Take a Walk to the Wild Side of Caenorhabditis elegans-Pathogen Interactions.

Microbiomes form intimate functional associations with their hosts. Much has been learned from correlating changes in microbiome composition to host organismal functions. However, in-depth functional studies require the manipulation of microbiome composition coupled with the precise interrogation of organismal physiology-features available in few host study systems. Caenorhabditis elegans has proven to be an excellent genetic model organism to study innate immunity and, more recently, microbiome interactions. The study of C. elegans-pathogen interactions has provided in depth understanding of innate immune pathways, many of which are conserved in other animals. However, many bacteria were chosen for these studies because of their convenience in the lab setting or their implication in human health rather than their native interactions with C. elegans In their natural environment, C. elegans feed on a variety of bacteria found in rotting organic matter, such as rotting fruits, flowers, and stems. Recent work has begun to characterize the native microbiome and has identified a common set of bacteria found in the microbiome of C. elegans While some of these bacteria are beneficial to C. elegans health, others are detrimental, leading to a complex, multifaceted understanding of bacterium-nematode interactions. Current research on nematode-bacterium interactions is focused on these native microbiome components, both their interactions with each other and with C. elegans We will summarize our knowledge of bacterial pathogen-host interactions in C. elegans, as well as recent work on the native microbiome, and explore the incorporation of these bacterium-nematode interactions into studies of innate immunity and pathogenesis.

Identification and characterization of differentially expressed genes in Caenorhabditis elegans in response to pathogenic and nonpathogenic Stenotrophomonas maltophilia.

BACKGROUND: Stenotrophomonas maltophilia is an emerging nosocomial pathogen that causes infection in immunocompromised patients. S. maltophilia isolates are genetically diverse, contain diverse virulence factors, and are variably pathogenic within several host species. Members of the Stenotrophomonas genus are part of the native microbiome of C. elegans, being found in greater relative abundance within the worm than its environment, suggesting that these bacteria accumulate within C. elegans. Thus, study of the C. elegans-Stenotrophomonas interaction is of both medical and ecological significance. To identify host defense mechanisms, we analyzed the C. elegans transcriptomic response to S. maltophilia strains of varying pathogenicity: K279a, an avirulent clinical isolate, JCMS, a virulent strain isolated in association with soil nematodes near Manhattan, KS, and JV3, an even more virulent environmental isolate. RESULTS: Overall, we found 145 genes that are commonly differentially expressed in response to pathogenic S. maltophilia strains, 89% of which are upregulated, with many even further upregulated in response to JV3 as compared to JCMS. There are many more JV3-specific differentially expressed genes (225, 11% upregulated) than JCMS-specific differentially expressed genes (14, 86% upregulated), suggesting JV3 has unique pathogenic mechanisms that could explain its increased virulence. We used connectivity within a gene network model to choose pathogen-specific and strain-specific differentially expressed candidate genes for functional analysis. Mutations in 13 of 22 candidate genes caused significant differences in C. elegans survival in response to at least one S. maltophilia strain, although not always the strain that induced differential expression, suggesting a dynamic response to varying levels of pathogenicity. CONCLUSIONS: Variation in observed pathogenicity and differences in host transcriptional responses to S. maltophilia strains reveal that strain-specific mechanisms play important roles in S. maltophilia pathogenesis. Furthermore, utilizing bacteria closely related to strains found in C. elegans natural environment provides a more realistic interaction for understanding host-pathogen response.

Transcriptomic, Functional, and Network Analyses Reveal Novel Genes Involved in the Interaction Between Caenorhabditis elegans and Stenotrophomonas maltophilia.

