A movable surface: formation of Yersinia sp. biofilms on motile Caenorhabditis elegans

Bubonic plague is transmitted by fleas whose feeding is blocked by a mass of Yersinia pestis in the digestive tract. Y. pestis and the closely related Y. pseudotuberculosis also block the feeding of Caenorhabditis elegans by forming a biofilm on the nematode head. C. elegans mutants with severe motility defects acquire almost no biofilm, indicating that normal animals accumulate the biofilm matrix as they move through a Yersinia lawn. Using the lectin wheat germ agglutinin as a probe, we show that the matrix on C. elegans contains carbohydrate produced by Yersinia. The carbohydrate is present in bacterial lawns prior to addition of nematodes, indicating that biofilm formation does not involve signaling between the two organisms. Furthermore, biofilm accumulation depends on continuous C. elegans exposure to a lawn of Yersinia bacteria.     

Muscle cell attachment in Caenorhabditis elegans

In the nematode Caenorhabditis elegans, the body wall muscles exert their force on the cuticle to generate locomotion. Interposed between the muscle cells and the cuticle are a basement membrane and a thin hypodermal cell. The latter contains bundles of filaments attached to dense plaques in the hypodermal cell membranes, which together we have called a fibrous organelle. In an effort to define the chain of molecules that anchor the muscle cells to the cuticle we have isolated five mAbs using preparations enriched in these components. Two antibodies define a 200-kD muscle antigen likely to be part of the basement membrane at the muscle/hypodermal interface. Three other antibodies probably identify elements of the fibrous organelles in the adjacent hypodermis. The mAb IFA, which reacts with mammalian intermediate filaments, also recognizes these structures. We suggest that the components recognized by these antibodies are likely to be involved in the transmission of tension from the muscle cell to the cuticle.     

Temperature-sensitive developmental mutants of Caenorhabditis elegans

About 200 temperature-sensitive mutants of the nematode Caenorhabditis elegans have been isolated. At restrictive temperature, the mutants are blocked in the reproductive life cycle. They have been placed into six broad categories based on their defective phenotypes. The six categories are: (1) mutants blocked in embryogenesis; (2) mutants defective in gonadogenesis; (3) mutants defective in spermatogenesis; (4) mutants that accumulate at an intermediate growth stage; (5) mutants that produce sterile adult progeny; (6) mutants that have a temperature-sensitive morphological defect that interrupts the reproductive life cycle. The critical times of temperature sensitivity have been measured using temperature-shift experiments. Most of the gonadogenesis and spermatogenesis mutants are temperature sensitive during the period of cellular differentiation rather than proliferation. The temperature responses of the gonadogenesis and zygote-defective mutants indicate a common association between functions in gonadogenesis and early embryogenesis. Many of the mutants placed in different categories share other temperature-sensitive phenotypes upon close examination. This implies that many of the functions required for development are general metabolic reactions under increased demand during differentiation and embryogenesis.     

Mutations affecting nerve attachment of Caenorhabditis elegans

Using a pan-neuronal GFP marker, a morphological screen was performed to detect Caenorhabditis elegans larval lethal mutants with severely disorganized major nerve cords. We recovered and characterized 21 mutants that displayed displacement or detachment of the ventral nerve cord from the body wall (Ven: ventral cord abnormal). Six mutations defined three novel genetic loci: ven-1, ven-2, and ven-3. Fifteen mutations proved to be alleles of previously identified muscle attachment/positioning genes, mup-4, mua-1, mua-5, and mua-6. All the mutants also displayed muscle attachment/positioning defects characteristic of mua/mup mutants. The pan-neuronal GFP marker also revealed that mutants of other mua/mup loci, such as mup-1, mup-2, and mua-2, exhibited the Ven defect. The hypodermis, the excretory canal, and the gonad were morphologically abnormal in some of the mutants. The pleiotropic nature of the defects indicates that ven and mua/mup genes are required generally for the maintenance of attachment of tissues to the body wall in C. elegans.     

Temperature-sensitive zygote defective mutants of Caenorhabditis elegans

Three genetically complementing temperature-sensitive mutants of Caenorhabditis elegans have been studied. Each of the three mutants has two critical times of temperature sensitivity and two distinctive corresponding phenotypes. Exposure to high temperature during gonadogenesis blocks the production of zygotes. Exposure of adults to high temperature interrupts embryogenesis of the zygotes being produced. Each of the mutants carries an autosomal mutation with a maternal effect. These mutants indicate that the individual temperature-sensitive functions are required at least twice during development and that early embryogenesis is dependent on the contribution of these functions from the maternal gonad.     

