Phenotypic covariance of longevity, immunity and stress resistance in the Caenorhabditis nematodes

Background \ud Ageing, immunity and stresstolerance are inherent characteristics of all organisms. In animals, these traits are regulated, at least in part, by forkhead transcription factors in response to upstream signals from the Insulin/Insulin– like growth factor signalling (IIS) pathway. In the nematode Caenorhabditis elegans, these phenotypes are molecularly linked such that activation of the forkhead transcription factor DAF-16 both extends lifespan and simultaneously increases immunity and stress resistance. It is known that lifespan varies significantly among the Caenorhabditis species but, although DAF-16 signalling is highly conserved, it is unclear whether this phenotypic linkage occurs in other species. Here we investigate this phenotypic covariance by comparing longevity, stress resistance and immunity in four \ud Caenorhabditis species. \ud \ud Methodology/Principal Findings \ud We show using phenotypic analysis of DAF-16 influenced phenotypes that among four closely related Caenorhabditis nematodes, the gonochoristic species (Caenorhabditis remanei and Caenorhabditis brenneri) have diverged \ud significantly with a longer lifespan, improved stress resistance and higher immunity than the hermaphroditic species (C. elegans and Caenorhabditis briggsae). Interestingly, we also observe significant differences in expression levels between the daf-16 homologues in these species using Real-Time PCR, which positively correlate with the observed phenotypes. Finally, we provide additional evidence in support of a role for DAF-16 in regulating phenotypic coupling by using a combination of wildtype isolates, constitutively active daf-16 mutants and bioinformatic analysis. \ud \ud Conclusions \ud The gonochoristic species display a significantly longer lifespan (p < 0.0001)and more robust immune and stress response (p<0.0001, thermal stress; p<0.01, heavy metal stress; p<0.0001, pathogenic stress) than the hermaphroditic species. Our data suggests that divergence in DAF-16 mediated phenotypes may underlie many of the differences observed between these four species of Caenorhabditis nematodes. These findings are further supported by the correlative higher daf-16 expression levels among the gonochoristic species and significantly higher lifespan, immunity and stress tolerance in the constitutively active daf-16 hermaphroditic mutants

Phenotypic covariance of Longevity, Immunity and Stress Resistance in the Caenorhabditis Nematodes

Background: Ageing, immunity and stresstolerance are inherent characteristics of all organisms. In animals, these traits are regulated, at least in part, by forkhead transcription factors in response to upstream signals from the Insulin/Insulin–like growth factor signalling (IIS) pathway. In the nematode Caenorhabditis elegans, these phenotypes are molecularly linked such that activation of the forkhead transcription factor DAF-16 both extends lifespan and simultaneously increases immunity and stress resistance. It is known that lifespan varies significantly among the Caenorhabditis species but, although DAF-16 signalling is highly conserved, it is unclear whether this phenotypic linkage occurs in other species. Here we investigate this phenotypic covariance by comparing longevity, stress resistance and immunity in four Caenorhabditis species. \ud \ud Methodology/Principal Findings: We show using phenotypic analysis of DAF-16 influenced phenotypes that among four closely related Caenorhabditis nematodes, the gonochoristic species (Caenorhabditis remanei and Caenorhabditis brenneri) have diverged significantly with a longer lifespan, improved stress resistance and higher immunity than the hermaphroditic species (C. elegans and Caenorhabditis briggsae). Interestingly, we also observe significant differences in expression levels between the daf-16 homologues in these species using Real-Time PCR, which positively correlate with the observed phenotypes. Finally, we provide additional evidence in support of a role for DAF-16 in regulating phenotypic coupling by using a combination of wildtype isolates, constitutively active daf-16 mutants and bioinformatic analysis. \ud \ud Conclusions: The gonochoristic species display a significantly longer lifespan (p<0.0001) and more robust immune and stress response (p<0.0001, thermal stress; p<0.01, heavy metal stress; p<0.0001, pathogenic stress) than the hermaphroditic species. Our data suggests that divergence in DAF-16 mediated phenotypes may underlie many of the differences observed between these four species of Caenorhabditis nematodes. These findings are further supported by the correlative higher daf-16 expression levels among the gonochoristic species and significantly higher lifespan, immunity and stress tolerance in the constitutively active daf-16 hermaphroditic mutants

