Stingray Venom Proteins: Mechanisms of Action Revealed Using a Novel Network Pharmacology Approach.

Animal venoms offer a valuable source of potent new drug leads, but their mechanisms of action are largely unknown. We therefore developed a novel network pharmacology approach based on multi-omics functional data integration to predict how stingray venom disrupts the physiological systems of target animals. We integrated 10 million transcripts from five stingray venom transcriptomes and 848,640 records from three high-content venom bioactivity datasets into a large functional data network. The network featured 216 signaling pathways, 29 of which were shared and targeted by 70 transcripts and 70 bioactivity hits. The network revealed clusters for single envenomation outcomes, such as pain, cardiotoxicity and hemorrhage. We carried out a detailed analysis of the pain cluster representing a primary envenomation symptom, revealing bibrotoxin and cholecystotoxin-like transcripts encoding pain-inducing candidate proteins in stingray venom. The cluster also suggested that such pain-inducing toxins primarily activate the inositol-3-phosphate receptor cascade, inducing intracellular calcium release. We also found strong evidence for synergistic activity among these candidates, with nerve growth factors cooperating with the most abundant translationally-controlled tumor proteins to activate pain signaling pathways. Our network pharmacology approach, here applied to stingray venom, can be used as a template for drug discovery in neglected venomous species.

Evaluation of high-throughput isomiR identification tools: illuminating the early isomiRome of Tribolium castaneum.

BACKGROUND: MicroRNAs carry out post-transcriptional gene regulation in animals by binding to the 3' untranslated regions of mRNAs, causing their degradation or translational repression. MicroRNAs influence many biological functions, and dysregulation can therefore disrupt development or even cause death. High-throughput sequencing and the mining of animal small RNA data has shown that microRNA genes can yield differentially expressed isoforms, known as isomiRs. Such isoforms are particularly relevant during early development, and the extension or truncation of the 5' end can change the profile of mRNA targets compared to the original mature sequence. We used the publicly available small RNA dataset of the model beetle Tribolium castaneum to create the first comparative isomiRome of early developmental stages in this species. Standard microRNA analysis software does not specifically account for isomiRs. We therefore carried out the first comparative evaluation of the specialized tools isomiRID, isomiR-SEA and miraligner, which can be downloaded for local use and can handle next generation sequencing data. RESULTS: We compared the performance of isomiRID, isomiR-SEA and miraligner using simulated Illumina HiSeq2000 and MiSeq data to test the impact of technical errors. We also created artificial microRNA isoforms to determine the effect of biological variants on the performance of each algorithm. We found that isomiRID achieved the best true positive rate among the three algorithms, but only accounted for one mutation at a time. In contrast, miraligner reported all variations simultaneously but with 78% sensitivity, yielding isomiRs with 3' or 5' deletions. Finally, isomiR-SEA achieved a sensitivity of 25-33% when the seed region was mutated or partly deleted, but was the only tool that could accommodate more than one mismatch. Using the best tool, we performed a complete isomiRome analysis of the early developmental stages of T. castaneum. CONCLUSIONS: Our findings will help researchers to select the most suitable isomiR analysis tools for their experiments. We confirmed the dynamic expression of 3' non-template isomiRs and expanded the isomiRome by all known isomiR modifications during the early development of T. castaneum.

Epigenetic Mechanisms Regulate Innate Immunity against Uropathogenic and Commensal-Like Escherichia coli in the Surrogate Insect Model Galleria mellonella.

