Molecular features of the cytolytic pore-forming bacterial protein toxins

The repertoire of the cytolytic pore-forming protein toxins (PFT) comprises 81 identified members. The essential feature of these cytolysins is their capacity to provoke the formation of hydrophilic pores in the cytoplasmic membranes of target eukaryotic cells. This process results from the binding of the proteins on the cell surface, followed by their oligomerization which leads to the insertion of the oligomers into the membrane and formation of protein-lined channels. It impairs the osmotic balance of the cell and causes cytolysis. In this review the molecular aspects of a number of important PFT and their respective encoding structural genes will be briefly described.     

Systemic regulation of autophagy in Caenorhabditis elegans

When no supply of environmental nutrients is available, cells induce autophagy, thereby generating a source of emergency metabolic substrates and energy to maintain the basal cellular activity needed for survival. This autophagy response to starvation has been well characterized in various multicellular organisms, including worms, flies, and mice. Although prosurvival effects of autophagy in response to starvation are well known in animals, the mechanisms by which animals regulate and coordinate autophagy systemically remain elusive. Using C. elegans as a model system, we found that specific amino acids could regulate starvation-induced autophagy, and that MGL-1 and MGL-2, Caenorhabditis elegans homologs of metabotropic glutamate receptors, were involved. MGL-1 and MGL-2 specifically acted in AIY and AIB neurons, respectively, to modulate the autophagy response in other tissues such as pharyngeal muscle. Our recent study suggests that the autophagy response to starvation, previously thought to be cell-autonomous, can be systemically regulated, and that there is a specific sensor for monitoring systemic amino acids levels in Caenorhabditis elegans.     

Mitochondrial electron transport is a key determinant of life span in Caenorhabditis elegans.

Increased protection from reactive oxygen species (ROS) is believed to increase life span. However, it has not been clearly demonstrated that endogenous ROS production actually limits normal life span. We have identified a mutation in the Caenorhabditis elegans iron sulfur protein (isp-1) of mitochondrial complex III, which results in low oxygen consumption, decreased sensitivity to ROS, and increased life span. Furthermore, combining isp-1(qm150) with a mutation (daf-2) that increases resistance to ROS does not result in any significant further increase in adult life span. These findings indicate that both isp-1 and daf-2 mutations increase life span by lowering oxidative stress and result in the maximum life span increase that can be produced in this way.     

Facultative Vivipary is a Life-History Trait in Caenorhabditis elegans

Organisms partition their resources among growth, maintenance, and reproduction and, when resources become limiting, the allocation to one process necessitates reduced allocation to others. When starved, Caenorhabditis elegans adults retain progeny internally which then consume the parent body contents, and some of those larvae use the resources to reach the resistant, long-lived dauer stage. If starved under similarly extreme conditions, larvae from eggs laid outside of the body are unable to develop into dauers. We interpret this switch from ovipary, or laying eggs, to bearing live young as facultative vivipary. This switch is induced by starvation of late fourth-stage larvae, young adults, or gravid adults. In C. elegans, vivipary is the altruistic allocation of all available parental energy and nutrients to progeny, with the associated costs to adult hermaphrodites of truncated life span and fecundity. As a life-history trait, facultative vivipary is a survival-enhancing response to stress that may provide insights into the evolution of reproduction and longevity.     

Caenorhabditis elegans development requires mitochondrial function in the nervous system

The mitochondrial respiratory chain (MRC) supplies the majority of the energy requirements of most eucaryotic cells. A null mutation in the Caenorhabditis elegans nuo-1 gene encoding a subunit of complex I (NADH-ubiquinone oxidoreductase) is lethal, leading to a developmental arrest at the third larval stage. To identify the tissues that regulate development in response to mitochondrial dysfunction, we restored nuo-1 expression with tissue-specific promoters. Only expression of nuo-1 ubiquitously or in the nervous system supported development to the adult stage. Pharyngeal expression of nuo-1 allowed development to proceed to the fourth larval stage. Expression of nuo-1 in the body muscles or in the germline had no effect. Furthermore, only ubiquitous or nervous system expression of nuo-1 allowed exit from the dauer state. Our results indicate that MRC function in the nervous system is needed to send and receive signals that control larval development and exit from dauer.     

Detection of deletions in the mitochondrial genome of Caenorhabditis elegans.

We have examined an aging population of Caenorhabditis elegans via a PCR assay to determine if deletions in the mitochondrial genome occur in the nematode. We detected eight such deletions, identified the breakpoints of four of these, and discovered direct repeats of 4-8 base pairs at the site of all four deletions. Six of the eight repeats involved in the deletions are located in or immediately adjacent to tRNAs. Without a biochemical bias, the probability of direct repeats being present at all four breakpoints was 4 x 10(-6).     

