Spread and Transmission of Bacterial Pathogens in Experimental Populations of the Nematode Caenorhabditis elegans

Caenorhabditis elegans is frequently used as a model species for the study of bacterial virulence and innate immunity. In recent years, diverse mechanisms contributing to the nematode''s immune response to bacterial infection have been discovered. Yet despite growing interest in the biochemical and molecular basis of nematode-bacterium associations, many questions remain about their ecology. Although recent studies have demonstrated that free-living nematodes could act as vectors of opportunistic pathogens in soil, the extent to which worms may contribute to the persistence and spread of these bacteria has not been quantified. We conducted a series of experiments to test whether colonization of and transmission between C. elegans nematodes could enable two opportunistic pathogens (Salmonella enterica and Pseudomonas aeruginosa) to spread on agar plates occupied by Escherichia coli. We monitored the transmission of S. enterica and P. aeruginosa from single infected nematodes to their progeny and measured bacterial loads both within worms and on the plates. In particular, we analyzed three factors affecting the dynamics of bacteria: (i) initial source of the bacteria, (ii) bacterial species, and (iii) feeding behavior of the host. Results demonstrate that worms increased the spread of bacteria through shedding and transmission. Furthermore, we found that despite P. aeruginosa''s relatively high transmission rate among worms, its pathogenic effects reduced the overall number of worms colonized. This study opens new avenues to understand the role of nematodes in the epidemiology and evolution of pathogenic bacteria in the environment.     

Chemosensory signal transduction in Caenorhabditis elegans

Chemosensory neurons translate perception of external chemical cues, including odorants, tastants, and pheromones, into information that drives attraction or avoidance motor programs. In the laboratory, robust behavioral assays, coupled with powerful genetic, molecular and optical tools, have made Caenorhabditis elegans an ideal experimental system in which to dissect the contributions of individual genes and neurons to ethologically relevant chemosensory behaviors. Here, we review current knowledge of the neurons, signal transduction molecules and regulatory mechanisms that underlie the response of C. elegans to chemicals, including pheromones. The majority of identified molecules and pathways share remarkable homology with sensory mechanisms in other organisms. With the development of new tools and technologies, we anticipate that continued study of chemosensory signal transduction and processing in C. elegans will yield additional new insights into the mechanisms by which this animal is able to detect and discriminate among thousands of chemical cues with a limited sensory neuron repertoire.     

The dauer hypothesis and the evolution of parasitism: 20 years on and still going strong

How any complex trait has evolved is a fascinating question, yet the evolution of parasitism among the nematodes is arguably one of the most arresting. How did free-living nematodes cross that seemingly insurmountable evolutionary chasm between soil dwelling and survival inside another organism? Which of the many finely honed responses to the varied and harsh environments of free-living nematodes provided the material upon which natural selection could act? Although several complementary theories explain this phenomenon, I will focus on the dauer hypothesis. The dauer hypothesis posits that the arrested third-stage dauer larvae of free-living nematodes such as Caenorhabditis elegans are, due to their many physiological similarities with infective third-stage larvae of parasitic nematodes, a pre-adaptation to parasitism. If so, then a logical extension of this hypothesis is that the molecular pathways which control entry into and recovery from dauer formation by free-living nematodes in response to environmental cues have been co-opted to control the processes of infective larval arrest and activation in parasitic nematodes. The molecular machinery that controls dauer entry and exit is present in a wide range of parasitic nematodes. However, the developmental outputs of the different pathways are both conserved and divergent, not only between populations of C. elegans or between C. elegans and parasitic nematodes but also between different species of parasitic nematodes. Thus the picture that emerges is more nuanced than originally predicted and may provide insights into the evolution of such an interesting and complex trait.     

Environmental-temperature and internal-state dependent thermotaxis plasticity of nematodes

Thermotaxis behavior of Caenorhabditis elegans is robust and highly plastic. A pair of sensory neurons, AFD, memorize environmental/cultivation temperature and communicate with a downstream neural circuit to adjust the temperature preference of the animal. This results in a behavioral bias where worms will move toward their cultivation temperature on a thermal gradient. Thermotaxis of C. elegans is also affected by the internal state and is temporarily abolished when worms are starved. Here I will discuss how C. elegans is able to modulate its behavior based on temperature by integrating environmental and internal information. Recent studies show that some parasitic nematodes have a similar thermosensory mechanism to C. elegans and exhibit cultivation-temperature-dependent thermotaxis. I will also discuss the common neural mechanisms that regulate thermosensation and thermotaxis in C. elegans and Strongyloides stercoralis.     