Innate-immunity-related genes in humans are activated during urinary tract infections (UTIs) caused by pathogenic strains of Escherichia coli but are suppressed by commensals. Epigenetic mechanisms play a pivotal role in the regulation of gene expression in response to environmental stimuli. To determine whether epigenetic mechanisms can explain the different behaviors of pathogenic and commensal bacteria, we infected larvae of the greater wax moth, Galleria mellonella, a widely used model insect host, with a uropathogenic E. coli (UPEC) strain that causes symptomatic UTIs in humans or a commensal-like strain that causes asymptomatic bacteriuria (ABU). Infection with the UPEC strain (CFT073) was more lethal to larvae than infection with the attenuated ABU strain (83972) due to the recognition of each strain by different Toll-like receptors, ultimately leading to differential DNA/RNA methylation and histone acetylation. We used next-generation sequencing and reverse transcription (RT)-PCR to correlate epigenetic changes with the induction of innate-immunity-related genes. Transcriptomic analysis of G. mellonella larvae infected with E. coli strains CFT073 and 83972 revealed strain-specific variations in the class and expression levels of genes encoding antimicrobial peptides, cytokines, and enzymes controlling DNA methylation and histone acetylation. Our results provide evidence for the differential epigenetic regulation of transcriptional reprogramming by UPEC and ABU strains of E. coli in G. mellonella larvae, which may be relevant to understanding the different behaviors of these bacterial strains in the human urinary tract.

Reassessment of the Listeria monocytogenes pan-genome reveals dynamic integration hotspots and mobile genetic elements as major components of the accessory genome.

BACKGROUND: Listeria monocytogenes is an important food-borne pathogen and model organism for host-pathogen interaction, thus representing an invaluable target considering research on the forces governing the evolution of such microbes. The diversity of this species has not been exhaustively explored yet, as previous efforts have focused on analyses of serotypes primarily implicated in human listeriosis. We conducted complete genome sequencing of 11 strains employing 454 GS FLX technology, thereby achieving full coverage of all serotypes including the first complete strains of serotypes 1/2b, 3c, 3b, 4c, 4d, and 4e. These were comparatively analyzed in conjunction with publicly available data and assessed for pathogenicity in the Galleria mellonella insect model. RESULTS: The species pan-genome of L. monocytogenes is highly stable but open, suggesting an ability to adapt to new niches by generating or including new genetic information. The majority of gene-scale differences represented by the accessory genome resulted from nine hyper variable hotspots, a similar number of different prophages, three transposons (Tn916, Tn554, IS3-like), and two mobilizable islands. Only a subset of strains showed CRISPR/Cas bacteriophage resistance systems of different subtypes, suggesting a supplementary function in maintenance of chromosomal stability. Multiple phylogenetic branches of the genus Listeria imply long common histories of strains of each lineage as revealed by a SNP-based core genome tree highlighting the impact of small mutations for the evolution of species L. monocytogenes. Frequent loss or truncation of genes described to be vital for virulence or pathogenicity was confirmed as a recurring pattern, especially for strains belonging to lineages III and II. New candidate genes implicated in virulence function were predicted based on functional domains and phylogenetic distribution. A comparative analysis of small regulatory RNA candidates supports observations of a differential distribution of trans-encoded RNA, hinting at a diverse range of adaptations and regulatory impact. CONCLUSIONS: This study determined commonly occurring hyper variable hotspots and mobile elements as primary effectors of quantitative gene-scale evolution of species L. monocytogenes, while gene decay and SNPs seem to represent major factors influencing long-term evolution. The discovery of common and disparately distributed genes considering lineages, serogroups, serotypes and strains of species L. monocytogenes will assist in diagnostic, phylogenetic and functional research, supported by the comparative genomic GECO-LisDB analysis server (http://bioinfo.mikrobio.med.uni-giessen.de/geco2lisdb).

The intracellular sRNA transcriptome of Listeria monocytogenes during growth in macrophages.

Small non-coding RNAs (sRNAs) are widespread effectors of post-transcriptional gene regulation in bacteria. Currently extensive information exists on the sRNAs of Listeria monocytogenes expressed during growth in extracellular environments. We used deep sequencing of cDNAs obtained from fractioned RNA (<500 nt) isolated from extracellularly growing bacteria and from L. monocytogenes infected macrophages to catalog the sRNA repertoire during intracellular bacterial growth. Here, we report on the discovery of 150 putative regulatory RNAs of which 71 have not been previously described. A total of 29 regulatory RNAs, including small non-coding antisense RNAs, are specifically expressed intracellularly. We validated highly expressed sRNAs by northern blotting and demonstrated by the construction and characterization of isogenic mutants of rli31, rli33-1 and rli50* for intracellular expressed sRNA candidates, that their expression is required for efficient growth of bacteria in macrophages. All three mutants were attenuated when assessed for growth in mouse and insect models of infection. Comparative genomic analysis revealed the presence of lineage specific sRNA candidates and the absence of sRNA loci in genomes of naturally occurring infection-attenuated bacteria, with additional loss in non-pathogenic listerial genomes. Our analyses reveal extensive sRNA expression as an important feature of bacterial regulation during intracellular growth.

