A P-glycoprotein protects Caenorhabditis elegans against natural toxins.

P-glycoproteins can cause resistance of mammalian tumor cells to chemotherapeutic drugs. They belong to an evolutionarily well-conserved family of ATP binding membrane transporters. Four P-glycoprotein gene homologs have been found in the nematode Caenorhabditis elegans; this report describes the functional analysis of two. We found that PGP-3 is expressed in both the apical membrane of the excretory cell and in the apical membrane of intestinal cells, whereas PGP-1 is expressed only in the apical membrane of the intestinal cells and the intestinal valve. By transposon-mediated deletion mutagenesis we generated nematode strains with deleted P-glycoprotein genes and found that the pgp-3 deletion mutant, but not the pgp-1 mutant, is sensitive to both colchicine and chloroquine. Our results suggest that soil nematodes have P-glycoproteins to protect themselves against toxic compounds made by plants and microbes in the rhizosphere.     

Histidine protects against zinc and nickel toxicity in Caenorhabditis elegans

Zinc is an essential trace element involved in a wide range of biological processes and human diseases. Zinc excess is deleterious, and animals require mechanisms to protect against zinc toxicity. To identify genes that modulate zinc tolerance, we performed a forward genetic screen for Caenorhabditis elegans mutants that were resistant to zinc toxicity. Here we demonstrate that mutations of the C. elegans histidine ammonia lyase (haly-1) gene promote zinc tolerance. C. elegans haly-1 encodes a protein that is homologous to vertebrate HAL, an enzyme that converts histidine to urocanic acid. haly-1 mutant animals displayed elevated levels of histidine, indicating that C. elegans HALY-1 protein is an enzyme involved in histidine catabolism. These results suggest the model that elevated histidine chelates zinc and thereby reduces zinc toxicity. Supporting this hypothesis, we demonstrated that dietary histidine promotes zinc tolerance. Nickel is another metal that binds histidine with high affinity. We demonstrated that haly-1 mutant animals are resistant to nickel toxicity and dietary histidine promotes nickel tolerance in wild-type animals. These studies identify a novel role for haly-1 and histidine in zinc metabolism and may be relevant for other animals.     

Hyperactivation of the proteasome in Caenorhabditis elegans protects against proteotoxic stress and extends lifespan

Virtually all age-related neurodegenerative diseases (NDs) can be characterized by the accumulation of proteins inside and outside the cell that are thought to significantly contribute to disease pathogenesis. One of the cell’s primary systems for the degradation of misfolded/damaged proteins is the ubiquitin proteasome system (UPS), and its impairment is implicated in essentially all NDs. Thus, upregulating this system to combat NDs has garnered a great deal of interest in recent years. Various animal models have focused on stimulating 26S activity and increasing 20S proteasome levels, but thus far, none have targeted intrinsic activation of the 20S proteasome itself. Therefore, we constructed an animal model that endogenously expresses a hyperactive, open gate proteasome in Caenorhabditis elegans. The gate-destabilizing mutation that we introduced into the nematode germline yielded a viable nematode population with enhanced proteasomal activity, including peptide, unstructured protein, and ubiquitin-dependent degradation activities. We determined these nematodes showed a significantly increased lifespan and substantial resistance to oxidative and proteotoxic stress but a significant decrease in fecundity. Our results show that introducing a constitutively active proteasome into a multicellular organism is feasible and suggests targeting the proteasome gating mechanism as a valid approach for future age-related disease research efforts in mammals.     