Yersinia enterocolitica Infection and tcaA-Dependent Killing of Caenorhabditis elegans

Caenorhabditis elegans is a validated model to study bacterial pathogenicity. We report that Yersinia enterocolitica strains {"type":"entrez-nucleotide","attrs":{"text":"W22703","term_id":"1299536","term_text":"W22703"}}W22703 (biovar 2, serovar O:9) and WA314 (biovar 1B, serovar O:8) kill C. elegans when feeding on the pathogens for at least 15 min before transfer to the feeding strain Escherichia coli OP50. The killing by Yersinia enterocolitica requires viable bacteria and, in contrast to that by Yersinia pestis and Yersinia pseudotuberculosis strains, is biofilm independent. The deletion of tcaA encoding an insecticidal toxin resulted in an OP50-like life span of C. elegans, indicating an essential role of TcaA in the nematocidal activity of Y. enterocolitica. TcaA alone is not sufficient for nematocidal activity because E. coli DH5α overexpressing TcaA did not result in a reduced C. elegans life span. Spatial-temporal analysis of C. elegans infected with green fluorescent protein-labeled Y. enterocolitica strains showed that Y. enterocolitica colonizes the nematode intestine, leading to an extreme expansion of the intestinal lumen. By low-dose infection with {"type":"entrez-nucleotide","attrs":{"text":"W22703","term_id":"1299536","term_text":"W22703"}}W22703 or DH5α followed by transfer to E. coli OP50, proliferation of Y. enterocolitica, but not E. coli, in the intestinal lumen of the nematode was observed. The titer of {"type":"entrez-nucleotide","attrs":{"text":"W22703","term_id":"1299536","term_text":"W22703"}}W22703 cells within the worm increased to over 10⁶ per worm 4 days after infection while a significantly lower number of a tcaA knockout mutant was recovered. A strong expression of tcaA was observed during the first 5 days of infection. Y. enterocolitica WA314 (biovar 1B, serovar O:8) mutant strains lacking the yadA, inv, yopE, and irp1 genes known to be important for virulence in mammals were not attenuated or only slightly attenuated in their toxicity toward the nematode, suggesting that these factors do not play a significant role in the colonization and persistence of this pathogen in nematodes. In summary, this study supports the hypothesis that C. elegans is a natural host and nutrient source of Y. enterocolitica.Yersinia enterocolitica belongs to the family of Enterobacteriaceae and is a psychrotolerant human pathogen that causes gastrointestinal syndromes ranging from acute enteritis to mesenteric lymphadenitis (5). It infects a number of mammals, and swine was identified as a major source for human infection (6). A multiphasic life cycle, which comprises a free-living phase and several host-associated phases, including cold-blooded and warm-blooded hosts, appears to be characteristic for biovars 1B and 2 to 5 of Y. enterocolitica (7, 24).Nonmammalian host organisms including Dictyostelium discoideum, Drosophila melanogaster, or Caenorhabditis elegans are increasingly used to study host-pathogen interactions (16, 26). Due to the obvious parallels between the mammalian and invertebrate defense mechanisms, it has been suggested that the bacteria-invertebrate interaction has shaped the evolution of microbial pathogenicity (53). Several human pathogens including Gram-positive and Gram-negative bacteria infect and kill the soil nematode C. elegans when they are supplied as a nutrient source (42). For example, Streptococcus pneumoniae (4), Listeria monocytogenes (50), extraintestinal Escherichia coli (15), and Staphylococcus aureus (43) but not Bacillus subtilis have been shown to kill the nematode. Upon infection of C. elegans with Enterococcus faecalis, Gram-positive virulence-related factors as well as putative antimicrobials have been identified (20, 35). The extensive conservation in virulence mechanisms directed against invertebrates as well as mammals was demonstrated using a screen with Pseudomonas aeruginosa (30). In this study, 10 of 13 genes whose knockout attenuated the nematode killing were also required for full virulence in a mouse model, confirming the suitability of the C. elegans model to study bacterial pathogenicity. C. elegans is also colonized by Salmonella enterica serovar Typhimurium (S. Typhimurium). This process requires Salmonella virulence factors and was used to study the innate immune response of the nematode (1, 2, 49).The effect of pathogenic Yersinia spp. on C. elegans has also been investigated. It could be demonstrated that both Yersinia pestis and Yersinia pseudotuberculosis block food intake by creating a biofilm around the worm''s mouth (13, 27). This biofilm formation requires the hemin storage locus (hms) and has been suggested to be responsible for the blockage of the digestive tract following uptake by fleas, thus acting as a bacterial defense against predation by invertebrates. In a study with 40 Y. pseudotuberculosis strains, one-quarter of them caused an infection of C. elegans by biofilm formation on the worm head (27). In contrast, a similar effect was not observed following nematode infection with 15 Y. enterocolitica strains. Using a Y. pestis strain lacking the hms genes, it could be demonstrated that this mutant can infect and kill the nematode by a biofilm-independent mechanism that includes the accumulation of Y. pestis in the intestine of the worm (47). This pathogenesis model was applied to show that putative virulence factors such as YapH, OmpT, or a metalloprotease, Y3857, but not the virulence plasmids pCD1 and pPCP1, are required for Y. pestis virulence in C. elegans. Six yet unknown genes required for full virulence in C. elegans were also identified, and one of them appeared to be a virulence factor in the mouse infection model.C. elegans has not been used to study the pathogenicity properties of Y. enterocolitica, mainly due to the fact that many of its virulence factors are upregulated at 37°C in comparison to growth at lower temperatures while C. elegans cannot be cultivated at temperatures above 25°C. In this study, we examined for the first time the infection of C. elegans by Y. enterocolitica strains, demonstrating that this pathogen colonizes and kills C. elegans and that the insecticidal toxin TcaA, which is expressed only at ambient temperature, is required for full nematocidal activity.     