Venom biotechnology: casting light on nature's deadliest weapons using synthetic biology.

Venoms are complex chemical arsenals that have evolved independently many times in the animal kingdom. Venoms have attracted the interest of researchers because they are an important innovation that has contributed greatly to the evolutionary success of many animals, and their medical relevance offers significant potential for drug discovery. During the last decade, venom research has been revolutionized by the application of systems biology, giving rise to a novel field known as venomics. More recently, biotechnology has also made an increasing impact in this field. Its methods provide the means to disentangle and study venom systems across all levels of biological organization and, given their tremendous impact on the life sciences, these pivotal tools greatly facilitate the coherent understanding of venom system organization, development, biochemistry, and therapeutic activity. Even so, we lack a comprehensive overview of major advances achieved by applying biotechnology to venom systems. This review therefore considers the methods, insights, and potential future developments of biotechnological applications in the field of venom research. We follow the levels of biological organization and structure, starting with the methods used to study the genomic blueprint and genetic machinery of venoms, followed gene products and their functional phenotypes. We argue that biotechnology can answer some of the most urgent questions in venom research, particularly when multiple approaches are combined together, and with other venomics technologies.

Complete sequences of plasmids from the hemolytic-uremic syndrome-associated Escherichia coli strain HUSEC41.

The complete and annotated sequences of four plasmids from a historical enteroaggregative Shiga toxin-producing Escherichia coli (HUSEC) serotype O104:H4 strain, HUSEC41/01-09591, isolated in 2001 in Germany are reported.

sRNAdb: a small non-coding RNA database for gram-positive bacteria.

BACKGROUND: The class of small non-coding RNA molecules (sRNA) regulates gene expression by different mechanisms and enables bacteria to mount a physiological response due to adaptation to the environment or infection. Over the last decades the number of sRNAs has been increasing rapidly. Several databases like Rfam or fRNAdb were extended to include sRNAs as a class of its own. Furthermore new specialized databases like sRNAMap (gram-negative bacteria only) and sRNATarBase (target prediction) were established. To the best of the authors' knowledge no database focusing on sRNAs from gram-positive bacteria is publicly available so far. DESCRIPTION: In order to understand sRNA's functional and phylogenetic relationships we have developed sRNAdb and provide tools for data analysis and visualization. The data compiled in our database is assembled from experiments as well as from bioinformatics analyses. The software enables comparison and visualization of gene loci surrounding the sRNAs of interest. To accomplish this, we use a client-server based approach. Offline versions of the database including analyses and visualization tools can easily be installed locally on the user's computer. This feature facilitates customized local addition of unpublished sRNA candidates and related information such as promoters or terminators using tab-delimited files. CONCLUSION: sRNAdb allows a user-friendly and comprehensive comparative analysis of sRNAs from available sequenced gram-positive prokaryotic replicons. Offline versions including analysis and visualization tools facilitate complex user specific bioinformatics analyses.

Identification of novel growth phase- and media-dependent small non-coding RNAs in Streptococcus pyogenes M49 using intergenic tiling arrays.