The bacterivorous nematode Caenorhabditis elegans is an excellent model for the study of innate immune responses to a variety of bacterial pathogens, including the emerging nosocomial bacterial pathogen Stenotrophomonas maltophilia. The study of this interaction has ecological and medical relevance as S. maltophilia is found in association with C. elegans and other nematodes in the wild and is an emerging opportunistic bacterial pathogen. We identified 393 genes that were differentially expressed when exposed to virulent and avirulent strains of S. maltophilia and an avirulent strain of E. coli. We then used a probabilistic functional gene network model (WormNet) to determine that 118 of the 393 differentially expressed genes formed an interacting network and identified a set of highly connected genes with eight or more predicted interactions. We hypothesized that these highly connected genes might play an important role in the defense against S. maltophila and found that mutations of six of seven highly connected genes have a significant effect on nematode survival in response to these bacteria. Of these genes, C48B4.1, mpk-2, cpr-4, clec-67, and lys-6 are needed for combating the virulent S. maltophilia JCMS strain, while dod-22 was solely involved in response to the avirulent S. maltophilia K279a strain. We further found that dod-22 and clec-67 were up regulated in response to JCMS vs. K279a, while C48B4.1, mpk-2, cpr-4, and lys-6 were down regulated. Only dod-22 had a documented role in innate immunity, which demonstrates the merit of our approach in the identification of novel genes that are involved in combating S. maltophilia infection.

A Stenotrophomonas maltophilia Strain Evades a Major Caenorhabditis elegans Defense Pathway.

Stenotrophomonas maltophilia is a ubiquitous bacterium and an emerging nosocomial pathogen. This bacterium is resistant to many antibiotics, associated with a number of infections, and a significant health risk, especially for immunocompromised patients. Given that Caenorhabditis elegans shares many conserved genetic pathways and pathway components with higher organisms, the study of its interaction with bacterial pathogens has biomedical implications. S. maltophilia has been isolated in association with nematodes from grassland soils, and it is likely that C. elegans encounters this bacterium in nature. We found that a local S. maltophilia isolate, JCMS, is more virulent than the other S. maltophilia isolates (R551-3 and K279a) tested. JCMS virulence correlates with intestinal distension and bacterial accumulation and requires the bacteria to be alive. Many of the conserved innate immune pathways that serve to protect C. elegans from various pathogenic bacteria also play a role in combating S. maltophilia JCMS. However, S. maltophilia JCMS is virulent to normally pathogen-resistant DAF-2/16 insulin-like signaling pathway mutants. Furthermore, several insulin-like signaling effector genes were not significantly differentially expressed between S. maltophilia JCMS and avirulent bacteria (Escherichia coli OP50). Taken together, these findings suggest that S. maltophilia JCMS evades the pathogen resistance conferred by the loss of DAF-2/16 pathway components. In summary, we have discovered a novel host-pathogen interaction between C. elegans and S. maltophilia and established a new animal model with which to study the mode of action of this emerging nosocomial pathogen.

Conversion of the LIN-1 ETS protein of Caenorhabditis elegans from a SUMOylated transcriptional repressor to a phosphorylated transcriptional activator.

The LIN-1 ETS transcription factor plays a pivotal role in controlling cell fate decisions during development of the Caenorhabditis elegans vulva. Prior to activation of the RTK/Ras/ERK-signaling pathway, LIN-1 functions as a SUMOylated transcriptional repressor that inhibits vulval cell fate. Here we demonstrate using the yeast two-hybrid system that SUMOylation of LIN-1 mediates interactions with a protein predicted to be involved in transcriptional repression: the RAD-26 Mi-2ß/CHD4 component of the nucleosome remodeling and histone deacetylation (NuRD) transcriptional repression complex. Genetic studies indicated that rad-26 functions to inhibit vulval cell fates in worms. Using the yeast two-hybrid system, we showed that the EGL-27/MTA1 component of the NuRD complex binds the carboxy-terminus of LIN-1 independently of LIN-1 SUMOylation. EGL-27 also binds UBC-9, an enzyme involved in SUMOylation, and MEP-1, a zinc-finger protein previously shown to bind LIN-1. Genetic studies indicate that egl-27 inhibits vulval cell fates in worms. These results suggest that LIN-1 recruits multiple proteins that repress transcription via both the SUMOylated amino-terminus and the unSUMOylated carboxy-terminus. Assays in cultured cells showed that the carboxy-terminus of LIN-1 was converted to a potent transcriptional activator in response to active ERK. We propose a model in which LIN-1 recruits multiple transcriptional repressors to inhibit the 1° vulval cell fate, and phosphorylation by ERK converts LIN-1 to a transcriptional activator that promotes the 1° vulval cell fate.