High-Throughput Isolation and Mapping of C. elegans Mutants Susceptible to Pathogen Infection

We present a novel strategy that uses high-throughput methods of isolating and mapping C. elegans mutants susceptible to pathogen infection. We show that C. elegans mutants that exhibit an enhanced pathogen accumulation (epa) phenotype can be rapidly identified and isolated using a sorting system that allows automation of the analysis, sorting, and dispensing of C. elegans by measuring fluorescent bacteria inside the animals. Furthermore, we validate the use of Amplifluor® as a new single nucleotide polymorphism (SNP) mapping technique in C. elegans. We show that a set of 9 SNPs allows the linkage of C. elegans mutants to a 5–8 megabase sub-chromosomal region

The Conserved Candida albicans CA3427 Gene Product Defines a New Family of Proteins Exhibiting the Generic Periplasmic Binding Protein Structural Fold

Nosocomial diseases due to Candida albicans infections are in constant rise in hospitals, where they cause serious complications to already fragile intensive care patients. Antifungal drug resistance is fast becoming a serious issue due to the emergence of strains resistant to currently available antifungal agents. Thus the urgency to identify new potential protein targets, the function and structure of which may guide the development of new antifungal drugs. In this context, we initiated a comparative genomics study in search of promising protein coding genes among the most conserved ones in reference fungal genomes. The CA3427 gene was selected on the basis of its presence among pathogenic fungi contrasting with its absence in the non pathogenic Saccharomyces cerevisiae. We report the crystal 3D-structure of the Candida albicans CA3427 protein at 2.1 Å resolution. The combined analysis of its sequence and structure reveals a structural fold originally associated with periplasmic binding proteins. The CA3427 structure highlights a binding site located between the two protein domains, corresponding to a sequence segment conserved among fungi. Two crystal forms of CA3427 were found, suggesting that the presence or absence of a ligand at the proposed binding site might trigger a “Venus flytrap” motion, coupled to the previously described activity of bacterial periplasmic binding proteins. The conserved binding site defines a new subfamily of periplasmic binding proteins also found in many bacteria of the bacteroidetes division, in a choanoflagellate (a free-living unicellular and colonial flagellate eukaryote) and in a placozoan (the closest multicellular relative of animals). A phylogenetic analysis suggests that this gene family originated in bacteria before its horizontal transfer to an ancestral eukaryote prior to the radiation of fungi. It was then lost by the Saccharomycetales which include Saccharomyces cerevisiae

C. elegans SWAN-1 Binds to EGL-9 and Regulates HIF-1-Mediated Resistance to the Bacterial Pathogen Pseudomonas aeruginosa PAO1

Pseudomonas aeruginosa is a nearly ubiquitous human pathogen, and infections can be lethal to patients with impaired respiratory and immune systems. Prior studies have established that strong loss-of-function mutations in the egl-9 gene protect the nematode C. elegans from P. aeruginosa PAO1 fast killing. EGL-9 inhibits the HIF-1 transcription factor via two pathways. First, EGL-9 is the enzyme that targets HIF-1 for oxygen-dependent degradation via the VHL-1 E3 ligase. Second, EGL-9 inhibits HIF-1-mediated gene expression through a VHL-1-independent mechanism. Here, we show that a loss-of-function mutation in hif-1 suppresses P. aeruginosa PAO1 resistance in egl-9 mutants. Importantly, we find stabilization of HIF-1 protein is not sufficient to protect C. elegans from P. aeruginosa PAO1 fast killing. However, mutations that inhibit both EGL-9 pathways result in higher levels of HIF-1 activity and confer resistance to the pathogen. Using forward genetic screens, we identify additional mutations that confer resistance to P. aeruginosa. In genetic backgrounds that stabilize C. elegans HIF-1 protein, loss-of-function mutations in swan-1 increase the expression of hypoxia response genes and protect C. elegans from P. aeruginosa fast killing. SWAN-1 is an evolutionarily conserved WD-repeat protein belonging to the AN11 family. Yeast two-hybrid and co-immunoprecipitation assays show that EGL-9 forms a complex with SWAN-1. Additionally, we present genetic evidence that the DYRK kinase MBK-1 acts downstream of SWAN-1 to promote HIF-1-mediated transcription and to increase resistance to P. aeruginosa. These data support a model in which SWAN-1, MBK-1 and EGL-9 regulate HIF-1 transcriptional activity and modulate resistance to P. aeruginosa PAO1 fast killing