BACKGROUND: Small non-coding RNAs (sRNAs) have attracted attention as a new class of gene regulators in both eukaryotes and bacteria. Genome-wide screening methods have been successfully applied in Gram-negative bacteria to identify sRNA regulators. Many sRNAs are well characterized, including their target mRNAs and mode of action. In comparison, little is known about sRNAs in Gram-positive pathogens. In this study, we identified novel sRNAs in the exclusively human pathogen Streptococcus pyogenes M49 (Group A Streptococcus, GAS M49), employing a whole genome intergenic tiling array approach. GAS is an important pathogen that causes diseases ranging from mild superficial infections of the skin and mucous membranes of the naso-pharynx, to severe toxic and invasive diseases. RESULTS: We identified 55 putative sRNAs in GAS M49 that were expressed during growth. Of these, 42 were novel. Some of the newly-identified sRNAs belonged to one of the common non-coding RNA families described in the Rfam database. Comparison of the results of our screen with the outcome of two recently published bioinformatics tools showed a low level of overlap between putative sRNA genes. Previously, 40 potential sRNAs have been reported to be expressed in a GAS M1T1 serotype, as detected by a whole genome intergenic tiling array approach. Our screen detected 12 putative sRNA genes that were expressed in both strains. Twenty sRNA candidates appeared to be regulated in a medium-dependent fashion, while eight sRNA genes were regulated throughout growth in chemically defined medium. Expression of candidate genes was verified by reverse transcriptase-qPCR. For a subset of sRNAs, the transcriptional start was determined by 5' rapid amplification of cDNA ends-PCR (RACE-PCR) analysis. CONCLUSIONS: In accord with the results of previous studies, we found little overlap between different screening methods, which underlines the fact that a comprehensive analysis of sRNAs expressed by a given organism requires the complementary use of different methods and the investigation of several environmental conditions. Despite a high conservation of sRNA genes within streptococci, the expression of sRNAs appears to be strain specific.

Comparative genomics and transcriptomics of lineages I, II, and III strains of Listeria monocytogenes.

BACKGROUND: Listeria monocytogenes is a food-borne pathogen that causes infections with a high-mortality rate and has served as an invaluable model for intracellular parasitism. Here, we report complete genome sequences for two L. monocytogenes strains belonging to serotype 4a (L99) and 4b (CLIP80459), and transcriptomes of representative strains from lineages I, II, and III, thereby permitting in-depth comparison of genome- and transcriptome -based data from three lineages of L. monocytogenes. Lineage III, represented by the 4a L99 genome is known to contain strains less virulent for humans. RESULTS: The genome analysis of the weakly pathogenic L99 serotype 4a provides extensive evidence of virulence gene decay, including loss of several important surface proteins. The 4b CLIP80459 genome, unlike the previously sequenced 4b F2365 genome harbours an intact inlB invasion gene. These lineage I strains are characterized by the lack of prophage genes, as they share only a single prophage locus with other L. monocytogenes genomes 1/2a EGD-e and 4a L99. Comparative transcriptome analysis during intracellular growth uncovered adaptive expression level differences in lineages I, II and III of Listeria, notable amongst which was a strong intracellular induction of flagellar genes in strain 4a L99 compared to the other lineages. Furthermore, extensive differences between strains are manifest at levels of metabolic flux control and phosphorylated sugar uptake. Intriguingly, prophage gene expression was found to be a hallmark of intracellular gene expression. Deletion mutants in the single shared prophage locus of lineage II strain EGD-e 1/2a, the lma operon, revealed severe attenuation of virulence in a murine infection model. CONCLUSION: Comparative genomics and transcriptome analysis of L. monocytogenes strains from three lineages implicate prophage genes in intracellular adaptation and indicate that gene loss and decay may have led to the emergence of attenuated lineages.

Venomics of the Central European Myrmicine Ants Myrmica rubra and Myrmica ruginodis.