The exopolysaccharide alginate protects Pseudomonas aeruginosa biofilm bacteria from IFN-gamma-mediated macrophage killing

The ability of Pseudomonas aeruginosa to form biofilms and cause chronic infections in the lungs of cystic fibrosis patients is well documented. Numerous studies have revealed that P. aeruginosa biofilms are highly refractory to antibiotics. However, dramatically fewer studies have addressed P. aeruginosa biofilm resistance to the host's immune system. In planktonic, unattached (nonbiofilm) P. aeruginosa, the exopolysaccharide alginate provides protection against a variety of host factors yet the role of alginate in protection of biofilm bacteria is unclear. To address this issue, we tested wild-type strains PAO1, PA14, the mucoid cystic fibrosis isolate, FRD1 (mucA22+), and the respective isogenic mutants which lacked the ability to produce alginate, for their susceptibility to human leukocytes in the presence and absence of IFN-gamma. Human leukocytes, in the presence of recombinant human IFN-gamma, killed biofilm bacteria lacking alginate after a 4-h challenge at 37 degrees C. Bacterial killing was dependent on the presence of IFN-gamma. Killing of the alginate-negative biofilm bacteria was mediated through mononuclear cell phagocytosis since treatment with cytochalasin B, which prevents actin polymerization, inhibited leukocyte-specific bacterial killing. By direct microscopic observation, phagocytosis of alginate-negative biofilm bacteria was significantly increased in the presence of IFN-gamma vs all other treatments. Addition of exogenous, purified alginate to the alginate-negative biofilms restored resistance to human leukocyte killing. Our results suggest that although alginate may not play a significant role in bacterial attachment, biofilm development, and formation, it may play an important role in protecting mucoid P. aeruginosa biofilm bacteria from the human immune system.     

Mesenchymal stem cell secretome protects against alpha-synuclein-induced neurodegeneration in a Caenorhabditis elegans model of Parkinson's disease

Background aimsThe capacity of the secretome from bone marrow-derived mesenchymal stem cells (BMSCs) to prevent dopaminergic neuron degeneration caused by overexpression of alpha-synuclein (α-syn) was explored using two Caenorhabditis elegans models of Parkinson's disease (PD).MethodsFirst, a more predictive model of PD that overexpresses α-syn in dopamine neurons was subjected to chronic treatment with secretome. This strain displays progressive dopaminergic neurodegeneration that is age-dependent. Following chronic treatment with secretome, the number of intact dopaminergic neurons was determined. Following these initial experiments, a C. elegans strain that overexpresses α-syn in body wall muscle cells was used to determine the impact of hBMSC secretome on α-syn inclusions. Lastly, in silico analysis of the components that constitute the secretome was performed.ResultsThe human BMSC (hBMSC) secretome induced a neuroprotective effect, leading to reduced dopaminergic neurodegeneration. Moreover, in animals submitted to chronic treatment with secretome, the number of α-syn inclusions was reduced, indicating that the secretome of MSCs was possibly contributing to the degradation of those structures. In silico analysis identified possible suppressors of α-syn proteotoxicity, including growth factors and players in the neuronal protein quality control mechanisms.ConclusionsThe present findings indicate that hBMSC secretome has the potential to be used as a disease-modifying strategy in future PD regenerative medicine approaches.     

Staphylococcal biofilm disassembly

Staphylococcus aureus and Staphylococcus epidermidis are a frequent cause of biofilm-associated infections that are a tremendous burden on our healthcare system. Staphylococcal biofilms exhibit extraordinary resistance to antimicrobial killing, limiting the efficacy of antibiotic therapy, and surgical intervention is often required to remove infected tissues or implanted devices. Recent work has provided new insight into the molecular basis of biofilm development in these opportunistic pathogens. Extracellular bacterial products, environmental conditions, and polymicrobial interactions have all been shown to influence profoundly the ability of these bacteria to colonize and disperse from clinically relevant surfaces. We review new developments in staphylococcal biofilm disassembly and set them in the context of potential strategies to control biofilm infections.     

