Katja Dierking

A Novel Metagenomic Short-Chain Dehydrogenase/ Reductase Attenuates Pseudomonas aeruginosa Biofilm Formation and Virulence on Caenorhabditis elegans

In Pseudomonas aeruginosa, the expression of a number of virulence factors, as well as biofilm formation, are controlled by quorum sensing (QS). N-Acylhomoserine lactones (AHLs) are an important class of signaling molecules involved in bacterial QS and in many pathogenic bacteria infection and host colonization are AHL-dependent. The AHL signaling molecules are subject to inactivation mainly by hydrolases (Enzyme Commission class number EC 3) (i.e. N-acyl-homoserine lactonases and N-acyl-homoserine-lactone acylases). Only little is known on quorum quenching mechanisms of oxidoreductases (EC 1). Here we report on the identification and structural characterization of the first NADP-dependent short-chain dehydrogenase/reductase (SDR) involved in inactivation of N-(3-oxo-dodecanoyl)-L-homoserine lactone (3-oxo-C12-HSL) and derived from a metagenome library. The corresponding gene was isolated from a soil metagenome and designated bpiB09. Heterologous expression and crystallographic studies established BpiB09 as an NADP-dependent reductase. Although AHLs are probably not the native substrate of this metagenome-derived enzyme, its expression in P. aeruginosa PAO1 resulted in significantly reduced pyocyanin production, decreased motility, poor biofilm formation and absent paralysis of Caenorhabditis elegans. Furthermore, a genome-wide transcriptome study suggested that the level of lasI and rhlI transcription together with 36 well known QS regulated genes was significantly (≥10-fold) affected in P. aeruginosa strains expressing the bpiB09 gene in pBBR1MCS-5. Thus AHL oxidoreductases could be considered as potent tools for the development of quorum quenching strategies.

The Janthinobacterium sp. HH01 Genome Encodes a Homologue of the V. cholerae CqsA and L. pneumophila LqsA Autoinducer Synthases

Janthinobacteria commonly form biofilms on eukaryotic hosts and are known to synthesize antibacterial and antifungal compounds. Janthinobacterium sp. HH01 was recently isolated from an aquatic environment and its genome sequence was established. The genome consists of a single chromosome and reveals a size of 7.10 Mb, being the largest janthinobacterial genome so far known. Approximately 80% of the 5,980 coding sequences (CDSs) present in the HH01 genome could be assigned putative functions. The genome encodes a wealth of secretory functions and several large clusters for polyketide biosynthesis. HH01 also encodes a remarkable number of proteins involved in resistance to drugs or heavy metals. Interestingly, the genome of HH01 apparently lacks the N-acylhomoserine lactone (AHL)-dependent signaling system and the AI-2-dependent quorum sensing regulatory circuit. Instead it encodes a homologue of the Legionella- and Vibrio-like autoinducer (lqsA/cqsA) synthase gene which we designated jqsA. The jqsA gene is linked to a cognate sensor kinase (jqsS) which is flanked by the response regulator jqsR. Here we show that a jqsA deletion has strong impact on the violacein biosynthesis in Janthinobacterium sp. HH01 and that a jqsA deletion mutant can be functionally complemented with the V. cholerae cqsA and the L. pneumophila lqsA genes.

High Innate Immune Specificity through Diversified C-Type Lectin-Like Domain Proteins in Invertebrates

A key question in current immunity research is how the innate immune system can generate high levels of specificity. Evidence is accumulating that invertebrates, which exclusively rely on innate defense mechanisms, can differentiate between pathogens on the species and even strain level. In this review, we identify and discuss the particular potential of C-type lectin-like domain (CTLD) proteins to generate high immune specificity. Whilst several CTLD proteins are known to act as pattern recognition receptors in the vertebrate innate immune system, the exact role of CTLD proteins in invertebrate immunity is much less understood. We show that CTLD genes are highly abundant in most metazoan genomes and summarize the current state of knowledge on CTLD protein function in insect, crustacean and nematode immune systems. We then demonstrate extreme CTLD gene diversification in the genomes of Caenorhabditis nematodes and provide an update of data from CTLD gene function studies in C. elegans, which indicate that the diversity of CTLD genes could contribute to immune specificity. In spite of recent achievements, the exact functions of the diversified invertebrate CTLD genes are still largely unknown. Our review therefore specifically discusses promising research approaches to rectify this knowledge gap.

Overlapping and unique signatures in the proteomic and transcriptomic responses of the nematode Caenorhabditis elegans toward pathogenic Bacillus thuringiensis

Pathogen infection can activate multiple signaling cascades that ultimately alter the abundance of molecules in cells. This change can be measured both at the transcript and protein level. Studies analyzing the immune response at both levels are, however, rare. Here, we compare transcriptome and proteome data generated after infection of the nematode and model organism Caenorhabditis elegans with the Gram-positive pathogen Bacillus thuringiensis. Our analysis revealed a high overlap between abundance changes of corresponding transcripts and gene products, especially for genes encoding C-type lectin domain-containing proteins, indicating their particular role in worm immunity. We additionally identified a unique signature at the proteome level, suggesting that the C. elegans response to infection is shaped by changes beyond transcription. Such effects appear to be influenced by AMP-activated protein kinases (AMPKs), which may thus represent previously unknown regulators of C. elegans immune defense.