Animal venoms are a rich source of novel biomolecules with potential applications in medicine and agriculture. Ants are one of the most species-rich lineages of venomous animals. However, only a fraction of their biodiversity has been studied so far. Here, we investigated the venom components of two myrmicine (subfamily Myrmicinae) ants: Myrmica rubra and Myrmica ruginodis. We applied a venomics workflow based on proteotranscriptomics and found that the venoms of both species are composed of several protein classes, including venom serine proteases, cysteine-rich secretory protein, antigen 5 and pathogenesis-related 1 (CAP) superfamily proteins, Kunitz-type serine protease inhibitors and venom acid phosphatases. Several of these protein classes are known venom allergens, and for the first time we detected phospholipase A1 in the venom of M. ruginodis. We also identified two novel epidermal growth factor (EGF) family toxins in the M. ruginodis venom proteome and an array of additional EGF-like toxins in the venom gland transcriptomes of both species. These are similar to known toxins from the related myrmicine ant, Manica rubida, and the myrmecine (subfamily Myrmeciinae) Australian red bulldog ant Myrmecia gullosa, and are possibly deployed as weapons in defensive scenarios or to subdue prey. Our work suggests that M.rubra and M. ruginodis venoms contain many enzymes and other high-molecular-weight proteins that cause cell damage. Nevertheless, the presence of EGF-like toxins suggests that myrmicine ants have also recruited smaller peptide components into their venom arsenal. Although little is known about the bioactivity and function of EGF-like toxins, their presence in myrmicine and myrmecine ants suggests they play a key role in the venom systems of the superfamily Formicoidea. Our work adds to the emerging picture of ant venoms as a source of novel bioactive molecules and highlights the need to incorporate such taxa in future venom bioprospecting programs.

Genomic and Chemical Decryption of the Bacteroidetes Phylum for Its Potential to Biosynthesize Natural Products.

With progress in genome sequencing and data sharing, 1,000s of bacterial genomes are publicly available. Genome mining-using bioinformatics tools in terms of biosynthetic gene cluster (BGC) identification, analysis, and rating-has become a key technology to explore the capabilities for natural product (NP) biosynthesis. Comprehensively, analyzing the genetic potential of the phylum Bacteroidetes revealed Chitinophaga as the most talented genus in terms of BGC abundance and diversity. Guided by the computational predictions, we conducted a metabolomics and bioactivity driven NP discovery program on 25 Chitinophaga strains. High numbers of strain-specific metabolite buckets confirmed the upfront predicted biosynthetic potential and revealed a tremendous uncharted chemical space. Mining this data set, we isolated the new iron chelating nonribosomally synthesized cyclic tetradeca- and pentadecalipodepsipeptide antibiotics chitinopeptins with activity against Candida, produced by C. eiseniae DSM 22224 and C. flava KCTC 62435, respectively. IMPORTANCE The development of pipelines for anti-infectives to be applied in plant, animal, and human health management are dried up. However, the resistance development against compounds in use calls for new lead structures. To fill this gap and to enhance the probability of success for the discovery of new bioactive natural products, microbial taxa currently underinvestigated must be mined. This study investigates the potential within the bacterial phylum Bacteroidetes. A combination of omics-technologies revealed taxonomical hot spots for specialized metabolites. Genome- and metabolome-based analyses showed that the phylum covers a new chemical space compared with classic natural product producers. Members of the Bacteroidetes may thus present a promising bioresource for future screening and isolation campaigns.

Highly specific and quick detection of Mycobacterium avium subsp. paratuberculosis in feces and gut tissue of cattle and humans by multiple real-time PCR assays.

Mycobacterium avium subsp. paratuberculosis is the causative agent of Johne's disease (JD) in cattle and may be associated with Crohn's disease (CD) in humans. It is the slowest growing of the cultivable mycobacteria, and culture from clinical, veterinary, food, or environmental specimens can take 4 months or even longer. Currently, the insertion element IS900 is used to detect M. avium subsp. paratuberculosis DNA. However, closely related IS900 elements are also present in other mycobacteria, thus limiting its specificity as a target. Here we describe the use of novel primer sets derived from the sequences of two highly specific single copy genes, MAP2765c and MAP0865, for the quantitative detection of M. avium subsp. paratuberculosis within 6 h by using real-time PCR. Specificity of the target was established using 40 M. avium subsp. paratuberculosis isolates, 67 different bacterial species, and two intestinal parasites. Using the probes and methods described, we detected 27 (2.09%) M. avium subsp. paratuberculosis-positive stool specimens from 1,293 individual stool samples by the use of either IS900 or probes deriving from the MAP2765c and MAP0865 genes described here. In general, bacterial load due to M. avium subsp. paratuberculosis was uniformly low in these samples and we estimated 500 to 5,000 M. avium subsp. paratuberculosis bacteria per gram of stool in assay-positive samples. Thus, the methods described here are useful for rapid and specific detection of M. avium subsp. paratuberculosis in clinical samples.

