Shapira M et al. (2008) Methods Mol Biol "Genetic analysis of Caenorhabditis elegans innate immunity."

Shapira M, Tan MW

[Methods Mol Biol, 2008]

Innate immunity is an ancient and conserved defense mechanism. The worm Caenorhabditis elegans provides a useful tool for studying the function of the innate immune system at the molecular and cellular levels within the context of a whole organism. The powerful genetics of the worm, combined with efficacy of gene knockdown by RNA interference (RNAi), offer complementary tools for analyzing the contribution of individual genes to innate immunity. It is important, however, to exclude pleiotropic effects that confound results. In this chapter, we will describe the procedures for performing both forward and reverse genetic screens and will discuss a number of techniques developed to resolve confounding effects, thus enhancing the power of this system.

Shapira M et al. (2006) Proc Natl Acad Sci U S A "A conserved role for a GATA transcription factor in regulating epithelial innate immune responses."

Hamlin BJ, Tan MW, Chen K, Ronen M, Rong J, Shapira M

[Proc Natl Acad Sci U S A, 2006]

Innate immunity is an ancient and conserved defense mechanism. Although host responses toward various pathogens have been delineated, how these responses are orchestrated in a whole animal is less understood. Through an unbiased genome-wide study performed in Caenorhabditis elegans, we identified a conserved function for endodermal GATA transcription factors in regulating local epithelial innate immune responses. Gene expression and functional RNAi-based analyses identified the tissue-specific GATA transcription factor ELT-2 as a major regulator of an early intestinal protective response to infection with the human bacterial pathogen Pseudomonas aeruginosa. In the adult worm, ELT-2 is required specifically for infection responses and survival on pathogen but makes no significant contribution to gene expression associated with intestinal maintenance or to resistance to cadmium, heat, and oxidative stress. We further demonstrate that this function is conserved, because the human endodermal transcription factor GATA6 has a protective function in lung epithelial cells exposed to P. aeruginosa. These findings expand the repertoire of innate immunity mechanisms and illuminate a yet-unknown function of endodermal GATA proteins.

Wypijewska A et al. (2012) Biochemistry "7-methylguanosine diphosphate (m(7)GDP) is not hydrolyzed but strongly bound by decapping scavenger (DcpS) enzymes and potently inhibits their activity."

Wypijewska A, Darzynkiewicz E, Davis RE, Jemielity J, Bojarska E, Lukaszewicz M, Stepinski J

[Biochemistry, 2012]

Decapping scavenger (DcpS) enzymes catalyze the cleavage of a residual cap structure following 3' 5' mRNA decay. Some previous studies suggested that both m(7)GpppG and m(7)GDP were substrates for DcpS hydrolysis. Herein, we show that mononucleoside diphosphates, m(7)GDP (7-methylguanosine diphosphate) and m(3)(2,2,7)GDP (2,2,7-trimethylguanosine diphosphate), resulting from mRNA decapping by the Dcp1/2 complex in the 5' 3' mRNA decay, are not degraded by recombinant DcpS proteins (human, nematode, and yeast). Furthermore, whereas mononucleoside diphosphates (m(7)GDP and m(3)(2,2,7)GDP) are not hydrolyzed by DcpS, mononucleoside triphosphates (m(7)GTP and m(3)(2,2,7)GTP) are, demonstrating the importance of a triphosphate chain for DcpS hydrolytic activity. m(7)GTP and m(3)(2,2,7)GTP are cleaved at a slower rate than their corresponding dinucleotides (m(7)GpppG and m(3)(2,2,7)GpppG, respectively), indicating an involvement of the second nucleoside for efficient DcpS-mediated digestion. Although DcpS enzymes cannot hydrolyze m(7)GDP, they have a high binding affinity for m(7)GDP and m(7)GDP potently inhibits DcpS hydrolysis of m(7)GpppG, suggesting that m(7)GDP may function as an efficient DcpS inhibitor. Our data have important implications for the regulatory role of m(7)GDP in mRNA metabolic pathways due to its possible interactions with different cap-binding proteins, such as DcpS or eIF4E.

Twumasi-Boateng K et al. (2012) PLoS One "Dissociation of immune responses from pathogen colonization supports pattern recognition in C. elegans."

Shapira M, Twumasi-Boateng K

[PLoS One, 2012]

Caenorhabditis elegans has been used for over a decade to characterize signaling cascades controlling innate immune responses. However, what initiates these responses in the worm has remained elusive. To gain a better understanding of the initiating events we delineated genome-wide immune responses to the bacterial pathogen Pseudomonas aeruginosa in worms heavily-colonized by the pathogen versus worms visibly not colonized. We found that infection responses in both groups were identical, suggesting that immune responses were not correlated with colonization and its associated damage. Quantitative RT-PCR measurements further showed that pathogen secreted factors were not able to induce an immune response, but exposure to a non-pathogenic Pseudomonas species was. These findings raise the possibility that the C.elegans immune response is initiated by recognition of microbe-associated molecular patterns. In the absence of orthologs of known pattern recognition receptors, C. elegans may rely on novel mechanisms, thus holding the potential to advance our understanding of evolutionarily conserved strategies for pathogen recognition.