Effect of prey richness on a consumer's intrinsic growth rate.

The intrinsic growth rate of non-selective microbivores increases asymptotically with increasing prey biomass, but we do not know how intrinsic growth rate is affected by prey richness. The objective of this experiment was to determine the effect of prey richness on the growth kinetics of nematode predators while grazing on mixed bacterial lawns. We found that the intrinsic growth rate of Caenorhabditis elegans in laboratory culture increased asymptotically with prey richness. The mechanism of this pattern was primarily due to the best available prey species in the mixture: the intrinsic growth rate of the consumer feeding on a mixture of prey was approximately equal to the intrinsic growth rate of the predator when feeding on the single best prey in monoculture. This was analogous to the selection effect observed in biodiversity-ecosystem functioning relationships. Generation time, and not reproductive output, was the life history trait component that was most consistent with the pattern of intrinsic growth rate. Our results suggest that in order to link invertebrate consumers' growth rates to their microbial species composition in the field, it will be necessary to determine the ability of microbivorous invertebrates to selectively forage in natural environments and to better understand the micro-scale distribution of microbial communities in their natural environments.

Long-term nitrogen amendment alters the diversity and assemblage of soil bacterial communities in tallgrass prairie.

Anthropogenic changes are altering the environmental conditions and the biota of ecosystems worldwide. In many temperate grasslands, such as North American tallgrass prairie, these changes include alteration in historically important disturbance regimes (e.g., frequency of fires) and enhanced availability of potentially limiting nutrients, particularly nitrogen. Such anthropogenically-driven changes in the environment are known to elicit substantial changes in plant and consumer communities aboveground, but much less is known about their effects on soil microbial communities. Due to the high diversity of soil microbes and methodological challenges associated with assessing microbial community composition, relatively few studies have addressed specific taxonomic changes underlying microbial community-level responses to different fire regimes or nutrient amendments in tallgrass prairie. We used deep sequencing of the V3 region of the 16S rRNA gene to explore the effects of contrasting fire regimes and nutrient enrichment on soil bacterial communities in a long-term (20 yrs) experiment in native tallgrass prairie in the eastern Central Plains. We focused on responses to nutrient amendments coupled with two extreme fire regimes (annual prescribed spring burning and complete fire exclusion). The dominant bacterial phyla identified were Proteobacteria, Verrucomicrobia, Bacteriodetes, Acidobacteria, Firmicutes, and Actinobacteria and made up 80% of all taxa quantified. Chronic nitrogen enrichment significantly impacted bacterial community diversity and community structure varied according to nitrogen treatment, but not phosphorus enrichment or fire regime. We also found significant responses of individual bacterial groups including Nitrospira and Gammaproteobacteria to long-term nitrogen enrichment. Our results show that soil nitrogen enrichment can significantly alter bacterial community diversity, structure, and individual taxa abundance, which have important implications for both managed and natural grassland ecosystems.

Several Grassland Soil Nematode Species Are Insensitive to RNA-Mediated Interference.

Phenotypic analysis of defects caused by RNA mediated interference (RNAi) in Caenorhabditis elegans has proven to be a powerful tool for determining gene function. In this study we investigated the effectiveness of RNAi in four non-model grassland soil nematodes, Oscheius sp FVV-2., Rhabditis sp, Mesorhabditis sp., and Acrobeloides sp. In contrast to reference experiments performed using C. elegans and Caenorhabditis briggsae, feeding bacteria expressing dsRNA and injecting dsRNA into the gonad did not produce the expected RNAi knockdown phenotypes in any of the grassland nematodes. Quantitative reverse-transcribed PCR (qRT-PCR) assays did not detect a statistically significant reduction in the mRNA levels of endogenous genes targeted by RNAi in Oscheius sp., and Mesorhabditis sp. From these studies we conclude that due to low effectiveness and inconsistent reproducibility, RNAi knockdown phenotypes in non-Caenorhabditis nematodes should be interpreted cautiously.