Specificity and complexity of the Caenorhabditis elegans innate immune response

In response to infection, Caenorhabditis elegans produces an array of antimicrobial proteins. To understand the C. elegans immune response, we have investigated the regulation of a large, representative sample of candidate antimicrobial genes. We found that all these putative antimicrobial genes are expressed in tissues exposed to the environment, a position from which they can ward off infection. Using RNA interference to inhibit the function of immune signaling pathways in C. elegans, we found that different immune response pathways regulate expression of distinct but overlapping sets of antimicrobial genes. We also show that different bacterial pathogens regulate distinct but overlapping sets of antimicrobial genes. The patterns of genes induced by pathogens do not coincide with any single immune signaling pathway. Thus, even in this simple model system for innate immunity, striking specificity and complexity exist in the immune response. The unique patterns of antimicrobial gene expression observed when C. elegans is exposed to different pathogens or when different immune signaling pathways are perturbed suggest that a large set of yet to be identified pathogen recognition receptors (PRRs) exist in the nematode. These PRRs must interact in a complicated fashion to induce a unique set of antimicrobial genes. We also propose the existence of an "antimicrobial fingerprint," which will aid in assigning newly identified C. elegans innate immunity genes to known immune signaling pathways.     

Priming of inducible defenses protects Norway spruce against tree-killing bark beetles

Plants can form an immunological memory known as defense priming, whereby exposure to a priming stimulus enables quicker or stronger response to subsequent attack by pests and pathogens. Such priming of inducible defenses provides increased protection and reduces allocation costs of defense. Defense priming has been widely studied for short-lived model plants such as Arabidopsis, but little is known about this phenomenon in long-lived plants like spruce. We compared the effects of pretreatment with sublethal fungal inoculations or application of the phytohormone methyl jasmonate (MeJA) on the resistance of 48-year-old Norway spruce (Picea abies) trees to mass attack by a tree-killing bark beetle beginning 35 days later. Bark beetles heavily infested and killed untreated trees but largely avoided fungus-inoculated trees and MeJA-treated trees. Quantification of defensive terpenes at the time of bark beetle attack showed fungal inoculation induced 91-fold higher terpene concentrations compared with untreated trees, whereas application of MeJA did not significantly increase terpenes. These results indicate that resistance in fungus-inoculated trees is a result of direct induction of defenses, whereas resistance in MeJA-treated trees is due to defense priming. This work extends our knowledge of defense priming from model plants to an ecologically important tree species.     

Autophagy protects against hypoxic injury in C. elegans

Macroautophagy has been implicated in a variety of pathological processes. Hypoxic/ischemic cellular injury is one such process in which autophagy has emerged as an important regulator. In general, autophagy is induced after an hypoxic/ischemic insult; however, whether the induction of autophagy promotes cell death or recovery is controversial and appears to be context dependent. We have developed C. elegans as a genetically tractable model for the study of hypoxic cell injury. Both necrosis and apoptosis are mechanisms of cell death following hypoxia in C. elegans. However, the role of autophagy in hypoxic injury in C. elegans has not been examined. Here, we found that RNAi knockdown of the C. elegans homologs of beclin 1/Atg6 (bec-1) and LC3/Atg8 (lgg-1, lgg-2), and mutation of Atg1 (unc-51) decreased animal survival after a severe hypoxic insult. Acute inhibition of autophagy by the type III phosphatidylinositol 3-kinase inhibitors, 3-methyladenine and Wortmannin, also sensitized animals to hypoxic death. Hypoxia-induced neuronal and myocyte injury as well as necrotic cellular morphology were increased by RNAi knockdown of BEC-1. Hypoxia increased the expression of a marker of autophagosomes in a bec-1-dependent manner. Finally, we found that the hypoxia hypersensitive phenotype of bec-1(RNAi) animals could be blocked by loss-of-function mutations in either the apoptosis or necrosis pathway. These results argue that inhibition of autophagy sensitizes C. elegans and its cells to hypoxic injury and that this sensitization is blocked or circumvented when either of the two major cell death mechanisms is inhibited.     