O'Connell EM et al. (2015) J Infect Dis "Targeting Filarial Abl-like Kinases: Orally Available, Food and Drug Administration-Approved Tyrosine Kinase Inhibitors Are Microfilaricidal and Macrofilaricidal."

Nutman TB, O'Connell EM, Bennuru S, Steel C, Dolan MA

[J Infect Dis, 2015]

BACKGROUND: Elimination of onchocerciasis and lymphatic filariasis is targeted for 2020. Given the coincident Loa loa infections in Central Africa and the potential for drug resistance development, the need for new microfilaricides and macrofilaricides has never been greater. With the genomes of L. loa, Onchocerca volvulus, Wuchereria bancrofti, and Brugia malayi available, new drug targets have been identified. METHODS: The effects of the tyrosine kinase inhibitors imatinib, nilotinib, and dasatinib on B. malayi adult males, adult females, L3 larvae, and microfilariae were assessed using a wide dose range (0-100 M) in vitro. RESULTS: For microfilariae, median inhibitory concentrations (IC50 values) on day 6 were 6.06 M for imatinib, 3.72 M for dasatinib, and 81.35 M for nilotinib; for L3 larvae, 11.27 M, 13.64 M, and 70.98 M, respectively; for adult males, 41.6 M, 3.87 M, and 68.22 M, respectively; and for adult females, 42.89 M, 9.8 M, and >100 M, respectively. Three-dimensional modeling suggests how these tyrosine kinase inhibitors bind and inhibit filarial protein activity. CONCLUSIONS: Given the safety of imatinib in humans, plans are underway for pilot clinical trials to assess its efficacy in patients with filarial infections.

Berg M et al. (2016) Front Microbiol "Host-Specific Functional Significance of Caenorhabditis Gut Commensals."

Berg M, Shapira M, Zhou XY

[Front Microbiol, 2016]

The gut microbiota is an important contributor to host health and fitness. Given its importance, microbiota composition should not be left to chance. However, what determines this composition is far from clear, with results supporting contributions of both environmental factors and host genetics. To gauge the relative contributions of host genetics and environment, specifically the microbial diversity, we characterized the gut microbiotas of Caenorhabditis species spanning 200-300 million years of evolution, and raised on different composted soil environments. Comparisons were based on 16S rDNA deep sequencing data, as well as on functional evaluation of gut isolates. Worm microbiotas were distinct from those in their respective soil environment, and included bacteria previously identified as part of the C. elegans core microbiota. Microbiotas differed between experiments initiated with different soil communities, but within each experiment, worm microbiotas clustered according to host identity, demonstrating a dominant contribution of environmental diversity, but also a significant contribution of host genetics. The dominance of environmental contributions hindered identification of host-associated microbial taxa from 16S data. Characterization of gut isolates from C. elegans and C. briggsae, focusing on the core family Enterobacteriaceae, were also unable to expose phylogenetic distinctions between microbiotas of the two species. However, functional evaluation of the isolates revealed host-specific contributions, wherein gut commensals protected their own host from infection, but not a non-host. Identification of commensal host-specificity at the functional level, otherwise overlooked in standard sequence-based analyses, suggests that the contribution of host genetics to shaping of gut microbiotas may be greater than previously realized.

Liu L et al. (2018) Genetics "Integration of Stress Signaling in Caenorhabditis elegans Through Cell Non-autonomous Contributions of the JNK Homolog KGB-1."

Shapira M, Ruediger C, Liu L

[Genetics, 2018]

Dealing with physiological stress is a necessity for all organisms, and the pathways charged with this task are highly conserved in metazoa. Accumulating evidence highlights cell non-autonomous activation as an important mode of integrating stress responses at the organism level, and work in <i>Caenorhabditis elegans</i> further highlighted the importance of such regulation for the unfolded protein response (UPR) and for DAF-16-dependent gene expression. Here we describe a role for the JNK homolog KGB-1 in cell non-autonomous regulation of these two response modules. KGB-1 protects developing larvae from protein folding stress (independent of canonical UPR pathways) and heavy metals, but has been shown to sensitize adult animals to the same stress, and to shorten lifespan, in association with an age-dependent antagonistic regulation of the longevity-associated transcription factor DAF-16. Using transgenic animals expressing KGB-1 from different tissue-specific promoters, focusing on tissues relevant for KGB-1-dependent phenotypes, we examined the effects of KGB-1 activation on gene regulation, stress resistance and lifespan. While cell autonomous contributions were observed, especially in the epidermis, KGB-1 largely operated through cell non-autonomous contributions, which mediated gene induction, age-dependent regulation of intestinal DAF-16, and stress resistance, and were mostly independent of KGB-1 expression in the target tissue. Additional genetic analysis revealed requirement for UNC-13 in mediating some of these contributions, indicating involvement of neurotransmission. Our results expand the role of KGB-1 in stress responses from providing local cellular protection, to integrating stress responses at the level of the whole-organism.

