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.