Molecular Characterization of a Soybean Cyst Nematode (Heterodera glycines) Homolog of unc-87

In Caenorhabditis elegans the unc-87 gene encodes a protein that binds to actin at the I band and is important in nematodes for maintenance of the body-wall muscle. Caenorhabditis elegans mutant phenotypes of unc-87 exhibit severe paralysis in larvae and limp paralysis in the adult. We cloned and characterized a full-length cDNA representing a Heterodera glycines homolog of the unc-87 gene from C. elegans that encodes a protein that contains a region of seven repeats similar to CLIK-23 from C-elegans and has 81% amino acid identity with that of C. elegans unc-87 variant A. In the EST database clones labeled "unc-87'''' encode mainly the 3'' portion of unc-87, while clones labeled "calponin homolog OV9M'''' contain mainly DNA sequence representing the 5'' and middle transcribed regions of unc-87. A 1770 nucleotide cDNA encoding H. glycines unc-87 was cloned and encodes a predicted UNC-87 protein product of 375 amino acids. The expression of unc-87 was determined using RT-PCR and, in comparison to its expression in eggs, unc-87 was expressed 6-fold higher in J2 juveniles and 20-fold and 13-fold (P = 0.05) higher in nematodes 15 and 30 days after inoculation, respectively. In situ hybridization patterns confirmed the expression patterns observed with RT-PCR.     

Regulation of Gap Junction Dynamics by UNC-44/ankyrin and UNC-33/CRMP through VAB-8 in C. elegans Neurons

Gap junctions are present in both vertebrates and invertebrates from nematodes to mammals. Although the importance of gap junctions has been documented in many biological processes, the molecular mechanisms underlying gap junction dynamics remain unclear. Here, using the C. elegans PLM neurons as a model, we show that UNC-44/ankyrin acts upstream of UNC-33/CRMP in regulation of a potential kinesin VAB-8 to control gap junction dynamics, and loss-of-function in the UNC-44/UNC-33/VAB-8 pathway suppresses the turnover of gap junction channels. Therefore, we first show a signal pathway including ankyrin, CRMP, and kinesin in regulating gap junctions.     

Discovery of Anthelmintic Drug Targets and Drugs Using Chokepoints in Nematode Metabolic Pathways

Parasitic roundworm infections plague more than 2 billion people (1/3 of humanity) and cause drastic losses in crops and livestock. New anthelmintic drugs are urgently needed as new drug resistance and environmental concerns arise. A “chokepoint reaction” is defined as a reaction that either consumes a unique substrate or produces a unique product. A chokepoint analysis provides a systematic method of identifying novel potential drug targets. Chokepoint enzymes were identified in the genomes of 10 nematode species, and the intersection and union of all chokepoint enzymes were found. By studying and experimentally testing available compounds known to target proteins orthologous to nematode chokepoint proteins in public databases, this study uncovers features of chokepoints that make them successful drug targets. Chemogenomic screening was performed on drug-like compounds from public drug databases to find existing compounds that target homologs of nematode chokepoints. The compounds were prioritized based on chemical properties frequently found in successful drugs and were experimentally tested using Caenorhabditis elegans. Several drugs that are already known anthelmintic drugs and novel candidate targets were identified. Seven of the compounds were tested in Caenorhabditis elegans and three yielded a detrimental phenotype. One of these three drug-like compounds, Perhexiline, also yielded a deleterious effect in Haemonchus contortus and Onchocerca lienalis, two nematodes with divergent forms of parasitism. Perhexiline, known to affect the fatty acid oxidation pathway in mammals, caused a reduction in oxygen consumption rates in C. elegans and genome-wide gene expression profiles provided an additional confirmation of its mode of action. Computational modeling of Perhexiline and its target provided structural insights regarding its binding mode and specificity. Our lists of prioritized drug targets and drug-like compounds have potential to expedite the discovery of new anthelmintic drugs with broad-spectrum efficacy.     