A new Caenorhabditis elegans model to study copper toxicity in Wilson disease

Wilson disease (WD) is caused by mutations in the ATP7B gene that encodes a copper (Cu) transporting ATPase whose trafficking from the Golgi to endo-lysosomal compartments drives sequestration of excess Cu and its further excretion from hepatocytes into the bile. Loss of ATP7B function leads to toxic Cu overload in the liver and subsequently in the brain, causing fatal hepatic and neurological abnormalities. The limitations of existing WD therapies call for the development of new therapeutic approaches, which require an amenable animal model system for screening and validation of drugs and molecular targets. To achieve this objective, we generated a mutant Caenorhabditis elegans strain with a substitution of a conserved histidine (H828Q) in the ATP7B ortholog cua-1 corresponding to the most common ATP7B variant (H1069Q) that causes WD. cua-1 mutant animals exhibited very poor resistance to Cu compared to the wild-type strain. This manifested in a strong delay in larval development, a shorter lifespan, impaired motility, oxidative stress pathway activation, and mitochondrial damage. In addition, morphological analysis revealed several neuronal abnormalities in cua-1 mutant animals exposed to Cu. Further investigation suggested that mutant CUA-1 is retained and degraded in the endoplasmic reticulum, similarly to human ATP7B-H1069Q. As a consequence, the mutant protein does not allow animals to counteract Cu toxicity. Notably, pharmacological correctors of ATP7B-H1069Q reduced Cu toxicity in cua-1 mutants indicating that similar pathogenic molecular pathways might be activated by the H/Q substitution and, therefore, targeted for rescue of ATP7B/CUA-1 function. Taken together, our findings suggest that the newly generated cua-1 mutant strain represents an excellent model for Cu toxicity studies in WD.     

Caspase-1 activation of lipid metabolic pathways in response to bacterial pore-forming toxins promotes cell survival

Many pathogenic organisms produce pore-forming toxins as virulence factors. Target cells however mount a response to such membrane damage. Here we show that toxin-induced membrane permeabilization leads to a decrease in cytoplasmic potassium, which promotes the formation of a multiprotein oligomeric innate immune complex, called the inflammasome, and the activation of caspase-1. Further, we find that when rendered proteolytic in this context caspase-1 induces the activation of the central regulators of membrane biogenesis, the Sterol Regulatory Element Binding Proteins (SREBPs), which in turn promote cell survival upon toxin challenge possibly by facilitating membrane repair. This study highlights that, in addition to its well-established role in triggering inflammation via the processing of the precursor forms of interleukins, caspase-1 has a broader role, in particular linking the intracellular ion composition to lipid metabolic pathways, membrane biogenesis, and survival.     

srf-3, a mutant of Caenorhabditis elegans, resistant to bacterial infection and to biofilm binding, is deficient in glycoconjugates

srf-3 is a mutant of C. elegans that is resistant to infection by Microbacterium nematophilum and to binding of the biofilm produced by Yersinia pseudotuberculosis and Yersinia pestis. Recently, SRF-3 was characterized as a nucleotide sugar transporter of the Golgi apparatus occurring exclusively in hypodermal seam cells, pharyngeal cells, and spermatheca. Based on the above observations, we hypothesized that srf-3 may have altered glyconjugates that may enable the mutant nematode to grow unaffected in the presence of the above pathogenic bacteria. Following analyses of N- and O-linked glycoconjugates of srf-3 and wild type nematodes using a combination of enzymatic degradation, permethylation, and mass spectrometry, we found in srf-3 a 65% reduction of acidic O-linked glycoconjugates containing glucuronic acid and galactose as well as a reduction of N-linked glycoconjugates containing galactose and fucose. These results are consistent with the specificity of SRF-3 for UDP-galactose and strongly suggest that the above glycoconjugates play an important role in allowing adhesion of M. nematophilum or Y. pseudotuberculosis biofilm to wild type C. elegans. Furthermore, because seam cells as well as pharyngeal cells secrete their glycoconjugates to the cuticle and surrounding surfaces, the results also demonstrate the critical role of these cells and their secreted glycoproteins in nematode-bacteria interactions and offer a mechanistic basis for strategies to block such recognition processes.     