Trang K et al. (2022) J Vis Exp "Compost Microcosms as Microbially Diverse, Natural-like Environments for Microbiome Research in Caenorhabditis elegans."

Shapira M, Bodkhe R, Trang K

[J Vis Exp, 2022]

The nematode Caenorhabditis elegans is emerging as a useful model for studying the molecular mechanisms underlying interactions between hosts and their gut microbiomes. While experiments with well-characterized bacteria or defined bacterial communities can facilitate the analysis of molecular mechanisms, studying nematodes in their natural microbial context is essential for exploring the diversity of such mechanisms. At the same time, the isolation of worms from the wild is not always feasible, and, even when possible, sampling from the wild restricts the use of the genetic toolkit otherwise available for C. elegans research. The following protocol describes a method for microbiome studies utilizing compost microcosms for the in-labgrowth in microbially diverse and natural-like environments. Locally sourced soil can be enriched with produce to diversify the microbial communities in which worms are raised and from which they are harvested, washed, and surface-sterilized for subsequent analyses. Representative experiments demonstrate the ability to modulate the microbial community in a common soil by enriching it with different produce and further demonstrate that worms raised in these distinct environments assemble similar gut microbiomes distinct from their respective environments, supporting the notion of a species-specific core gut microbiome. Overall, compost microcosms provide natural-like in-lab environments for microbiome research as an alternative to synthetic microbial communities or to the isolation of wild nematodes.

Twumasi-Boateng K et al. (2014) J Vis Exp "Automated separation of C. elegans variably colonized by a bacterial pathogen."

Berg M, Shapira M, Twumasi-Boateng K

[J Vis Exp, 2014]

The wormsorter is an instrument analogous to a FACS machine that is used in studies of Caenorhabditis elegans, typically to sort worms based on expression of a fluorescent reporter. Here, we highlight an alternative usage of this instrument, for sorting worms according to their degree of colonization by a GFP-expressing pathogen. This new usage allowed us to address the relationship between colonization of the worm intestine and induction of immune responses. While C. elegans immune responses to different pathogens have been documented, it is still unknown what initiates them. The two main possibilities (which are not mutually exclusive) are recognition of pathogen-associated molecular patterns, and detection of damage caused by infection. To differentiate between the two possibilities, exposure to the pathogen must be dissociated from the damage it causes. The wormsorter enabled separation of worms that were extensively-colonized by the Gram-negative pathogen Pseudomonas aeruginosa, with the damage likely caused by pathogen load, from worms that were similarly exposed, but not, or marginally, colonized. These distinct populations were used to assess the relationship between pathogen load and the induction of transcriptional immune responses. The results suggest that the two are dissociated, supporting the possibility of pathogen recognition.

Lemire BD et al. (2009) Mech Ageing Dev "C. elegans longevity pathways converge to decrease mitochondrial membrane potential."

Behrendt M, Decorby A, Lemire BD, Gaskova D

[Mech Ageing Dev, 2009]

Energy production via oxidative phosphorylation generates a mitochondrial membrane potential (DeltaPsi(m)) across the inner membrane. In this work, we show that a lower DeltaPsi(m) is associated with increased lifespan in Caenorhabditis elegans. The long-lived mutants daf-2(e1370), age-1(hx546), clk-1(qm30), isp-1(qm150) and eat-2(ad465) all have a lower DeltaPsi(m) than wild type animals. The lower DeltaPsi(m) of daf-2(e1370) is daf-16 dependent, indicating that the insulin-like signaling pathway not only regulates lifespan but also mitochondrial energetics. RNA interference (RNAi) against 17 genes shown to extend lifespan also decrease DeltaPsi(m). Furthermore, lifespan can be significantly extended with the uncoupler carbonylcyanide-3-chlorophenylhydrazone (CCCP), which dissipates DeltaPsi(m). We conclude that longevity pathways converge on the mitochondria and lead to a decreased DeltaPsi(m). Our results are consistent with the 'uncoupling to survive' hypothesis, which states that dissipation of the DeltaPsi(m) will extend lifespan.