Egg-laying defective mutants of the nematode Caenorhabditis elegans

We have isolated 145 fertile mutants of C. elegans that are defective in egg laying and have characterized 59 of them genetically, behaviorally and pharmacologically. These 59 mutants define 40 new genes called egl. for egg-laying abnormal. Most of the other mutants are defective in previously identified genes. The egl mutants differ with respect to the severity of their egg-laying defects and the presence of behavioral or morphological pleiotropies. We have defined four distinct categories of mutants based on their responses to the pharmacological agents serotonin and imipramine, which stimulate egg laying by wild-type hermaphrodites. These drugs test the functioning of the vulva, the vulval and uterine muscles and the hermaphrodite-specific neurons (HSNs), which innervate the vulval muscles. Mutants representing 14 egl genes fail to respond to serotonin and to imipramine and are likely to be defective in the functioning of the vulva or the vulval and uterine muscles. Four mutants (representing four different genes) lay eggs in response to serotonin but not to imipramine and appear to be egg-laying defective because of defects in the HSNs; three of these four were selected specifically for these drug responses. Mutants representing seven egl genes lay eggs in response to serotonin and to imipramine. One egl mutant responds to imipramine but not to serotonin. The remaining egl mutants show variable or intermediate responses to the drugs. Two of the HSN-defective mutants, egl-1 and her-1(n695), lack HSN cell bodies and are likely to be expressing the normally male-specific program of HSN cell death. Whereas egl-1 animals appear to be defective specifically in HSN development, her-1(n695) animals exhibit multiple morphological pleiotropies, displaying partial transformation of the sexual phenotype of many cells and tissues. At least two of the egl mutants appear to be defective in the processing of environmental signals that modulate egg laying and may define new components of the neural circuitry that control egg laying.     

Metaphase to anaphase (mat) transition-defective mutants in Caenorhabditis elegans

The metaphase to anaphase transition is a critical stage of the eukaryotic cell cycle, and, thus, it is highly regulated. Errors during this transition can lead to chromosome segregation defects and death of the organism. In genetic screens for temperature-sensitive maternal effect embryonic lethal (Mel) mutants, we have identified 32 mutants in the nematode Caenorhabditis elegans in which fertilized embryos arrest as one-cell embryos. In these mutant embryos, the oocyte chromosomes arrest in metaphase of meiosis I without transitioning to anaphase or producing polar bodies. An additional block in M phase exit is evidenced by the failure to form pronuclei and the persistence of phosphohistone H3 and MPM-2 antibody staining. Spermatocyte meiosis is also perturbed; primary spermatocytes arrest in metaphase of meiosis I and fail to produce secondary spermatocytes. Analogous mitotic defects cause M phase delays in mitotic germline proliferation. We have named this class of mutants "mat" for metaphase to anaphase transition defective. These mutants, representing six different complementation groups, all map near genes that encode subunits of the anaphase promoting complex or cyclosome, and, here, we show that one of the genes, emb-27, encodes the C. elegans CDC16 ortholog.     