Hc-daf-2 encodes an insulin-like receptor kinase in the barber’s pole worm, Haemonchus contortus, and restores partial dauer regulation

Infective L3s (iL3s) of parasitic nematodes share common behavioural, morphological and developmental characteristics with the developmentally arrested (dauer) larvae of the free-living nematode Caenorhabditis elegans. It is proposed that similar molecular mechanisms regulate entry into or exit from the dauer stage in C. elegans, and the transition from free-living to parasitic forms of parasitic nematodes. In C. elegans, one of the key factors regulating the dauer transition is the insulin-like receptor (designated Ce-DAF-2) encoded by the gene Ce-daf-2. However, nothing is known about DAF-2 homologues in most parasitic nematodes. Here, using a PCR-based approach, we identified and characterised a gene (Hc-daf-2) and its inferred product (Hc-DAF-2) in Haemonchus contortus (a socioeconomically important parasitic nematode of ruminants). The sequence of Hc-DAF-2 displays significant sequence homology to insulin receptors (IR) in both vertebrates and invertebrates, and contains conserved structural domains. A sequence encoding an important proteolytic motif (RKRR) identified in the predicted peptide sequence of Hc-DAF-2 is consistent with that of the human IR, suggesting that it is involved in the formation of the IR complex. The Hc-daf-2 gene was transcribed in all life stages of H. contortus, with a significant up-regulation in the iL3 compared with other stages. To compare patterns of expression between Hc-daf-2 and Ce-daf-2, reporter constructs fusing the Ce-daf-2 or Hc-daf-2 promoter to sequence encoding GFP were microinjected into the N2 strain of C. elegans, and transgenic lines were established and examined. Both genes showed similar patterns of expression in amphidial (head) neurons, which relate to sensation and signal transduction. Further study by heterologous genetic complementation in a daf-2-deficient strain of C. elegans (CB1370) showed partial rescue of function by Hc-daf-2. Taken together, these findings provide a first insight into the roles of Hc-daf-2/Hc-DAF-2 in the biology and development of H. contortus, particularly in the transition to parasitism.     

Temperature sensing and context-dependent thermal behavior in nematodes

As small ectotherms, whose temperature equilibrates almost instantly with that of their environment, free-living nematodes rely on their behavior for thermoregulation. Caenorhabditis elegans has been extensively used as a model to address the fundamental mechanisms involved in thermosensation and the production of temperature-dependent behaviors. Behavioral responses include avoidance of acute noxious heat or cold stimuli and thermotactic responses to innocuous temperatures to produce oriented navigation in spatial thermogradients. In order to produce these behaviors, C. elegans relies on its ability to detect thermal cues with exquisite sensitivity, orchestrate a set of specific behavioral responses and adapt these responses in specific contexts, including according to past sensory experience and current internal states. The present review focuses on recent advances in our understanding of the processes occurring at the molecular, cellular, and circuit levels that enable thermosensory information processing and plasticity.     

The multiple faces of calcineurin signaling in Caenorhabditis elegans: Development, behaviour and aging

Calcineurin, a well-conserved protein phosphatase 2B (PP2B), is a Ca²⁺-calmodulin–dependent serine/threonine protein phosphatase that is known to be involved in a myriad of cellular processes and signal transduction pathways. The biological role of calcineurin has been extensively studied in diverse groups of organisms. Homologues of mammalian and Drosophila calcineurin subunits exist in the nematode, Caenorhabditis elegans. The C. elegans counterpart of the catalytic subunit, calcineurin A, cna-1/tax-6, and the regulatory subunit, calcineurin B, cnb-1, are known to express ubiquitously in multiple tissues including neurons. The characterization of C. elegans calcineurin mutants facilitates identification of its physiological functions and signaling pathways. Genetic interactions between cna-1/tax-6 and cnb-1 mutants with a number of mutants involved in several signaling pathways have exemplified the pivotal role of calcineurin in regulating nematode development, behaviour and lifespan (aging). The present review has been aimed to provide a succinct summary of the multiple functions of calcineurin in C. elegans relating to its development, fertility, proliferation, behaviour and lifespan. Analyses of cna-1/tax-6 and cnb-1 interacting proteins and regulators of the phosphatase in this fascinating worm model have an immense scope to identify potential drug targets in various parasitic nematodes, which cause many diseases inflicting huge economic loss; and also for many human diseases, particularly neurodegenerative and myocardial diseases.     

