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.     

Mos1-mediated insertional mutagenesis in Caenorhabditis elegans

We describe a protocol for mutating genes in the nematode Caenorhabditis elegans using the Mos1 transposon of Drosophila mauritiana. Mutated genes containing a Mos1 insertion are molecularly tagged by this heterologous transposable element. Mos1 insertions can therefore be identified in as little as 3 weeks using only basic molecular biology techniques. Mutagenic efficiency of Mos1 is tenfold lower than classical chemical mutagens. However, the ease and speed with which mutagenic insertions can be mapped compares favorably with the vast amount of work involved in classical genetic mapping. Therefore, Mos1 could be the tool of choice when screening procedures are efficient. In addition, Mos1 mutagenesis can greatly simplify the mapping of mutations that exhibit low penetrance, subtle or synthetic phenotypes. The recent development of targeted engineering of C. elegans loci carrying Mos1 insertions further increases the attractiveness of Mos1-mediated mutagenesis.     

Genome characterization, analysis of virulence and transformation of Microbacterium nematophilum, a coryneform pathogen of the nematode Caenorhabditis elegans

A coryneform bacterium designated Microbacterium nematophilum has previously been reported to act as a pathogen for Caenorhabditis elegans. This bacterium is able to colonize the rectum of infected worms and cause localized swelling, constipation and slowed growth. Additional isolates and analysis of this bacterium are described here. Tests of pathogenicity on other Caenorhabditis nematodes show that M. nematophilum infection is lethal to most species in the genus, in contrast to its relatively mild effects on C. elegans. The size and geometry of the pathogen genome have been determined as a closed circular molecule of 2.85 Mb with high G+C content. Bacteria also harbor a 55 kb plasmid, pMN1, which is largely composed of a lysogenic bacteriophage genome. Mutagenesis experiments have yielded stable avirulent mutants of M. nematophilum. As a first step towards molecular genetic analysis, methods for low-efficiency transformation of M. nematophilum have been developed.     

Characterization of Mos1-mediated mutagenesis in Caenorhabditis elegans: a method for the rapid identification of mutated genes

Insertional mutagenesis with a heterologous transposon provides a method to rapidly determine the molecular identity of mutated genes. The Drosophila transposon Mos1 can be mobilized to cause mutations in Caenorhabditis elegans (Bessereau et al. 2001); however, the mutagenic rate was initially too low for use in most forward genetic screens. To increase the effectiveness of Mos1-mediated mutagenesis we examined the conditions influencing Mos1 transposition. First, optimal transposition occurs 24 hr after expression of the transposase and is unlikely to occur in differentiated sperm or oocytes. Second, transposition is limited to germ-cell nuclei that contain donor elements, but the transposase enzyme can diffuse throughout the gonad syncytium. Third, silencing of transposition is caused by changes in the donor array that occur over time. Finally, multiple transposition events occur in individual germ cells. By using screening techniques based on these results, Mos1 mutagenicity was increased to within an order of magnitude of chemical mutagens.     

Mos as a tool for genome-wide insertional mutagenesis in Caenorhabditis elegans: results of a pilot study

The sequence of the Caenorhabditis elegans genome contains approximately 19 000 genes. Available mutants currently exist for <20% of these genes. The existence of a Mos-based inducible transposon system in C.elegans could theoretically serve as a tool to saturate the genome with insertions. We report here the results of a pilot study aimed at assaying this strategy. We generated 914 independent random Mos insertions and determined their location by inverse PCR. The distribution of the insertions throughout the genome does not reveal any gross distortion, with the exception of a major hotspot on chromosome I (rDNA locus). Transposons are evenly distributed between the genic and intergenic regions. Within genes, transposons insert preferentially into the introns. We derived the consensus target site for Mos in C.elegans (ATATAT), which is common to Tc1, another mariner element. Finally, we assayed the mutagenic properties of insertions located in exons by comparing the phenotype of homozygous strains to that of known mutations or RNAi of the same gene. This pilot experiment shows that a Mos-based approach is a viable strategy that can contribute to the constitution of genome-wide collections of identified C.elegans mutants.     

