Tissue- and paralogue-specific functions of acyl-CoA-binding proteins in lipid metabolism in Caenorhabditis elegans.

ACBP (acyl-CoA-binding protein) is a small primarily cytosolic protein that binds acyl-CoA esters with high specificity and affinity. ACBP has been identified in all eukaryotic species, indicating that it performs a basal cellular function. However, differential tissue expression and the existence of several ACBP paralogues in many eukaryotic species indicate that these proteins serve distinct functions. The nematode Caenorhabditis elegans expresses seven ACBPs: four basal forms and three ACBP domain proteins. We find that each of these paralogues is capable of complementing the growth of ACBP-deficient yeast cells, and that they exhibit distinct temporal and tissue expression patterns in C. elegans. We have obtained loss-of-function mutants for six of these forms. All single mutants display relatively subtle phenotypes; however, we find that functional loss of ACBP-1 leads to reduced triacylglycerol (triglyceride) levels and aberrant lipid droplet morphology and number in the intestine. We also show that worms lacking ACBP-2 show a severe decrease in the ß-oxidation of unsaturated fatty acids. A quadruple mutant, lacking all basal ACBPs, is slightly developmentally delayed, displays abnormal intestinal lipid storage, and increased ß-oxidation. Collectively, the present results suggest that each of the ACBP paralogues serves a distinct function in C. elegans.

Gap junctions in C. elegans.

As in other multicellular organisms, the nematode Caenorhabditis elegans uses gap junctions to provide direct cell-to-cell contact. The nematode gap junctions are formed by innexins (invertebrate analogs of the connexins); a family of proteins that surprisingly share no primary sequence homology, but do share structural and functional similarity with connexins. The model organism C. elegans contains 25 innexin genes and innexins are found in virtually all cell types and tissues. Additionally, many innexins have dynamic expression patterns during development, and several innexins are essential genes in the nematode. C. elegans is a popular invertebrate model due to several features including a simple anatomy, a complete cell lineage, sequenced genome and an array of genetic resources. Thus, the worm has potential to offer valuable insights into the various functions of gap junction mediated intercellular communication.

Strength in numbers: "Omics" studies of C. elegans innate immunity.

For more than ten years the nematode Caenorhabditis elegans has proven to be a valuable model for studies of the host response to various bacterial and fungal pathogens. When exposed to a pathogenic organism, a clear response is elicited in the nematode, which is characterized by specific alterations on the transcriptional and translational levels. Early on, researchers took advantage of the possibility to conduct large-scale investigations of the C. elegans immune response. Multiple studies demonstrated that C. elegans does indeed mount a protective response against invading pathogens, thus rendering this small nematode a very useful and simple host model for the study of innate immunity and host-pathogen interactions. Here, we provide an overview of key aspects of innate immunity in C. elegans revealed by recent whole-genome transcriptomics and proteomics studies of the global response of C. elegans to various bacterial and fungal pathogens.

A role for the RNA chaperone Hfq in controlling adherent-invasive Escherichia coli colonization and virulence.

Adherent-invasive Escherichia coli (AIEC) has been linked with the onset and perpetuation of inflammatory bowel diseases. The AIEC strain LF82 was originally isolated from an ileal biopsy from a patient with Crohn's disease. The pathogenesis of LF82 results from its abnormal adherence to and subsequent invasion of the intestinal epithelium coupled with its ability to survive phagocytosis by macrophages once it has crossed the intestinal barrier. To gain further insight into AIEC pathogenesis we employed the nematode Caenorhabditis elegans as an in vivo infection model. We demonstrate that AIEC strain LF82 forms a persistent infection in C. elegans, thereby reducing the host lifespan significantly. This host killing phenotype was associated with massive bacterial colonization of the nematode intestine and damage to the intestinal epithelial surface. C. elegans killing was independent of known LF82 virulence determinants but was abolished by deletion of the LF82 hfq gene, which encodes an RNA chaperone involved in mediating posttranscriptional gene regulation by small non-coding RNAs. This finding reveals that important aspects of LF82 pathogenesis are controlled at the posttranscriptional level by riboregulation. The role of Hfq in LF82 virulence was independent of its function in regulating RpoS and RpoE activity. Further, LF82Δhfq mutants were non-motile, impaired in cell invasion and highly sensitive to various chemical stress conditions, reinforcing the multifaceted function of Hfq in mediating bacterial adaptation. This study highlights the usefulness of simple non-mammalian infection systems for the identification and analysis of bacterial virulence factors.