Multiple genes affect sensitivity of Caenorhabditis elegans to the bacterial pathogen Microbacterium nematophilum

Interactions with bacteria play a major role in immune responses, ecology, and evolution of all animals, but they have been neglected until recently in the case of C. elegans. We report a genetic investigation of the interaction of C. elegans with the nematode-specific pathogen Microbacterium nematophilum, which colonizes the rectum and causes distinctive tail swelling in its host. A total of 121 mutants with altered response to infection were isolated from selections or screens for a bacterially unswollen (Bus) phenotype, using both chemical and transposon mutagenesis. Some of these correspond to known genes, affecting either bacterial adhesion or colonization (srf-2, srf-3, srf-5) or host swelling response (sur-2, egl-5). Most mutants define 15 new genes (bus-1-bus-6, bus-8, bus-10, bus-12-bus-18). The majority of these mutants exhibit little or no rectal infection when challenged with the pathogen and are probably altered in surface properties such that the bacteria can no longer infect worms. A number have corresponding alterations in lectin staining and cuticle fragility. Most of the uninfectable mutants grow better than wild type in the presence of the pathogen, but the sur-2 mutant is hypersensitive, indicating that the tail-swelling response is associated with a specific defense mechanism against this pathogen.     

Two modes of mitochondrial dysfunction lead independently to lifespan extension in Caenorhabditis elegans

In Caenorhabditis elegans, longevity is increased by a partial loss‐of‐function mutation in the mitochondrial complex III subunit gene isp‐1. Longevity is also increased by RNAi against the expression of a variety of mitochondrial respiratory chain genes, including isp‐1, but it is unknown whether the isp‐1(qm150) mutation and the RNAi treatments trigger the same underlying mechanisms of longevity. We have identified nuo‐6(qm200), a mutation in a conserved subunit of mitochondrial complex I (NUDFB4). The mutation reduces the function of complex I and, like isp‐1(qm150), results in low oxygen consumption, slow growth, slow behavior, and increased lifespan. We have compared the phenotypes of nuo‐6(qm200) to those of nuo‐6(RNAi) and found them to be distinct in crucial ways, including patterns of growth and fertility, behavioral rates, oxygen consumption, ATP levels, autophagy, and resistance to paraquat, as well as expression of superoxide dismutases, mitochondrial heat‐shock proteins, and other gene expression markers. RNAi treatments appear to generate a stress and autophagy response, while the genomic mutation alters electron transport and reactive oxygen species metabolism. For many phenotypes, we also compared isp‐1(qm150) to isp‐1(RNAi) and found the same pattern of differences. Most importantly, we found that, while the lifespan of nuo‐6, isp‐1 double mutants is not greater than that of the single mutants, the lifespan increase induced by nuo‐6(RNAi) is fully additive to that induced by isp‐1(qm150), and the increase induced by isp‐1(RNAi) is fully additive to that induced by nuo‐6(qm200). Our results demonstrate that distinct and separable aspects of mitochondrial biology affect lifespan independently.     

Lipase-like 5 enzyme controls mitochondrial activity in response to starvation in Caenorhabditis elegans

The C. elegans lipase-like 5 (lipl-5) gene is predicted to code for a lipase homologous to the human gastric acid lipase. Its expression was previously shown to be modulated by nutritional or immune cues, but nothing is known about its impact on the lipid landscape and ensuing functional consequences. In the present work, we used mutants lacking LIPL-5 protein and found that lipl-5 is important for normal lipidome composition as well as its remodeling in response to food deprivation. Particularly, lipids with signaling functions such as ceramides and mitochondrial lipids were affected by lipl-5 silencing. In comparison with wild type worms, animals lacking LIPL-5 were enriched in cardiolipins linked to polyunsaturated C20 fatty acids and coenzyme Q-9. Differences in mitochondrial lipid composition were accompanied by differences in mitochondrial activity as mitochondria from well-fed lipl-5 mutants were significantly more able to oxidize respiratory substrates when compared with mitochondria from well-fed wild type worms. Strikingly, starvation elicited important changes in mitochondrial activity in wild type worms, but not in lipl-5 worms. This indicates that this lipase is a determinant of mitochondrial functional remodeling in response to food withdrawal.     

The mitochondrial prohibitin complex is essential for embryonic viability and germline function in Caenorhabditis elegans

Prohibitins in eukaryotes consist of two subunits (PHB1 and PHB2) that together form a high molecular weight complex in the mitochondrial inner membrane. The evolutionary conservation and the ubiquitous expression in mammalian tissues of the prohibitin complex suggest an important function among eukaryotes. The PHB complex has been shown to play a role in the stabilization of newly synthesized subunits of mitochondrial respiratory enzymes in the yeast Saccharomyces cerevisiae. We have used Caenorhabditis elegans as model system to study the role of the PHB complex during development of a multicellular organism. We demonstrate that prohibitins in C. elegans form a high molecular weight complex in the mitochondrial inner membrane similar to that of yeast and humans. By using RNA-mediated gene inactivation, we show that PHB proteins are essential during embryonic development and are required for somatic and germline differentiation in the larval gonad. We further demonstrate that a deficiency in PHB proteins results in altered mitochondrial biogenesis in body wall muscle cells. This paper reports a strong loss of function phenotype for prohibitin gene inactivation in a multicellular organism and shows for the first time that prohibitins serve an essential role in mitochondrial function during organismal development.     