Autophagy protects against hypoxic injury in C. elegans

Macroautophagy has been implicated in a variety of pathological processes. Hypoxic/ischemic cellular injury is one such process in which autophagy has emerged as an important regulator. In general, autophagy is induced after a hypoxic/ischemic insult; however, whether the induction of autophagy promotes cell death or recovery is controversial and appears to be context dependent. We have developed C. elegans as a genetically tractable model for the study of hypoxic cell injury. Both necrosis and apoptosis are mechanisms of cell death following hypoxia in C. elegans. However, the role of autophagy in hypoxic injury in C. elegans has not been examined. Here, we found that RNAi knockdown of the C. elegans homologs of beclin 1/Atg6 (bec-1) and LC3/Atg8 (lgg-1, lgg-2), and mutation of Atg1 (unc-51) decreased animal survival after a severe hypoxic insult. Acute inhibition of autophagy by the type III phosphatidylinositol 3-kinase inhibitors, 3-methyladenine and Wortmannin, also sensitized animals to hypoxic death. Hypoxia-induced neuronal and myocyte injury as well as necrotic cellular morphology were increased by RNAi knockdown of BEC-1. Hypoxia increased the expression of a marker of autophagosomes in a bec-1-dependent manner. Finally, we found that the hypoxia hypersensitive phenotype of bec-1(RNAi) animals could be blocked by loss-of-function mutations in either the apoptosis or necrosis pathway. These results argue that inhibition of autophagy sensitizes C. elegans and its cells to hypoxic injury and that this sensitization is blocked or circumvented when either of the two major cell-death mechanisms is inhibited.     

Selection against males in Caenorhabditis elegans under two mutational treatments

Within populations with mixed mating systems, selfing is expected to be favoured over outcrossing unless a countervailing process such as severe inbreeding depression is present. In this study, we consider the relationship between the expression of deleterious alleles and the maintenance of outcrossing in the nematode species, Caenorhabditis elegans. This species is characterized by an androdioecious breeding system composed of males at low frequency and self-fertilizing hermaphrodites that can only outcross via males. Here, we find that experimentally increasing the mutational load in four different isogenic wild isolates using 10 generations of Ethylmethane sulphonate (EMS) and UV irradiation mutagenesis significantly diminishes the cost of males. Males are maintained at higher frequencies in mutagenized versus non-mutagenized populations. Nevertheless, males still tend to be driven to low frequencies within isolates that are known to be prone to lose males. Further, we determine the viability effects of a single round of mutagen exposure and find that, for EMS, outcrossing overcomes the almost completely recessive and nearly lethal effects generated. We briefly interpret our results in light of current evolutionary theory of outcrossing rates.     

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.     

Amphibian immune defenses against chytridiomycosis: impacts of changing environments

Eco-immunology is the field of study that attempts to understand the functions of the immune system in the context of the host's environment. Amphibians are currently suffering devastating declines and extinctions in nearly all parts of the world due to the emerging infectious disease chytridiomycosis caused by the chytrid fungus, Batrachochytrium dendrobatidis. Because chytridiomycosis is a skin infection and remains confined to the skin, immune defenses of the skin are critical for survival. Skin defenses include secreted antimicrobial peptides and immunoglobulins as well as antifungal metabolites produced by symbiotic skin bacteria. Low temperatures, toxic chemicals, and stress inhibit the immune system and may impair natural defenses against B. dendrobatidis. Tadpoles' mouth parts can be infected by B. dendrobatidis. Damage to the mouth parts can impair growth, and the affected tadpoles maintain the pathogen in the environment even when adults have dispersed. Newly metamorphosing frogs appear to be especially vulnerable to infection and to the lethal effects of this pathogen because the immune system undergoes a dramatic reorganization at metamorphosis, and postmetamorphic defenses are not yet mature. Here we review our current understanding of amphibian immune defenses against B. dendrobatidis and the ability of the pathogen to resist those defenses. We also briefly review what is known about the impacts of temperature, environmental chemicals, and stress on the host-pathogen interactions and suggest future directions for research.