Targeted engineering of the Caenorhabditis elegans genome following Mos1-triggered chromosomal breaks

The Drosophila element Mos1 is a class II transposon, which moves by a 'cut-and-paste' mechanism and can be experimentally mobilized in the Caenorhabditis elegans germ line. Here, we triggered the excision of identified Mos1 insertions to create chromosomal breaks at given sites and further manipulate the broken loci. Double-strand break (DSB) repair could be achieved by gene conversion using a transgene containing sequences homologous to the broken chromosomal region as a repair template. Consequently, mutations engineered in the transgene could be copied to a specific locus at high frequency. This pathway was further characterized to develop an efficient tool--called MosTIC--to manipulate the C. elegans genome. Analysis of DSB repair during MosTIC experiments demonstrated that DSBs could also be sealed by end-joining in the germ line, independently from the evolutionarily conserved Ku80 and ligase IV factors. In conjunction with a publicly available Mos1 insertion library currently being generated, MosTIC will provide a general tool to customize the C. elegans genome.     

Sensitivity of Caenorhabditis elegans clk-1 mutants to ubiquinone side-chain length reveals multiple ubiquinone-dependent processes

Ubiquinone (coenzyme Q, or Q) is a membrane constituent, whose head group is capable of accepting and donating electrons and whose lipidic side chain is composed of a variable number of isoprene subunits. A possible role for Q as a dietary antioxidant for treating conditions that involve altered cellular redox states is being intensely studied. Mutations in the clk-1 gene of the nematode Caenorhabditis elegans affect numerous physiological rates including behavioral rates, developmental rates, reproduction, and life span. clk-1 encodes a protein associated with the inner mitochondrial membrane that is necessary for Q biosynthesis in C. elegans. clk-1 mutants do not synthesize Q but accumulate demethoxyubiquinone, a Q synthesis intermediate that is able to partially sustain mitochondrial respiration in worms as well as in mammals. Recently, we and others have found that exogenous Q is necessary for the fertility and development of clk-1 mutants. Here, we take advantage of the clk-1 genetic model to identify structural features of Q that are functionally important in vivo. We show that clk-1 mutants are exquisitely sensitive to the length of the side chain of the Q they consume. We also identified differential sensitivity to Q side-chain length between null alleles of clk-1 (qm30 and qm51) and the weaker allele e2519. This allows us to propose a model where we distinguish several types of Q-dependent processes in vivo: processes that are very sensitive to Q side-chain length and processes that are permissive to Q with shorter chains.     

Caenorhabditis elegans reveals a FxNPxY-independent low-density lipoprotein receptor internalization mechanism mediated by epsin1

Low-density lipoprotein receptor (LDLR) internalization clears cholesterol-laden LDL particles from circulation in humans. Defects in clathrin-dependent LDLR endocytosis promote elevated serum cholesterol levels and can lead to atherosclerosis. However, our understanding of the mechanisms that control LDLR uptake remains incomplete. To identify factors critical to LDLR uptake, we pursued a genome-wide RNA interference screen using Caenorhabditis elegans LRP-1/megalin as a model for LDLR transport. In doing so, we discovered an unanticipated requirement for the clathrin-binding endocytic adaptor epsin1 in LDLR endocytosis. Epsin1 depletion reduced LDLR internalization rates in mammalian cells, similar to the reduction observed following clathrin depletion. Genetic and biochemical analyses of epsin in C. elegans and mammalian cells uncovered a requirement for the ubiquitin-interaction motif (UIM) as critical for receptor transport. As the epsin UIM promotes the internalization of some ubiquitinated receptors, we predicted LDLR ubiquitination as necessary for endocytosis. However, engineered ubiquitination-impaired LDLR mutants showed modest internalization defects that were further enhanced with epsin1 depletion, demonstrating epsin1-mediated LDLR endocytosis is independent of receptor ubiquitination. Finally, we provide evidence that epsin1-mediated LDLR uptake occurs independently of either of the two documented internalization motifs (FxNPxY or HIC) encoded within the LDLR cytoplasmic tail, indicating an additional internalization mechanism for LDLR.     

