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 (2017) Curr Opin Microbiol "Host-microbiota interactions in Caenorhabditis elegans and their significance."

Shapira M

[Curr Opin Microbiol, 2017]

As a useful genetic model, C. elegans can facilitate investigation of the genetic underpinnings of host-microbiota interactions. However, decades of feeding it with Escherichia coli left a gap in our understanding of its interactions with microbes, hindering such use. This is changing, with recent studies characterizing the gut microbiota of worms in their natural habitats, comparing them to those in their environment, and evaluating the significance of gut and environmental commensals. This work defined a shared core gut microbiota significantly influenced by host genetics, and unraveled bacterial contributions to life history traits. Establishing C. elegans as a new model of host-microbiota interactions will benefit from existing knowledge about bacterial modulation of worm physiology, and could draw mechanistic insights from characterized interactions between parasitic nematodes and their symbionts.

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.

Block DH et al. (2015) Worm "GATA transcription factors as tissue-specific master regulators for induced responses."

Block DH, Shapira M

[Worm, 2015]

GATA transcription factors play important roles in directing developmental genetic programs and cell differentiation, and are conserved in animals, plants and fungi. C. elegans has 11 GATA-type transcription factors that orchestrate development of the gut, epidermis and vulva. However, the expression of certain GATA proteins persists into adulthood, where their function is less understood. Accumulating evidence demonstrates contributions of 2 terminal differentiation GATA transcription factors, ELT-2 and ELT-3, to epithelial immune responses in the adult intestine and epidermis (hypodermis), respectively. Involvement in other stress responses has also been documented. We recently showed that ELT-2 acted as a tissue-specific master regulator, cooperating with 2 transcription factors activated by the p38 pathway, ATF-7 and SKN-1, to control immune responses in the adult C. elegans intestine. Here, we discuss the broader implications of these findings for understanding the involvement of GATA transcription factors in adult stress responses, and draw parallels between ELT-2 and ELT-3 to speculate that the latter may fulfill similar tissue-specific functions in the epidermis.

Tan MW et al. (2011) Cell Microbiol "Genetic and molecular analysis of nematode-microbe interactions."

Tan MW, Shapira M

[Cell Microbiol, 2011]

Symbiosis, the living together of unlike organisms, such as between microbes and their multicellular eukaryotic hosts, can be categorized as parasitic, commensal or mutualistic. The establishment of symbiosis and the outcome of microbe-host interactions are dictated largely by both microbe- and host-derived factors. Over the last decade, the nematode Caenorhabditis elegans has provided a facile experimental system to study such interactions, with parasitic interactions being the primary focus. The myriad of genetic and molecular tools available has made C. elegans a powerful model system to interrogate the interactions between a host and its pathogens, and has provided a greater understanding of the molecular underpinnings of these interactions, many of which were found to be conserved across other taxa. Commensal and mutualistic interactions between worms and their microbes, although less studied, have the potential to enhance our understanding of genetic and molecular features underlying host-microbe interactions. Here, we highlight new insights obtained in delineating the signalling pathways that function within and between host cells in combating assaults from extracellular and intracellular pathogens. We also discuss potential new insights that could be gained from further studies into commensal and mutualistic relationships between nematodes and microbes.

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.

Kissoyan, K.A.B. et al. (2019) International Worm Meeting "Natural C. elegans Microbiota Protects against Infection via Production of a Cyclic Lipopeptide of the Viscosin Group."

Dierking, K., Kissoyan, K.A.B.

[International Worm Meeting, 2019]

C. elegans is associated in nature with a species-rich, distinct microbiota, which was characterized only recently [1]. Our understanding of C. elegans microbiota function is thus still in its infancy. Here, we identify natural C. elegans microbiota isolates of the Pseudomonas fluorescens subgroup that increase C. elegans resistance to pathogen infection. We show that different Pseudomonas isolates provide paramount protection from infection with the natural C. elegans pathogen Bacillus thuringiensis through distinct mechanisms [2] . The P. lurida isolates MYb11 and MYb12 (members of the P. fluorescens subgroup) protect C. elegans against B. thuringiensis infection by directly inhibiting growth of the pathogen both in vitro and in vivo. Using genomic and biochemical approaches, we demonstrate that MYb11 and MYb12 produce massetolide E, a cyclic lipopeptide biosurfactant of the viscosin group, which is active against pathogenic B. thuringiensis. In contrast to MYb11 and MYb12, P. fluorescens MYb115-mediated protection involves increased resistance without inhibition of pathogen growth and most likely depends on indirect, host-mediated mechanisms. We are currently investigating the molecular basis of P. fluorescens MYb115-mediated protection using a multi-omics approach to identify C. elegans candidate genes involved in microbiota-mediated protection. Moreover, we are further exploring the antagonistic interactions between C. elegans microbiota and pathogens. This work provides new insight into the functional significance of the C. elegans natural microbiota and expands our knowledge of immune-protective mechanisms. 1. Zhang, F., Berg, M., Dierking, K., Felix, M.A., Shapira, M., Samuel, B.S., and Schulenburg, H. (2017). Caenorhabditis elegans as a model for microbiome research. Front. Microbiol. 8:485. 2. Kissoyan, K.A.B., Drechsler, M., Stange, E.-L., Zimmermann, J., Kaleta, C., Bode, H.B., and Dierking, K. (2019). Natural C. elegans Microbiota Protects against Infection via Production of a Cyclic Lipopeptide of the Viscosin Group. Curr. Biol. 29.

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.