Quinones in long-lived clk-1 mutants of Caenorhabditis elegans

Ubiquinone (UQ) (coenzyme Q) is a lipophilic redox-active molecule that functions as an electron carrier in the mitochondrial electron transport chain. Electron transfer via UQ involves the formation of semiubiquinone radicals, which causes the generation of superoxide radicals upon reaction with oxygen. In the reduced form, UQ functions as a lipid-soluble antioxidant, and protects cells from lipid peroxidation. Thus, UQ is also important as a lipophilic regulator of oxidative stress. Recently, a study on long-lived clk-1 mutants of Caenorhabditis elegans demonstrated that biosynthesis of UQ is dramatically altered in mutant mitochondria. Demethoxy ubiquinone (DMQ), that accumulates in clk-1 mutants in place of UQ, may contribute to the extension of life span. Here we elucidate the possible mechanisms of life span extension in clk-1 mutants, with particular emphasis on the electrochemical property of DMQ. Recent findings on the biochemical function of CLK-1 are also discussed.     

Mutator phenotype of Caenorhabditis elegans DNA damage checkpoint mutants

DNA damage response proteins identify sites of DNA damage and signal to downstream effectors that orchestrate either apoptosis or arrest of the cell cycle and DNA repair. The C. elegans DNA damage response mutants mrt-2, hus-1, and clk-2(mn159) displayed 8- to 15-fold increases in the frequency of spontaneous mutation in their germlines. Many of these mutations were small- to medium-sized deletions, some of which had unusual sequences at their breakpoints such as purine-rich tracts or direct or inverted repeats. Although DNA-damage-induced apoptosis is abrogated in the mrt-2, hus-1, and clk-2 mutant backgrounds, lack of the apoptotic branch of the DNA damage response pathway in cep-1/p53, ced-3, and ced-4 mutants did not result in a Mutator phenotype. Thus, DNA damage checkpoint proteins suppress the frequency of mutation by ensuring that spontaneous DNA damage is accurately repaired in C. elegans germ cells. Although DNA damage response defects that predispose humans to cancer are known to result in large-scale chromosome aberrations, our results suggest that small- to medium-sized deletions may also play roles in the development of cancer.     

Tissue specificity of Caenorhabditis elegans enhanced RNA interference mutants

Gene knockdown by RNA interference (RNAi) in Caenorhabditis elegans is readily achieved by feeding bacteria expressing double-stranded RNA (dsRNA). Enhanced RNAi (Eri) mutants facilitate RNAi due to their hypersensitivity to dsRNA. Here, we compare eight Eri mutants for sensitivity to ingested dsRNA, targeting a variety of tissue-specific genes.     

Trehalose renders the dauer larva of Caenorhabditis elegans resistant to extreme desiccation

Water is essential for life on Earth. In its absence, however, some organisms can interrupt their life cycle and temporarily enter an ametabolic state, known as anhydrobiosis [1]. It is assumed that sugars (in particular trehalose) are instrumental for survival under anhydrobiotic conditions [2]. However, the role of trehalose remained obscure because the corresponding evidence was purely correlative and based mostly on in vitro studies without any genetic manipulations of trehalose metabolism. In this study, we used C. elegans as a genetic model to investigate molecular mechanisms of anhydrobiosis. We show that the C. elegans dauer larva is a true anhydrobiote: under defined conditions it can survive even after losing 98% of its body water. This ability is correlated with a several fold increase in the amount of trehalose. Mutants unable to synthesize trehalose cannot survive even mild dehydration. Light and electron microscopy indicate that one of the major functions of trehalose is the preservation of membrane organization. Fourier-transform infrared spectroscopy of whole worms suggests that this is achieved by preserving homogeneous and compact packing of lipid acyl chains. By means of infrared spectroscopy, we can now distinguish a "dry, yet alive" larva from a "dry and dead" one.     