Caenorhabditis elegans as a Model for Microbiome Research

The nematode Caenorhabditis elegans is used as a central model system across biological disciplines. Surprisingly, almost all research with this worm is performed in the absence of its native microbiome, possibly affecting generality of the obtained results. In fact, the C. elegans microbiome had been unknown until recently. This review brings together results from the first three studies on C. elegans microbiomes, all published in 2016. Meta-analysis of the data demonstrates a considerable conservation in the composition of the microbial communities, despite the distinct geographical sample origins, study approaches, labs involved and perturbations during worm processing. The C. elegans microbiome is enriched and in some cases selective for distinct phylotypes compared to corresponding substrate samples (e.g., rotting fruits, decomposing plant matter, and compost soil). The dominant bacterial groups include several Gammaproteobacteria (Enterobacteriaceae, Pseudomonaceae, and Xanthomonodaceae) and Bacteroidetes (Sphingobacteriaceae, Weeksellaceae, Flavobacteriaceae). They are consistently joined by several rare putative keystone taxa like Acetobacteriaceae. The bacteria are able to enhance growth of nematode populations, as well as resistance to biotic and abiotic stressors, including high/low temperatures, osmotic stress, and pathogenic bacteria and fungi. The associated microbes thus appear to display a variety of effects beneficial for the worm. The characteristics of these effects, their relevance for C. elegans fitness, the presence of specific co-adaptations between microbiome members and the worm, and the molecular underpinnings of microbiome-host interactions represent promising areas of future research, for which the advantages of C. elegans as an experimental system should prove of particular value.

Contrasting invertebrate immune defense behaviors caused by a single gene, the Caenorhabditis elegans neuropeptide receptor gene npr-1

BackgroundThe invertebrate immune system comprises physiological mechanisms, physical barriers and also behavioral responses. It is generally related to the vertebrate innate immune system and widely believed to provide nonspecific defense against pathogens, whereby the response to different pathogen types is usually mediated by distinct signalling cascades. Recent work suggests that invertebrate immune defense can be more specific at least at the phenotypic level. The underlying genetic mechanisms are as yet poorly understood.ResultsWe demonstrate in the model invertebrate Caenorhabditis elegans that a single gene, a homolog of the mammalian neuropeptide Y receptor gene, npr-1, mediates contrasting defense phenotypes towards two distinct pathogens, the Gram-positive Bacillus thuringiensis and the Gram-negative Pseudomonas aeruginosa. Our findings are based on combining quantitative trait loci (QTLs) analysis with functional genetic analysis and RNAseq-based transcriptomics. The QTL analysis focused on behavioral immune defense against B. thuringiensis, using recombinant inbred lines (RILs) and introgression lines (ILs). It revealed several defense QTLs, including one on chromosome X comprising the npr-1 gene. The wildtype N2 allele for the latter QTL was associated with reduced defense against B. thuringiensis and thus produced an opposite phenotype to that previously reported for the N2 npr-1 allele against P. aeruginosa. Analysis of npr-1 mutants confirmed these contrasting immune phenotypes for both avoidance behavior and nematode survival. Subsequent transcriptional profiling of C. elegans wildtype and npr-1 mutant suggested that npr-1 mediates defense against both pathogens through p38 MAPK signaling, insulin-like signaling, and C-type lectins. Importantly, increased defense towards P. aeruginosa seems to be additionally influenced through the induction of oxidative stress genes and activation of GATA transcription factors, while the repression of oxidative stress genes combined with activation of Ebox transcription factors appears to enhance susceptibility to B. thuringiensis.ConclusionsOur findings highlight the role of a single gene, npr-1, in fine-tuning nematode immune defense, showing the ability of the invertebrate immune system to produce highly specialized and potentially opposing immune responses via single regulatory genes.

GATA transcription factor as a likely key regulator of the Caenorhabditis elegans innate immune response against gut pathogens.

Invertebrate defence against pathogens exclusively relies on components of the innate immune system. Comprehensive information has been collected over the last years on the molecular components of invertebrate immunity and the involved signalling processes, especially for the main invertebrate model species, the fruitfly Drosophila melanogaster and the nematode Caenorhabditis elegans. Yet, the exact regulation of general and specific defences is still not well understood. In the current study, we take advantage of a recently established database, WormExp, which combines all available gene expression studies for C. elegans, in order to explore commonalities and differences in the regulation of nematode immune defence against a large variety of pathogens versus food microbes. We identified significant overlaps in the transcriptional response towards microbes, especially pathogenic bacteria. We also found that the GATA motif is overrepresented in many microbe-induced gene sets and in targets of other previously identified regulators of worm immunity. Moreover, the activated targets of one of the known C. elegans GATA transcription factors, ELT-2, are significantly enriched in the gene sets, which are differentially regulated by gut-infecting pathogens. These findings strongly suggest that GATA transcription factors and particularly ELT-2 play a central role in regulating the C. elegans immune response against gut pathogens. More specific responses to distinct pathogens may be mediated by additional transcription factors, either acting alone or jointly with GATA transcription factors. Taken together, our analysis of the worms transcriptional response to microbes provides a new perspective on the C. elegans immune system, which we propose to be coordinated by GATA transcription factor ELT-2 in the gut.