Alcaligenes faecalis ZD02, a Novel Nematicidal Bacterium with an Extracellular Serine Protease Virulence Factor

Root knot nematodes (RKNs) are the world''s most damaging plant-parasitic nematodes (PPNs), and they can infect almost all crops. At present, harmful chemical nematicides are applied to control RKNs. Using microbial nematicides has been proposed as a better management strategy than chemical control. In this study, we describe a novel nematicidal bacterium named Alcaligenes faecalis ZD02. A. faecalis ZD02 was isolated from Caenorhabditis elegans cadavers and has nematostatic and nematicidal activity, as confirmed by C. elegans growth assay and life span assay. In addition, A. faecalis ZD02 fermentation broth showed toxicity against C. elegans and Meloidogyne incognita. To identify the nematicidal virulence factor, the genome of strain ZD02 was sequenced. By comparing all of the predicted proteins of strain ZD02 to reported nematicidal virulence factors, we determined that an extracellular serine protease (Esp) has potential to be a nematicidal virulence factor, which was confirmed by bioassay on C. elegans and M. incognita. Using C. elegans as the target model, we found that both A. faecalis ZD02 and the virulence factor Esp can damage the intestines of C. elegans. The discovery that A. faecalis ZD02 has nematicidal activity provides a novel bacterial resource for the control of RKNs.     

Together or alone? Foraging strategies in Caenorhabditis elegans

A central goal in Life Sciences is to understand how genes encode behaviour and how environmental factors influence the expression of the genes concerned. To reach this goal a combined ecological, molecular biological and physiological approach is required in combination with a suitable model organism. Such an approach allows the elucidation of all parts of the complicated chain of events that lead from induction of gene expression to behaviour, i.e. from environmental stimulus, sensory organs and extracellular and intracellular neuronal signal processing to activation of effector organs. A particularly good model species with which to take this approach is the nematode Caenorhabditis elegans, as it has been described in great detail at the genomic, cellular and behavioural levels. Different strains of C. elegans display prominent behavioural variation in foraging behaviour. Some strains will form social feeding groups when subjected to certain environmental stimuli, while others do not. This variation is due to the existence of just two isoforms of the gene npr‐1, namely 215F and 215V. Here, we describe these behavioural variations at the molecular and cellular levels to attempt to determine the environmental inputs that cause aggregation of these small nematodes. As many different stimuli affect aggregation either positively or negatively, aggregation behaviour seems to be displayed when it improves survival chances. However, not much is known about the ecological context in which C. elegans lives. Investigation of the habitats of different strains of C. elegans would help us to understand why and how a specific foraging strategy enhances survival. The relatively well‐understood molecular pathways that direct its social feeding behaviour make C. elegans a highly suitable model organism to test ecological and behavioural hypotheses about the mechanisms that differentiate between aggregation and solitary behaviours.     

In silico analyses of neuropeptide-like protein (NLP) profiles in parasitic nematodes

Nematode parasite infections cause disease in humans and animals and threaten global food security by reducing productivity in livestock and crop farming. The escalation of anthelmintic resistance in economically important nematode parasites underscores the need for the identification of novel drug targets in these worms. Nematode neuropeptide signalling is an attractive system for chemotherapeutic exploitation, with neuropeptide G-protein coupled receptors (NP-GPCRs) representing the lead targets. In order to successfully validate NP-GPCRs for parasite control it is necessary to characterise their function and importance to nematode biology. This can be aided through identification of receptor activating ligand(s) via deorphanisation. Such efforts require the identification of all neuropeptide ligands within parasites. Here we mined the genomes of nine therapeutically relevant pathogenic nematodes to characterise the neuropeptide-like protein complements and demonstrate that: (i) parasitic nematodes possess a reduced complement of neuropeptide-like protein-encoding genes relative to Caenorhabditis elegans; (ii) parasite neuropeptide-like protein profiles are broadly conserved between nematode clades; (iii) five Ce-nlps are completely conserved across the nematode species examined; (iv) the extent and position of neuropeptide-like protein-motif conservation is variable; (v) novel RPamide-encoding genes are present in parasitic nematodes; (vi) novel Allatostatin-C-like peptide encoding genes are present in both C. elegans and parasitic nematodes; (vii) novel neuropeptide-like protein families are absent in C. elegans; and (viii) highly conserved nematode neuropeptide-like proteins are bioactive. These data highlight the complexity of nematode neuropeptide-like proteins and reveal the need for nomenclature revision in this diverse neuropeptide family. The identification of neuropeptide-like protein ligands, and characterisation of those with functional relevance, advance our understanding of neuropeptide signalling to support exploitation of the neuropeptidergic system as an anthelmintic target.     

