify pattern.

Ellis RE et al. (1991) International C. elegans Meeting "PROGRESS IN CLONING fog- 1"

Kimble JE, Ellis RE

[International C. elegans Meeting, 1991]

The fog-l gene plays an important role in the decision of germ cells to differentiate as either spermatocytes or oocytes (Doniach 1986, Barton and Kimble 1990). In fog-l mutants, all germ cells develop into oocytes. This is true for both males and hermaphrodites. However, these fog-l mutations do not appear to affect other aspects of sexual development. Furthermore, the fog-l mutant phenotype is not suppressed by mutations in any of the other genes known to control sex- determination. Thus fog-l might function to initiate spermatogenesis in response to other genes that regulate general sexdetermination. There are 42 fog-l mutations. These mutations arise at a frequency typical for knockout mutations in other C. elegans genes, and four of these fog-l alleles were isolated from a screen capable of recovering deletions. Thus it is likely that these mutations cause a loss of fog- l function. In order to study the mechanisms by which fog-l functions, and to determine how fog-l expression is regulated by the genes that control sex-determination, we are now cloning this gene. The fog-l gene is located on the left arm of LGI, between sup-ll and unc-ll, a region spanned by a large contig of about one megabase in size. By mapping DNA polymorphisms with respect to fog-l, we have narrowed down the region in which fog-l must be located, and are now injecting YAC DNA that covers this region in an attempt to rescue fog-l mutants.

Abraham D et al. (1989) Am J Trop Med Hyg "Immunity to larval Brugia malayi in BALB/c mice: protective immunity and inhibition of larval development."

Grieve RB, Christensen BM, Abraham D, Holy JM

[Am J Trop Med Hyg, 1989]

The objective of this study was to analyze the immune response of mice to the larval stages of Brugia malayi. Male BALB/c mice were inoculated with 3 doses of irradiated third-stage larvae (L-3) of B. malayi and were subsequently challenged with L-3 implanted ip within diffusion chambers. After 3 weeks, larvae were recovered to determine their viability, length, and stage of development. A significant reduction in parasite survival was observed in immunized mice. Furthermore, larvae recovered from immunized mice were significantly shorter than larvae recovered from control mice. All larvae recovered from immunized mice were L-3, whereas 96% of larvae recovered from controls were fourth-stage larvae (L-4). Sera collected from control and immunized mice were tested for the presence of antibodies reactive with L-3 and L-4 antigens using an indirect fluorescent antibody assay employing frozen larval cross-sections as antigen. Sera recovered after challenge of control mice reacted with internal, but not surface, antigens of L-3 and L-4. Alternatively, sera from immunized mice reacted with both internal and external antigens of both L-3 and L-4.

Possik, E. et al. (2017) International Worm Meeting "FLCN and AMPK confer resistance to bacterial pathogens via TFEB/TFE3 mediated innate immune response."

Possik, E., Kapelanski-Lamoureux, A, Paquette, M, Pause, A, Vijayaraghavan, T, El-Houjeiri, L

[International Worm Meeting, 2017]

Humans and animals are constantly exposed to pathogenic microorganisms that can enter the body and cause disease. Mechanisms of defense against invading pathogens are essential for all living organisms to propagate and survive. The transcription factor EB (TFEB), a master transcriptional regulator of lysosomal and autophagic pathways, has been recently shown to play a critical role in innate immunity both in Caenorhabditis elegans (C. elegans) and mammals. Despite the important role (s) of TFEB in physiological and pathological states including neurodegenerative diseases, immunity, and cancer, its upstream regulation is not fully elucidated. Here, we show that FLCN-1 and its binding partner 5'AMP-activated protein kinase (AMPK) regulate HLH-30, the TFEB worm homolog. FLCN-1 is the nematode ortholog of the tumor suppressor FLCN, responsible for the Birt-Hogg-Dube (BHD) tumor syndrome. Using C. elegans, we show that flcn-1 mutant animals exhibit an increased resistance to bacterial pathogens in an hlh-30-dependent manner. Loss of flcn-1 induces the nuclear translocation of HLH-30 upon infection with Staphylococcus aureus, which increases the transcription of antimicrobial genes. We further show that this phenotype is independent of mTOR. Using RNA-Seq technology, we demonstrate that AMPK regulates a subset of the hlh-30-dependent antimicrobial peptide genes. Accordingly, loss of AMPK decreases the HLH-30 nuclear translocation and suppresses the increased resistance of flcn-1 mutant animals to Staphylococcus aureus and Pseudomonas aeruginosa. Importantly, our findings highlight that this pathway is evolutionarily conserved. Specifically, we show that loss of Flcn in mouse embryonic fibroblasts also induces the nuclear translocation of TFEB/TFE3 and the upregulation of inflammatory cytokines in an mTOR independent manner. Overall, our findings support an important role for FLCN and AMPK in the regulation of TFEB, which is a central mechanism of resistance to pathogenic agents.