Normalization and centering of array-based heterologous genome hybridization based on divergent control probes.

BACKGROUND: Hybridization of heterologous (non-specific) nucleic acids onto arrays designed for model-organisms has been proposed as a viable genomic resource for estimating sequence variation and gene expression in non-model organisms. However, conventional methods of normalization that assume equivalent distributions (such as quantile normalization) are inappropriate when applied to non-specific (heterologous) hybridization. We propose an algorithm for normalizing and centering intensity data from heterologous hybridization that makes no prior assumptions of distribution, reduces the false appearance of homology, and provides a way for researchers to confirm whether heterologous hybridization is suitable. RESULTS: Data are normalized by adjusting for Gibbs free energy binding, and centered by adjusting for the median of a common set of control probes assumed to be equivalently dissimilar for all species. This procedure was compared to existing approaches and found to be as successful as Loess normalization at detecting sequence variations (deletions) and even more successful than quantile normalization at reducing the accumulation of false positive probe matches between two related nematode species, Caenorhabditis elegans and C. briggsae. Despite the improvements, we still found that probe fluorescence intensity was too poorly correlated with sequence similarity to result in reliable detection of matching probe sequence. CONCLUSIONS: Cross-species hybridizations can be a way to adapt genome-enabled tools for closely related non-model organisms, but data must be appropriately normalized and centered in a way that accommodates hybridization of nucleic acids with diverged sequence. For short, 25-mer probes, hybridization intensity alone may be insufficiently correlated with sequence similarity to allow reliable inference of homology at the probe level.

The nuclear receptor NHR-25 cooperates with the Wnt/beta-catenin asymmetry pathway to control differentiation of the T seam cell in C. elegans.

Asymmetric cell divisions produce new cell types during animal development. Studies in Caenorhabditis elegans have identified major signal-transduction pathways that determine the polarity of cell divisions. How these relatively few conserved pathways interact and what modulates them to ensure the diversity of multiple tissue types is an open question. The Wnt/beta-catenin asymmetry pathway governs polarity of the epidermal T seam cell in the C. elegans tail. Here, we show that the asymmetry of T-seam-cell division and morphogenesis of the male sensory rays require NHR-25, an evolutionarily conserved nuclear receptor. NHR-25 ensures the neural fate of the T-seam-cell descendants in cooperation with the Wnt/beta-catenin asymmetry pathway. Loss of NHR-25 enhances the impact of mutated nuclear effectors of this pathway, POP-1 (TCF) and SYS-1 (beta-catenin), on T-seam-cell polarity, whereas it suppresses the effect of the same mutations on asymmetric division of the somatic gonad precursor cells. Therefore, NHR-25 can either synergize with or antagonize the Wnt/beta-catenin asymmetry pathway depending on the tissue context. Our findings define NHR-25 as a versatile modulator of Wnt/beta-catenin-dependent cell-fate decisions.

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.

Wnt and EGF pathways act together to induce C. elegans male hook development.