High genetic diversity in the chemoreceptor superfamily of Caenorhabditis elegans

We investigated genetic polymorphism in the Caenorhabditis elegans srh and str chemoreceptor gene families, each of which consists of approximately 300 genes encoding seven-pass G-protein-coupled receptors. Almost one-third of the genes in each family are annotated as pseudogenes because of apparent functional defects in N2, the sequenced wild-type strain of C. elegans. More than half of these "pseudogenes" have only one apparent defect, usually a stop codon or deletion. We sequenced the defective region for 31 such genes in 22 wild isolates of C. elegans. For 10 of the 31 genes, we found an apparently functional allele in one or more wild isolates, suggesting that these are not pseudogenes but instead functional genes with a defective allele in N2. We suggest the term "flatliner" to describe genes whose functional vs. pseudogene status is unclear. Investigations of flatliner gene positions, d(N)/d(S) ratios, and phylogenetic trees indicate that they are not readily distinguished from functional genes in N2. We also report striking heterogeneity in the frequency of other polymorphisms among these genes. Finally, the large majority of polymorphism was found in just two strains from geographically isolated islands, Hawaii and Madeira. This suggests that our sampling of wild diversity in C. elegans is narrow and that identification of additional strains from similarly isolated regions will greatly expand the diversity available for study.     

A mutant affecting the heavy chain of myosin in Caenorhabditis elegans

A set of non-complementing, closely linked, ethyl methanesulphonate-induced mutations in Caenorhabditis elegans specifically affects the structure and function of body-wall muscle cells but not the pharyngeal musculature. One of these mutations, e675, is semidominant and results in the production of a new protein of about 203,000 molecular weight in addition to normal myosin at about 210,000 M_r. The abnormal polypeptide chain is structurally very similar to normal myosin heavy chain when maps of iodinated peptides are compared.The E675 mutant shows a clear relation between defective movement, disruption of the body-wall muscle structure, and the molecular defect in the myosin heavy chains. The altered chain is synthesized in heterozygotes, suggesting that the e675 mutation is either in a structural gene for the heavy chain or in a cis acting control element. The hypothesis that there are two classes of myosin heavy chain within the same cells is discussed.     

Topological cluster analysis reveals the systemic organization of the Caenorhabditis elegans connectome

The modular organization of networks of individual neurons interwoven through synapses has not been fully explored due to the incredible complexity of the connectivity architecture. Here we use the modularity-based community detection method for directed, weighted networks to examine hierarchically organized modules in the complete wiring diagram (connectome) of Caenorhabditis elegans (C. elegans) and to investigate their topological properties. Incorporating bilateral symmetry of the network as an important cue for proper cluster assignment, we identified anatomical clusters in the C. elegans connectome, including a body-spanning cluster, which correspond to experimentally identified functional circuits. Moreover, the hierarchical organization of the five clusters explains the systemic cooperation (e.g., mechanosensation, chemosensation, and navigation) that occurs among the structurally segregated biological circuits to produce higher-order complex behaviors.     

The response of Caenorhabditis elegans to hydrogen sulfide and hydrogen cyanide

Hydrogen sulfide (H2S), an endogenously produced small molecule, protects animals from various stresses. Recent studies demonstrate that animals exposed to H2S are long lived, resistant to hypoxia, and resistant to ischemia-reperfusion injury. We performed a forward genetic screen to gain insights into the molecular mechanisms Caenorhabditis elegans uses to appropriately respond to H2S. At least two distinct pathways appear to be important for this response, including the H2S-oxidation pathway and the hydrogen cyanide (HCN)-assimilation pathway. The H2S-oxidation pathway requires two distinct enzymes important for the oxidation of H2S: the sulfide:quinone reductase sqrd-1 and the dioxygenase ethe-1. The HCN-assimilation pathway requires the cysteine synthase homologs cysl-1 and cysl-2. A low dose of either H2S or HCN can activate hypoxia-inducible factor 1 (HIF-1), which is required for C. elegans to respond to either gas. sqrd-1 and cysl-2 represent the entry points in the H2S-oxidation and HCN-assimilation pathways, respectively, and expression of both of these enzymes is highly induced by HIF-1 in response to both H2S and HCN. In addition to their role in appropriately responding to H2S and HCN, we found that cysl-1 and cysl-2 are both essential mediators of innate immunity against fast paralytic killing by Pseudomonas. Furthermore, in agreement with these data, we showed that growing worms in the presence of H2S is sufficient to confer resistance to Pseudomonas fast paralytic killing. Our results suggest the hypoxia-independent hif-1 response in C. elegans evolved to respond to the naturally occurring small molecules H2S and HCN.     