WormExp: a web-based application for a Caenorhabditis elegans-specific gene expression enrichment analysis

MOTIVATION A particular challenge of the current omics age is to make sense of the inferred differential expression of genes and proteins. The most common approach is to perform a gene ontology (GO) enrichment analysis, thereby relying on a database that has been extracted from a variety of organisms and that can therefore only yield reliable information on evolutionary conserved functions. RESULTS We here present a web-based application for a taxon-specific gene set exploration and enrichment analysis, which is expected to yield novel functional insights into newly determined gene sets. The approach is based on the complete collection of curated high-throughput gene expression data sets for the model nematode Caenorhabditis elegans, including 1786 gene sets from more than 350 studies. AVAILABILITY AND IMPLEMENTATION WormExp is available at http://wormexp.zoologie.uni-kiel.de CONTACTS hschulenburg@zoologie.uni-kiel.de SUPPLEMENTARY INFORMATION Supplementary data are available at Bioinformatics online.

Enhanced behavioral immune defenses in a C. elegans C-type lectin-like domain gene mutant

&NA; C‐type lectin‐like domain (CTLD) proteins occupy crucial functions in the immune system of vertebrates, but their role in invertebrate immunity is much less understood. The nematode Caenorhabditis elegans possesses a highly diverse CTLD protein encoding (clec) gene repertoire. A role of C. elegans clec genes in pathogen defense is always assumed, yet experimental evidence for clec immune function is rare. To systematically test the potential function of clec genes in the C. elegans defense against pathogens, we screened 39 clec mutants for survival on the Gram‐positive pathogen Bacillus thuringiensis (BT18247) and 37 clec mutants on the Gram‐negative pathogen Pseudomonas aeruginosa (PA14). We found that clec mutants can exhibit either decreased or, unexpectedly, increased resistance to infection. Since we observed high escape behavior for some of the clec mutants on BT18247 during the initial screen, we then asked if increased pathogen avoidance behavior underlies the increased resistance of some clec mutants. We thus tested lawn leaving behavior of the resistant clec‐29(ok3181), clec‐34(ok2120), clec‐151(ok2264), and C54G4.4(ok2110) mutant on BT18247. We found that C54G4.4(ok2110) mutant animals exhibit a particularly strong lawn leaving behavior, in addition to prolonged feeding cessation when exposed to BT18247. Together, our results indicate that clec genes mediate both resistance and susceptibility to infection. Further, behavioral analyses of the C54G4.4(ok2110) mutant implicate C54G4.4 in the regulation of pathogen avoidance behavior towards BT18247. We conclude that C. elegans clec genes may act both as positive and negative regulators of physiological as well as behavioral immune defense responses. HighlightsC. elegans clec mutants can exhibit either decreased or increased resistance to bacterial infection.The CTLD gene mutant C54G4.4(ok2110) displays enhanced pathogen avoidance behavior towards Bacillus thuringiensis.C. elegans clec genes may act both as positive and negative regulators of physiological as well as behavioral immune defense responses.

High instability of a nematicidal Cry toxin plasmid in Bacillus thuringiensis

In bacterial pathogens, virulence factors are often carried on plasmids and other mobile genetic elements, and as such, plasmid evolution is central in understanding pathogenicity. Bacillus thuringiensis is an invertebrate pathogen that uses plasmid-encoded crystal (Cry) toxins to establish infections inside the host. Our study aimed to quantify stability of two Cry toxin-encoding plasmids, BTI_23p and BTI_16p, under standard laboratory culturing conditions. These two plasmids are part of the genome of the B. thuringiensis strain MYBT18679, which is of particular interest because of its high pathogenicity towards nematodes. One of the plasmids, BTI_23p, was found to be highly unstable, with substantial loss occurring within a single growth cycle. Nevertheless, longer term experimental evolution in the absence of a host revealed maintenance of the plasmid at low levels in the bacterial populations. BTI_23p encodes two nematicidal Cry toxins, Cry21Aa2 and Cry14Aa1. Consistent with previous findings, loss of the plasmid abolished pathogenicity towards the nematode Caenorhabditis elegans, which could be rescued by addition of Cry21Aa2-expressing Escherichia coli. These results implicate BTI_23p as a plasmid that is required for successful infection, yet unstable when present at high frequency in the population, consistent with the role of Cry toxins as public goods.