Direct isolation of longevity mutants in the nematode Caenorhabditis elegans

We have isolated several new EMS-induced, long-lived mutants of Caenorhabditis elegans, using a novel screen that eliminates the need for replica plating. Three new alleles of age-1 (z10, z12, and z25) were identified by failure to complement age-1 (hx546) for life span extension; these alleles had life spans ranging from 18.9 to 25.9 days at 25°C, with an average 46% increase in life span. After backcrossing, alleles were examined in a wild-type background for resistance to several environmental stresses: heat (35°C), ultraviolet (UV) light (20 J/m²), and hydrogen peroxide (H₂O₂) (0.5 M). Two replicates of the test of thermotolerance were completed on each strain, giving mean survivals of 842 min (hx546), 810 min (z10), 862 min (z12), and 860 min (z25), compared to 562 min for wild type. All the age-1 alleles were significantly tolerant, compared with wild type (P < 0.001). Two replicates for UV resistance were also completed with mean survivals of 103, 118, 108, and 89 hr, respectively, compared to 72 hr for wild type. One test of hydrogen peroxide resistance has shown that z12 and N2 had a mean survival of 41 hr, while the other age-1 alleles had mean survival of 54 hr (z10), and 62 hr (z25); H₂O₂ resistance is the only environmental stress that differentiates among the age-1 alleles. © 1996 Wiley-Liss, Inc.     

Isolation of long-lived mutants in Caenorhabditis elegans using selection for resistance to juglone

The accumulation of molecular damage from attack by reactive oxygen species is one cause of aging. Therefore, some mutant organisms showing increased resistance to reactive oxygen species should live longer. We show that selection for Caenorhabditis elegans mutants that are resistant to juglone, a reactive oxygen species-generating compound, leads to the identification of long-lived mutants. Indeed, four of six resistant mutants isolated were also long-lived. This study illustrates once more the strong relationship between oxidative stress and the aging processes.     

The hmsHFRS operon of Xenorhabdus nematophila is required for biofilm attachment to Caenorhabditis elegans

The bacterium Xenorhabdus nematophila is an insect pathogen and an obligate symbiont of the nematode Steinernema carpocapsae. X. nematophila makes a biofilm that adheres to the head of the model nematode Caenorhabditis elegans, a capability X. nematophila shares with the biofilms made by Yersinia pestis and Yersinia pseudotuberculosis. As in Yersinia spp., the X. nematophila biofilm requires a 4-gene operon, hmsHFRS. Also like its Yersinia counterparts, the X. nematophila biofilm is bound by the lectin wheat germ agglutinin, suggesting that beta-linked N-acetyl-D-glucosamine or N-acetylneuraminic acid is a component of the extracellular matrix. C. elegans mutants with aberrant surfaces that do not permit Yersinia biofilm attachment also are resistant to X. nematophila biofilms. An X. nematophila hmsH mutant that failed to make biofilms on C. elegans had no detectable defect in symbiotic association with S. carpocapsae, nor was virulence reduced against the insect Manduca sexta.     

Interactions of anthelmintic drugs in Caenorhabditis elegans neuro-muscular ion channel mutants

Due to the increasing development of anthelmintic resistance in nematodes worldwide, it is important to search for anthelmintic compounds with new modes of action and also to investigate the possibility to combine compounds with possible synergistic effects. There might also be the chance to take advantage of the fact that nematode populations which have developed resistance against one anthelmintic class might respond hypersusceptibly to another drug class. The aim of this study was to investigate responses of Caenorhabditis elegans populations with mutations in neuro-muscular ion channels to different anthelmintic classes. Furthermore, potential synergistic effects between two anthelmintic compounds from different classes, i.e. emodepside and tribendimidine, were studied. Although there was neither a synergistic nor an antagonistic effect between emodepside and tribendimidine, other types of interactions could be identified. The C. elegans GABA_A-receptor (GABA_A-R) unc-49 mutants, showing decreased emodepside susceptibility, were more susceptible to tribendimidine than wild-type C. elegans. In contrast, the reverse phenomenon – hypersusceptibility to emodepside in tribendimidine resistant acetylcholine-receptor (AChR) loss of function mutants – was not observed. Moreover, the slo-1 mutant strain (completely emodepside resistant) also showed hypersusceptibility to piperazine. Interestingly, neither the GABA_A-R unc-49 mutants nor the AChR mutants showed decreased susceptibility against piperazine, although there were some studies that indicated an involvement of GABA_A-R or AChR in the piperazine mode of action. In conclusion, the present study provides evidence suggesting that interactions between commercially available anthelmintic drugs with different modes of action might be a relatively common phenomenon but this has to be carefully worked out for each anthelmintic and each anthelmintic drug combination. Moreover, results obtained in C. elegans will have to be confirmed using parasitic nematodes in the future.