Quantitative proteomics identifies ferritin in the innate immune response of C. elegans.

When encountering a pathogen, all organisms evoke a protective response by inducing defense mechanisms to help fight off the invader. The invertebrate model organism Caenorhabditis elegans has proven to be valuable for studies of the host response and the small nematode mounts a substantial transcriptional response to numerous pathogens. Here, we use global quantitative proteomics to profile the response to infection with E. coli strain LF82 isolated from patients suffering from Crohn's disease, an inflammatory bowel disease. We show that LF82 infection induces more than one hundred proteins. The response share many functional categories with other innate immunity studies in C. elegans, but also identifies novel host immune effector proteins. We demonstrate functional relevance for four LF82 induced proteins, including a lysozyme and a C-type lectin. The ferritin homolog FTN-2 was shown to be necessary for the full protective response against the Gram-negative LF82 and the Gram-positive pathogen Staphylococcus aureus. This study is the first to demonstrate a role for ferritin in the innate immune response of C. elegans, and our results suggests that quantitative proteomics is an attractive approach for identifying additional components in the complex immune response of the nematode.

RNAi screen of DAF-16/FOXO target genes in C. elegans links pathogenesis and dauer formation.

The DAF-16/FOXO transcription factor is the major downstream output of the insulin/IGF1R signaling pathway controlling C. elegans dauer larva development and aging. To identify novel downstream genes affecting dauer formation, we used RNAi to screen candidate genes previously identified to be regulated by DAF-16. We used a sensitized genetic background [eri-1(mg366); sdf-9(m708)], which enhances both RNAi efficiency and constitutive dauer formation (Daf-c). Among 513 RNAi clones screened, 21 displayed a synthetic Daf-c (SynDaf) phenotype with sdf-9. One of these genes, srh-100, was previously identified to be SynDaf, but twenty have not previously been associated with dauer formation. Two of the latter genes, lys-1 and cpr-1, are known to participate in innate immunity and six more are predicted to do so, suggesting that the immune response may contribute to the dauer decision. Indeed, we show that two of these genes, lys-1 and clc-1, are required for normal resistance to Staphylococcus aureus. clc-1 is predicted to function in epithelial cohesion. Dauer formation exhibited by daf-8(m85), sdf-9(m708), and the wild-type N2 (at 27°C) were all enhanced by exposure to pathogenic bacteria, while not enhanced in a daf-22(m130) background. We conclude that knockdown of the genes required for proper pathogen resistance increases pathogenic infection, leading to increased dauer formation in our screen. We propose that dauer larva formation is a behavioral response to pathogens mediated by increased dauer pheromone production.

Expression of ceramide glucosyltransferases, which are essential for glycosphingolipid synthesis, is only required in a small subset of C. elegans cells.

Glycosphingolipids (GSLs) are glycosylated derivatives of ceramide in the lipid bilayer. Their ubiquitous distribution and complexity suggest that they have important functions, but what these are in vivo is still poorly understood. Here, we characterize the phenotype of Caenorhabditis elegans mutants with essentially no GSLs. The C. elegans genome encodes three ceramide glucosyltransferase (CGT) genes, which encode enzymes required for GSL biosynthesis. Animals lacking CGT do not synthesize GSLs, arrest growth at the first larval stage, and display defects in a subset of cells in their digestive tract; these defects impair larval feeding, resulting in a starvation-induced growth arrest. Restoring CGT function in these digestive tract cells - but not in a variety of other tissues - is sufficient to rescue the phenotypes associated with loss of CGT function. These unexpected findings suggest that GSLs are dispensable in most C. elegans cells, including those of the nervous system.