Structural and functional diversity in the neuronal microtubules of Caenorhabditis elegans

Tannic acid fixation reveals differences in the number of protofilaments between microtubules (MTs) in the nematode Caenorhabditis elegans. Most cells have MTs with 11 protofilaments but the six touch receptor neurons (the microtubule cells) have MTs with 15 protofilaments. No 13-protofilament (13-p) MT has been seen. The modified cilia of sensory neurons also possess unusual structures. The cilia contain nine outer doublets with A subfibers of 13 protofilaments and B subfibers of 11 protofilaments and a variable number of inner singlet MTs containing 11 protofilaments. The 15-p MTs but not the 11-p MTs are eliminated by colchicine-treatment or by mutation of the gene mec-7. Concomitantly, touch sensitivity is also lost. However, whereas colchicine treatment leads to the loss of all MTs from the microtubule cells, mutations in mec-7 result in the partial replacement of the 15-p MTs with 11-p MTs. Benzimidazoles (benomyl and nocodazole) have more general effects on C. elegans (slow growth, severe uncoordination, and loss of processes from the ventral cord) but do not affect the 15-p MTs. Benomyl will, however, disrupt the replacement 11-p MTs found in the microtubule cells of mec-7 mutants. The 11-p and 15-p MTs also respond differently to temperature and fixation conditions. It is likely that either type of MT will suffice for the proper outgrowth of the microtubule cell process, but only the 15-p MT can function in the specialized role of sensory transduction of the microtubule cells.     

Trans-cellular introduction of HIV-1 protein Nef induces pathogenic response in Caenorhabditis elegans

Background

Caenorhabditis elegans has emerged as a very powerful model for studying the host pathogen interactions. Despite the absence of a naturally occurring viral infection for C. elegans, the model is now being exploited experimentally to study the basic aspects of virus-host interplay. The data generated from recent studies suggests that the virus that infects mammalian cells does infect, replicate and accumulate in C. elegans.

Methodology/Principal Findings

We took advantage of the easy-to-achieve protein introduction in C. elegans and employing the methodology, we administered HIV-1 protein Nef into live worms. Nef is known to be an important protein for exacerbating HIV-1 pathogenesis in host by enhancing viral replication. The deletion of nef from the viral genome has been reported to inhibit its replication in the host, thereby leading to delayed pathogenesis. Our studies, employing Nef introduction into C. elegans, led to creation of an in-vivo model that allowed us to study, whether or not, the protein induces effect in the whole organism. We observed a marked lipodystrophy, effect on neuromuscular function, impaired fertility and reduced longevity in the worms exposed to Nef. The observed effects resemble to those observed in Nef transgenic mice and most interestingly the effects also relate to some of the pathogenic aspects exhibited by human AIDS patients.

Conclusions/Significance

Our studies underline the importance of this in vivo model for studying the interactions of Nef with host proteins, which could further be used for identifying possible inhibitors of such interactions.     

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.     

Single-molecule force spectroscopy reveals a stepwise unfolding of Caenorhabditis elegans giant protein kinase domains

Myofibril assembly and disassembly are complex processes that regulate overall muscle mass. Titin kinase has been implicated as an initiating catalyst in signaling pathways that ultimately result in myofibril growth. In titin, the kinase domain is in an ideal position to sense mechanical strain that occurs during muscle activity. The enzyme is negatively regulated by intramolecular interactions occurring between the kinase catalytic core and autoinhibitory/regulatory region. Molecular dynamics simulations suggest that human titin kinase acts as a force sensor. However, the precise mechanism(s) resulting in the conformational changes that relieve the kinase of this autoinhibition are unknown. Here we measured the mechanical properties of the kinase domain and flanking Ig/Fn domains of the Caenorhabditis elegans titin-like proteins twitchin and TTN-1 using single-molecule atomic force microscopy. Our results show that these kinase domains have significant mechanical resistance, unfolding at forces similar to those for Ig/Fn β-sandwich domains (30-150 pN). Further, our atomic force microscopy data is consistent with molecular dynamic simulations, which show that these kinases unfold in a stepwise fashion, first an unwinding of the autoinhibitory region, followed by a two-step unfolding of the catalytic core. These data support the hypothesis that titin kinase may function as an effective force sensor.