Caenorhabditis elegans Genomic Response to Soil Bacteria Predicts Environment-Specific Genetic Effects on Life History Traits

With the post-genomic era came a dramatic increase in high-throughput technologies, of which transcriptional profiling by microarrays was one of the most popular. One application of this technology is to identify genes that are differentially expressed in response to different environmental conditions. These experiments are constructed under the assumption that the differentially expressed genes are functionally important in the environment where they are induced. However, whether differential expression is predictive of functional importance has yet to be tested. Here we have addressed this expectation by employing Caenorhabditis elegans as a model for the interaction of native soil nematode taxa and soil bacteria. Using transcriptional profiling, we identified candidate genes regulated in response to different bacteria isolated in association with grassland nematodes or from grassland soils. Many of the regulated candidate genes are predicted to affect metabolism and innate immunity suggesting similar genes could influence nematode community dynamics in natural systems. Using mutations that inactivate 21 of the identified genes, we showed that most contribute to lifespan and/or fitness in a given bacterial environment. Although these bacteria may not be natural food sources for C. elegans, we show that changes in food source, as can occur in environmental disturbance, can have a large effect on gene expression, with important consequences for fitness. Moreover, we used regression analysis to demonstrate that for many genes the degree of differential gene expression between two bacterial environments predicted the magnitude of the effect of the loss of gene function on life history traits in those environments

Comparative Developmental Expression Profiling of Two C. elegans Isolates

Gene expression is known to change during development and to vary among genetically diverse strains. Previous studies of temporal patterns of gene expression during C. elegans development were incomplete, and little is known about how these patterns change as a function of genetic background. We used microarrays that comprehensively cover known and predicted worm genes to compare the landscape of genetic variation over developmental time between two isolates of C. elegans. We show that most genes vary in expression during development from egg to young adult, many genes vary in expression between the two isolates, and a subset of these genes exhibit isolate-specific changes during some developmental stages. This subset is strongly enriched for genes with roles in innate immunity. We identify several novel motifs that appear to play a role in regulating gene expression during development, and we propose functional annotations for many previously unannotated genes. These results improve our understanding of gene expression and function during worm development and lay the foundation for linkage studies of the genetic basis of developmental variation in gene expression in this important model organism

Pseudomonas aeruginosa Suppresses Host Immunity by Activating the DAF-2 Insulin-Like Signaling Pathway in Caenorhabditis elegans

Some pathogens have evolved mechanisms to overcome host immune defenses by inhibiting host defense signaling pathways and suppressing the expression of host defense effectors. We present evidence that Pseudomonas aeruginosa is able to suppress the expression of a subset of immune defense genes in the animal host Caenorhabditis elegans by activating the DAF-2/DAF-16 insulin-like signaling pathway. The DAF-2/DAF-16 pathway is important for the regulation of many aspects of organismal physiology, including metabolism, stress response, longevity, and immune function. We show that intestinal expression of DAF-16 is required for resistance to P. aeruginosa and that the suppression of immune defense genes is dependent on the insulin-like receptor DAF-2 and the FOXO transcription factor DAF-16. By visualizing the subcellular localization of DAF-16::GFP fusion protein in live animals during infection, we show that P. aeruginosa–mediated downregulation of a subset of immune genes is associated with the ability to translocate DAF-16 from the nuclei of intestinal cells. Suppression of DAF-16 is mediated by an insulin-like peptide, INS-7, which functions upstream of DAF-2. Both the inhibition of DAF-16 and downregulation of DAF-16–regulated genes, such as thn-2, lys-7, and spp-1, require the P. aeruginosa two-component response regulator GacA and the quorum-sensing regulators LasR and RhlR and are not observed during infection with Salmonella typhimurium or Enterococcus faecalis. Our results reveal a new mechanism by which P. aeruginosa suppresses host immune defense