Adaptation of Listeria monocytogenes to oxidative and nitrosative stress in IFN-γ-activated macrophages.

IFN-γ-activated macrophages are considered to be the primary effector cells in host defense against Listeria monocytogenes infections. However despite the induction of the complex host defense mechanisms, survival of L. monocytogenes in activated macrophages is still observed. Here we used a whole genome-based transcriptome approach to examine for bacterial genes specifically induced in IFN-γ-activated macrophages. We demonstrated that cells activated by IFN-γ had elevated oxidative and nitrosative stress levels in both the activated macrophages as well as in the intracellular replicating bacteria isolated from these infected cells. We found that a subset of 21 transcripts were specifically differentially regulated in bacteria growing in cells pretreated with IFN-γ. Bioinformatics and functional analysis revealed that many of these genes have roles involved in overcoming oxidative stress and contribute to bacterial survival within activated macrophages. We detected increased transcription of the putative trpE gene of L. monocytogenes, encoding an anthranilate synthase, in bacteria growing in IFN-γ cells indicating host cell metabolic restriction of bacterial growth. Indeed we found enhanced activation of host cell genes involved in the kynurenine pathway indicating an increased need of L. monocytogenes for tryptophan during replication in IFN-γ-activated macrophages.

A Spider Toxin Exemplifies the Promises and Pitfalls of Cell-Free Protein Production for Venom Biodiscovery.

Arthropod venoms offer a promising resource for the discovery of novel bioactive peptides and proteins, but the limited size of most species translates into minuscule venom yields. Bioactivity studies based on traditional fractionation are therefore challenging, so alternative strategies are needed. Cell-free synthesis based on synthetic gene fragments is one of the most promising emerging technologies, theoretically allowing the rapid, laboratory-scale production of specific venom components, but this approach has yet to be applied in venom biodiscovery. Here, we tested the ability of three commercially available cell-free protein expression systems to produce venom components from small arthropods, using U2-sicaritoxin-Sdo1a from the six-eyed sand spider Hexophtalma dolichocephala as a case study. We found that only one of the systems was able to produce an active product in low amounts, as demonstrated by SDS-PAGE, mass spectrometry, and bioactivity screening on murine neuroblasts. We discuss our findings in relation to the promises and limitations of cell-free synthesis for venom biodiscovery programs in smaller invertebrates.

Elucidation of the MicroRNA Transcriptome in Western Corn Rootworm Reveals Its Dynamic and Evolutionary Complexity.

Diabrotica virgifera virgifera (western corn rootworm, WCR) is one of the most destructive agricultural insect pests in North America. It is highly adaptive to environmental stimuli and crop protection technologies. However, little is known about the underlying genetic basis of WCR behavior and adaptation. More specifically, the involvement of small RNAs (sRNAs), especially microRNAs (miRNAs), a class of endogenous small non-coding RNAs that regulate various biological processes, has not been examined, and the datasets of putative sRNA sequences have not previously been generated for WCR. To achieve a comprehensive collection of sRNA transcriptomes in WCR, we constructed, sequenced, and analyzed sRNA libraries from different life stages of WCR and northern corn rootworm (NCR), and identified 101 conserved precursor miRNAs (pre-miRNAs) in WCR and other Arthropoda. We also identified 277 corn rootworm specific pre-miRNAs. Systematic analyses of sRNA populations in WCR revealed that its sRNA transcriptome, which includes PIWI-interacting RNAs (piRNAs) and miRNAs, undergoes a dynamic change throughout insect development. Phylogenetic analysis of miRNA datasets from model species reveals that a large pool of species-specific miRNAs exists in corn rootworm; these are potentially evolutionarily transient. Comparisons of WCR miRNA clusters to other insect species highlight conserved miRNA-regulated processes that are common to insects. Parallel Analysis of RNA Ends (PARE) also uncovered potential miRNA-guided cleavage sites in WCR. Overall, this study provides a new resource for studying the sRNA transcriptome and miRNA-mediated gene regulation in WCR and other Coleopteran insects.