Comparative studies of vulva development between Caenorhabditis elegans and other nematode species have provided some insight into the evolution of patterning networks. However, molecular genetic details are available only in C. elegans and Pristionchus pacificus. To extend our knowledge on the evolution of patterning networks, we studied the C. elegans male hook competence group (HCG), an equivalence group that has similar developmental origins to the vulval precursor cells (VPCs), which generate the vulva in the hermaphrodite. Similar to VPC fate specification, each HCG cell adopts one of three fates (1 degree, 2 degrees, 3 degrees), and 2 degrees HCG fate specification is mediated by LIN-12/Notch. We show that 2 degrees HCG specification depends on the presence of a cell with the 1 degree fate. We also provide evidence that Wnt signaling via the Frizzled-like Wnt receptor LIN-17 acts to specify the 1 degree and 2 degrees HCG fate. A requirement for EGF signaling during 1 degree fate specification is seen only when LIN-17 activity is compromised. In addition, activation of the EGF pathway decreases dependence on LIN-17 and causes ectopic hook development. Our results suggest that WNT plays a more significant role than EGF signaling in specifying HCG fates, whereas in VPC specification EGF signaling is the major inductive signal. Nonetheless, the overall logic is similar in the VPCs and the HCG: EGF and/or WNT induce a 1 degree lineage, and LIN-12/NOTCH induces a 2 degrees lineage. Wnt signaling is also required for execution of the 1 degree and 2 degrees HCG lineages. lin-17 and bar-1/beta-catenin are preferentially expressed in the presumptive 1 degree cell P11.p. The dynamic subcellular localization of BAR-1-GFP in P11.p is concordant with the timing of HCG fate determination.

Analysis of Wnt signaling during Caenorhabditis elegans postembryonic development.

Wnts play a central role in the development of many cells and tissue types in all species studied to date. Like many other extracellular signaling pathways, secreted Wnt proteins are involved in many different processes; in C. elegans these include: cell proliferation, differentiation, cell migration, control of cell polarity, axon outgrowth and control of the stem cell niche. Perturbations in Wnt signaling are also key factors in cancer formation, and therefore of interest to oncobiologists. Wnts are secreted glycoproteins, which bind to Frizzled transmembrane receptors and signal either through, or independently of beta-catenin. Both beta-catenin-dependant (Wnt/beta-catenin) and -independent pathways function during postembryonic development in C. elegans and allow Wnt researchers to explore aspects of Wnt signaling both in common with other organisms and unique to the nematode. Chapter 9 in Volume 2 discusses various processes controlled by Wnt signaling during C. elegans embryonic development; this chapter discusses Wnt controlled processes that occur during postembryonic development, including an overview of methods used to observe their function.

Microarray challenges in ecology.

Microarrays are used to measure simultaneously the amount of mRNAs transcribed from many genes. They were originally designed for gene expression profiling in relatively simple biological systems, such as cell lines and model systems under constant laboratory conditions. This poses a challenge to ecologists who increasingly want to use microarrays to unravel the genetic mechanisms underlying complex interactions among organisms and between organisms and their environment. Here, we discuss typical experimental and statistical problems that arise when analyzing genome-wide expression profiles in an ecological context. We show that experimental design and environmental confounders greatly influence the identification of candidate genes in ecological microarray studies, and that following several simple recommendations could facilitate the analysis of microarray data in ecological settings.

Asymmetric localizations of LIN-17/Fz and MIG-5/Dsh are involved in the asymmetric B cell division in C. elegans.

LIN-44/Wnt and LIN-17/Frizzled (Fz) function in a planar cell polarity (PCP)-like pathway to regulate the asymmetric B cell division in Caenorhabditis elegans. We observed asymmetric localization of LIN-17/Frizzled (Fz) and MIG-5/Dishevelled (Dsh) during the B cell division. LIN-17::GFP was asymmetrically localized within the B cell prior to and after the B cell division and correlated with B cell polarity. Asymmetric localization of LIN-17::GFP was dependent upon LIN-44/Wnt and MIG-5/Dsh function. The LIN-17 transmembrane domain and a portion of the cysteine-rich domain (CRD) were required for LIN-17 function and asymmetric distribution to the B cell daughters, while the conserved KTXXXW motif was only required for function. MIG-5::GFP was also asymmetrically localized within the B cell prior to and after the B cell division in a LIN-17- and LIN-44-dependent manner. Functions of the MIG-5 DEP, PDZ and DIX domains were also conserved. Thus, a novel PCP-like pathway, in which LIN-17 and MIG-5 are asymmetrically localized, is involved in the regulation of B cell polarity.