Pseudomonas fluorescens NZI7 repels grazing by C. elegans,a natural predator

The bacteriovorous nematode Caenorhabditis elegans has been used to investigate many aspects of animal biology, including interactions with pathogenic bacteria. However, studies examining C. elegans interactions with bacteria isolated from environments in which it is found naturally are relatively scarce. C. elegans is frequently associated with cultivation of the edible mushroom Agaricus bisporus, and has been reported to increase the severity of bacterial blotch of mushrooms, a disease caused by bacteria from the Pseudomonas fluorescens complex. We observed that pseudomonads isolated from mushroom farms showed differential resistance to nematode predation. Under nutrient poor conditions, in which most pseudomonads were consumed, the mushroom pathogenic isolate P. fluorescens NZI7 was able to repel C. elegans without causing nematode death. A draft genome sequence of NZI7 showed it to be related to the biocontrol strain P. protegens Pf-5. To identify the genetic basis of nematode repellence in NZI7, we developed a grid-based screen for mutants that lacked the ability to repel C. elegans. The mutants isolated in this screen included strains with insertions in the global regulator GacS and in a previously undescribed GacS-regulated gene cluster, ‘EDB'' (‘edible''). Our results suggest that the product of the EDB cluster is a poorly diffusible or cell-associated factor that acts together with other features of NZI7 to provide a novel mechanism to deter nematode grazing. As nematodes interact with NZI7 colonies before being repelled, the EDB factor may enable NZI7 to come into contact with and be disseminated by C. elegans without being subject to intensive predation.     

SLO-1-channels of parasitic nematodes reconstitute locomotor behaviour and emodepside sensitivity in Caenorhabditis elegans slo-1 loss of function mutants

The calcium-gated potassium channel SLO-1 in Caenorhabditis elegans was recently identified as key component for action of emodepside, a new anthelmintic drug with broad spectrum activity. In this study we identified orthologues of slo-1 in Ancylostoma caninum, Cooperia oncophora, and Haemonchus contortus, all important parasitic nematodes in veterinary medicine. Furthermore, functional analyses of these slo-1 orthologues were performed using heterologous expression in C. elegans. We expressed A. caninum and C. oncophora slo-1 in the emodepside-resistant genetic background of the slo-1 loss-of-function mutant NM1968 slo-1(js379). Transformants expressing A. caninum slo-1 from C. elegans slo-1 promoter were highly susceptible (compared to the fully emodepside-resistant slo-1(js379)) and showed no significant difference in their emodepside susceptibility compared to wild-type C. elegans (p = 0.831). Therefore, the SLO-1 channels of A. caninum and C. elegans appear to be completely functionally interchangeable in terms of emodepside sensitivity. Furthermore, we tested the ability of the 5′ flanking regions of A. caninum and C. oncophora slo-1 to drive expression of SLO-1 in C. elegans and confirmed functionality of the putative promoters in this heterologous system. For all transgenic lines tested, expression of either native C. elegans slo-1 or the parasite-derived orthologue rescued emodepside sensitivity in slo-1(js379) and the locomotor phenotype of increased reversal frequency confirming the reconstitution of SLO-1 function in the locomotor circuits. A potent mammalian SLO-1 channel inhibitor, penitrem A, showed emodepside antagonising effects in A. caninum and C. elegans. The study combined the investigation of new anthelmintic targets from parasitic nematodes and experimental use of the respective target genes in C. elegans, therefore closing the gap between research approaches using model nematodes and those using target organisms. Considering the still scarcely advanced techniques for genetic engineering of parasitic nematodes, the presented method provides an excellent opportunity for examining the pharmacofunction of anthelmintic targets derived from parasitic nematodes.