The pore-forming protein Cry5B elicits the pathogenicity of Bacillus sp. against Caenorhabditis elegans

The soil bacterium Bacillus thuringiensis is a pathogen of insects and nematodes and is very closely related to, if not the same species as, Bacillus cereus and Bacillus anthracis. The defining characteristic of B. thuringiensis that sets it apart from B. cereus and B. anthracis is the production of crystal (Cry) proteins, which are pore-forming toxins or pore-forming proteins (PFPs). Although it is known that PFPs are important virulence factors since their elimination results in reduced virulence of many pathogenic bacteria, the functions by which PFPs promote virulence are incompletely understood. Here we study the effect of Cry proteins in B. thuringiensis pathogenesis of the nematode Caenorhabditis elegans. We find that whereas B. thuringiensis on its own is not able to infect C. elegans, the addition of the PFP Cry protein, Cry5B, results in a robust lethal infection that consumes the nematode host in 1-2 days, leading to a "Bob" or bag-of-bacteria phenotype. Unlike other infections of C. elegans characterized to date, the infection by B. thuringiensis shows dose-dependency based on bacterial inoculum size and based on PFP concentration. Although the infection process takes 1-2 days, the PFP-instigated infection process is irreversibly established within 15 minutes of initial exposure. Remarkably, treatment of C. elegans with Cry5B PFP is able to instigate many other Bacillus species, including B. anthracis and even "non-pathogenic" Bacillus subtilis, to become lethal and infectious agents to C. elegans. Co-culturing of Cry5B-expressing B. thuringiensis with B. anthracis can result in lethal infection of C. elegans by B. anthracis. Our data demonstrate that one potential property of PFPs is to sensitize the host to bacterial infection and further that C. elegans and probably other roundworms can be common hosts for B. cereus-group bacteria, findings with important ecological and research implications.     

Caenorhabditis elegans,a pluricellular model organism to screen new genes involved in mitochondrial genome maintenance

The inheritance of functional mitochondria depends on faithful replication and transmission of mitochondrial DNA (mtDNA). A large and heterogeneous group of human disorders is associated with mitochondrial genome quantitative and qualitative anomalies. Several nuclear genes have been shown to account for these severe OXPHOS disorders. However, in several cases, the disease-causing mutations still remain unknown.Caenorhabditis elegans has been largely used for studying various biological functions because this multicellular organism has short life cycle and is easy to grow in the laboratory. Mitochondrial functions are relatively well conserved between human and C. elegans, and heteroplasmy exists in this organism as in human. C. elegans therefore represents a useful tool for studying mtDNA maintenance. Suppression by RNA interference of genes involved in mtDNA replication such as polg-1, encoding the mitochondrial DNA polymerase, results in reduced mtDNA copy number but in a normal phenotype of the F1 worms. By combining RNAi of genes involved in mtDNA maintenance and EtBr exposure, we were able to reveal a strong and specific phenotype (developmental larval arrest) associated to a severe decrease of mtDNA copy number. Moreover, we tested and validated the screen efficiency for human orthologous genes encoding mitochondrial nucleoid proteins. This allowed us to identify several genes that seem to be closely related to mtDNA maintenance in C. elegans.This work reports a first step in the further development of a large-scale screening in C. elegans that should allow to identify new genes of mtDNA maintenance whose human orthologs will obviously constitute new candidate genes for patients with quantitative or qualitative mtDNA anomalies.     

Genetic impairment of autophagy intensifies expanded polyglutamine toxicity in Caenorhabditis elegans

Neuronal homeostasis requires a balance between anabolic and catabolic processes. Eukaryotic cells use two distinct systems for the degradation of unused proteins: the ubiquitin-proteasome system and the autophagic system. The autophagic system is also necessary for the degradation of bulk amounts of proteins and organelles. We have searched for new autophagy-related genes in the Caenorhabditis elegans genome and investigated their role in a polyglutamine (polyQ) disease model. Here, we have shown that inactivation of these genes intensified the toxicity of expanded polyQ in C. elegans neurons and muscles, and at the same time inactivation of CeTor reduced the polyQ toxicity.