Complete genome sequence of Listeria seeligeri, a nonpathogenic member of the genus Listeria.

We report the complete and annotated genome sequence of the nonpathogenic Listeria seeligeri SLCC3954 serovar 1/2b type strain harboring the smallest completely sequenced genome of the genus Listeria.

MicroRNAs regulate innate immunity against uropathogenic and commensal-like Escherichia coli infections in the surrogate insect model Galleria mellonella.

Uropathogenic Escherichia coli (UPEC) strains cause symptomatic urinary tract infections in humans whereas commensal-like E. coli strains in the urinary bladder cause long-term asymptomatic bacteriuria (ABU). We previously reported that UPEC and ABU strains differentially regulate key DNA methylation and histone acetylation components in the surrogate insect host Galleria mellonella to epigenetically modulate innate immunity-related gene expression, which in turn controls bacterial growth. In this follow-up study, we infected G. mellonella larvae with UPEC strain CFT073 or ABU strain 83972 to identify differences in the expression of microRNAs (miRNAs), a class of non-coding RNAs that regulate gene expression at the post-transcriptional level. Our small RNA sequencing analysis showed that UPEC and ABU infections caused significant changes in the abundance of miRNAs in the larvae, and highlighted the differential expression of 147 conserved miRNAs and 95 novel miRNA candidates. We annotated the G. mellonella genome sequence to investigate the miRNA-regulated expression of genes encoding antimicrobial peptides, signaling proteins, and enzymatic regulators of DNA methylation and histone acetylation in infected larvae. Our results indicate that miRNAs play a role in the epigenetic reprograming of innate immunity in G. mellonella larvae to distinguish between pathogenic and commensal strains of E. coli.

An Economic Dilemma Between Molecular Weapon Systems May Explain an Arachno-atypical Venom in Wasp Spiders (Argiope bruennichi).

Spiders use venom to subdue their prey, but little is known about the diversity of venoms in different spider families. Given the limited data available for orb-weaver spiders (Araneidae), we selected the wasp spider Argiope bruennichi for detailed analysis. Our strategy combined a transcriptomics pipeline based on multiple assemblies with a dual proteomics workflow involving parallel mass spectrometry techniques and electrophoretic profiling. We found that the remarkably simple venom of A. bruennichi has an atypical composition compared to other spider venoms, prominently featuring members of the cysteine-rich secretory protein, antigen 5 and pathogenesis-related protein 1 (CAP) superfamily and other, mostly high-molecular-weight proteins. We also detected a subset of potentially novel toxins similar to neuropeptides. We discuss the potential function of these proteins in the context of the unique hunting behavior of wasp spiders, which rely mostly on silk to trap their prey. We propose that the simplicity of the venom evolved to solve an economic dilemma between two competing yet metabolically expensive weapon systems. This study emphasizes the importance of cutting-edge methods to encompass the lineages of smaller venomous species that have yet to be characterized in detail, allowing us to understand the biology of their venom systems and to mine this prolific resource for translational research.

Novel bacterial artificial chromosome vector pUvBBAC for use in studies of the functional genomics of Listeria spp.

Bacterial artificial chromosome (BAC) vectors are important tools for microbial genome research. We constructed a novel BAC vector, pUvBBAC, for replication in both gram-negative and gram-positive bacterial hosts. The pUvBBAC vector was used to generate a BAC library for the facultative intracellular pathogen Listeria monocytogenes EGD-e. The library had insert sizes ranging from 68 to 178 kb. We identified two recombinant BACs from the L. monocytogenes pUvBBAC library that each contained the entire virulence gene cluster (vgc) of L. monocytogenes and transferred them to a nonpathogenic Listeria innocua strain. Recombinant L. innocua strains harboring pUvBBAC+vgc1 and pUvBBAC+vgc2 produced the vgc-specific listeriolysin (LLO) and actin assembly protein ActA and represent the first reported cloning of the vgc locus in its entirety. The use of the novel broad-host-range BAC vector pUvBBAC extends the versatility of this technology and provides a powerful platform for detailed functional genomics of gram-positive bacteria as well as its use in explorative functional metagenomics.