Wnt signaling and a Hox protein cooperatively regulate psa-3/Meis to determine daughter cell fate after asymmetric cell division in C. elegans.

Asymmetric cell division is a mechanism for achieving cellular diversity. In C. elegans, many asymmetric cell divisions are controlled by the Wnt-MAPK pathway through POP-1/TCF. It is poorly understood, however, how POP-1 determines the specific fates of daughter cells. We found that nob-1/Hox, ceh-20/Pbx, and a Meis-related gene, psa-3, are required for asymmetric division of the T hypodermal cell. psa-3 expression was asymmetric between the T cell daughters, and it was regulated by POP-1 through a POP-1 binding site in the psa-3 gene. psa-3 expression was also regulated by NOB-1 and CEH-20 through a NOB-1 binding sequence in a psa-3 intron. PSA-3 can bind CEH-20 and function after the T cell division to promote the proper fate of the daughter cell. These results indicate that cooperation between Wnt signaling and a Hox protein functions to determine the specific fate of a daughter cell.

Molecular approach for assessing responses of microbial-feeding nematodes to burning and chronic nitrogen enrichment in a native grassland.

A substantial proportion of the primary productivity in grassland ecosystems is allocated belowground, sustaining an abundant and diverse community of microbes and soil invertebrates. These belowground communities drive many important ecosystem functions and are responsive to a variety of environmental changes. Nematodes, an abundant and diverse component of grassland soil communities, are particularly responsive to altered environmental conditions, such as those associated with reduced fire frequency and nitrogen enrichment, with the most consistent responses displayed by microbial-feeding nematodes. However, much of the available research characterizing nematode responses to environmental change has been carried out at the taxonomic level of family or by broad trophic categories (e.g. fungivores, bacterivores). The extent to which differential responses to environmental change occurs at the genus level or below is unclear. Therefore, the objective of this study was to use molecular methods to quantify the response of microbial-feeding nematodes, at the lowest levels of taxonomic resolution, to nitrogen enrichment and changes in fire frequency. Using sequencing and quantitative polymerase chain reaction (PCR) probes for the 18S ribosomal RNA gene and the ITS1 region, we identified 19 microbial-feeding nematode taxa across four families. When nematodes were sampled across treatments, we found that some nematode taxa within a family responded similarly to nitrogen and burning treatments, while other taxa within the same family respond quite differently. Additionally, although nematodes from different families on average responded differently to nitrogen enrichment and burning, similar responses were seen in nematode taxa that span three taxonomic families. Thus, if nematodes are to be used as indicators of environmental change, care should be taken to assess the response at the lowest taxonomic level possible.

A novel noncanonical Wnt pathway is involved in the regulation of the asymmetric B cell division in C. elegans.

The polarities of several cells that divide asymmetrically during Caenorhabditis elegans development are controlled by Wnt signaling. LIN-44/Wnt and LIN-17/Fz control the polarities of cells in the tail of developing C. elegans larvae, including the male-specific blast cell, B, that divides asymmetrically to generate a larger anterior daughter and a smaller posterior daughter. We determined that WRM-1 and the major canonical Wnt pathway components: BAR-1, SGG-1/GSK-3 and PRY-1/Axin were not involved in the control of B cell polarity. However, POP-1/Tcf is involved and is asymmetrically distributed to the B daughter nuclei, as it is in many cell divisions during C. elegans development. Aspects of the B cell division are reminiscent of the divisions controlled by the planar cell polarity (PCP) pathway that has been described in both Drosophila and vertebrate systems. We identified C. elegans homologs of Wnt/PCP signaling components and have determined that many of them appear to be involved in the regulation of B cell polarity. Specifically, MIG-5/Dsh, RHO-1/RhoA and LET-502/ROCK appear to play major roles, while other PCP components appear to play minor roles. We conclude that a noncanonical Wnt pathway, which is different from other Wnt pathways in C. elegans, regulates B cell polarity.