Microbiome-derived antimicrobial peptides offer therapeutic solutions for the treatment of Pseudomonas aeruginosa infections.

Microbiomes are rife for biotechnological exploitation, particularly the rumen microbiome, due to their complexicity and diversity. In this study, antimicrobial peptides (AMPs) from the rumen microbiome (Lynronne 1, 2, 3 and P15s) were assessed for their therapeutic potential against seven clinical strains of Pseudomonas aeruginosa. All AMPs exhibited antimicrobial activity against all strains, with minimum inhibitory concentrations (MICs) ranging from 4-512 µg/mL. Time-kill kinetics of all AMPs at 3× MIC values against strains PAO1 and LES431 showed complete kill within 10 min to 4 h, although P15s was not bactericidal against PAO1. All AMPs significantly inhibited biofilm formation by strains PAO1 and LES431, and induction of resistance assays showed no decrease in activity against these strains. AMP cytotoxicity against human lung cells was also minimal. In terms of mechanism of action, the AMPs showed affinity towards PAO1 and LES431 bacterial membrane lipids, efficiently permeabilising the P. aeruginosa membrane. Transcriptome and metabolome analysis revealed increased catalytic activity at the cell membrane and promotion of ß-oxidation of fatty acids. Finally, tests performed with the Galleria mellonella infection model showed that Lynronne 1 and 2 were efficacious in vivo, with a 100% survival rate following treatment at 32 mg/kg and 128 mg/kg, respectively. This study illustrates the therapeutic potential of microbiome-derived AMPs against P. aeruginosa infections.

Modulation of Rumen Microbes Through Extracellular Vesicle Released by the Rumen Fluke Calicophoron daubneyi.

Parasite derived extracellular vesicles (EVs) have been proposed to play key roles in the establishment and maintenance of infection. Calicophoron daubneyi is a newly emerging parasite of livestock with many aspects of its underpinning biology yet to be resolved. This research is the first in-depth investigation of EVs released by adult C. daubneyi. EVs were successfully isolated using both differential centrifugation and size exclusion chromatography (SEC), and morphologically characterized though transmission electron microscopy (TEM). EV protein components were characterized using a GeLC approach allowing the elucidation of comprehensive proteomic profiles for both their soluble protein cargo and surface membrane bound proteins yielding a total of 378 soluble proteins identified. Notably, EVs contained Sigma-class GST and cathepsin L and B proteases, which have previously been described in immune modulation and successful establishment of parasitic flatworm infections. SEC purified C. daubneyi EVs were observed to modulate rumen bacterial populations by likely increasing microbial species diversity via antimicrobial activity. This data indicates EVs released from adult C. daubneyi have a role in establishment within the rumen through the regulation of microbial populations offering new routes to control rumen fluke infection and to develop molecular strategies to improve rumen efficiency.

The importance of extracellular vesicle purification for downstream analysis: A comparison of differential centrifugation and size exclusion chromatography for helminth pathogens.

BACKGROUND: Robust protocols for the isolation of extracellular vesicles (EVs) from the rest of their excretory-secretory products are necessary for downstream studies and application development. The most widely used purification method of EVs for helminth pathogens is currently differential centrifugation (DC). In contrast, size exclusion chromatography (SEC) has been included in the purification pipeline for EVs from other pathogens, highlighting there is not an agreed research community 'gold standard' for EV isolation. In this case study, Fasciola hepatica from natural populations were cultured in order to collect EVs from culture media and evaluate a SEC or DC approach to pathogen helminth EV purification. METHODOLOGY/PRINCIPAL FINDINGS: Transmission electron and atomic force microscopy demonstrated that EVs prepared by SEC were both smaller in size and less diverse than EV resolved by DC. Protein quantification and Western blotting further demonstrated that SEC purification realised a higher EV purity to free excretory-secretory protein (ESP) yield ratio compared to DC approaches as evident by the reduction of soluble free cathepsin L proteases in SEC EV preparations. Proteomic analysis further highlighted DC contamination from ESP as shown by an increased diversity of protein identifications and unique peptide hits in DC EVs as compared to SEC EVs. In addition, SEC purified EVs contained less tegumental based proteins than DC purified EVs. CONCLUSIONS/SIGNIFICANCE: The data suggests that DC and SEC purification methods do not isolate equivalent EV population profiles and caution should be taken in the choice of EV purification utilised, with certain protocols for DC preparations including more free ES proteins and tegumental artefacts. We propose that SEC methods should be used for EV purification prior to downstream studies.

Using 'Omic Approaches to Compare Temporal Bacterial Colonization of Lolium perenne, Lotus corniculatus, and Trifolium pratense in the Rumen.

Understanding rumen plant-microbe interactions is central for development of novel methodologies allowing improvements in ruminant nutrient use efficiency. This study investigated rumen bacterial colonization of fresh plant material and changes in plant chemistry over a period of 24 h period using three different fresh forages: Lolium perenne (perennial ryegrass; PRG), Lotus corniculatus (bird's foot trefoil; BFT) and Trifolium pratense (red clover; RC). We show using 16S rRNA gene ion torrent sequencing that plant epiphytic populations present pre-incubation (0 h) were substantially different to those attached post incubations in the presence of rumen fluid on all forages. Thereafter primary and secondary colonization events were evident as defined by changes in relative abundances of attached bacteria and changes in plant chemistry, as assessed using Fourier transform infrared (FTIR) spectroscopy. For PRG colonization, primary colonization occurred for up to 4 h and secondary colonization from 4 h onward. The changes from primary to secondary colonization occurred significantly later with BFT and RC, with primary colonization being up to 6 h and secondary colonization post 6 h of incubation. Across all 3 forages the main colonizing bacteria present at all time points post-incubation were Prevotella, Pseudobutyrivibrio, Ruminococcus, Olsenella, Butyrivibrio, and Anaeroplasma (14.2, 5.4, 1.9, 2.7, 1.8, and 2.0% on average respectively), with Pseudobutyrivibrio and Anaeroplasma having a higher relative abundance during secondary colonization. Using CowPI, we predict differences between bacterial metabolic function during primary and secondary colonization. Specifically, our results infer an increase in carbohydrate metabolism in the bacteria attached during secondary colonization, irrespective of forage type. The CowPI data coupled with the FTIR plant chemistry data suggest that attached bacterial function is similar irrespective of forage type, with the main changes occurring between primary and secondary colonization. These data suggest that the sward composition of pasture may have major implications for the temporal availability of nutrients for animal.

CowPI: A Rumen Microbiome Focussed Version of the PICRUSt Functional Inference Software.

Metataxonomic 16S rDNA based studies are a commonplace and useful tool in the research of the microbiome, but they do not provide the full investigative power of metagenomics and metatranscriptomics for revealing the functional potential of microbial communities. However, the use of metagenomic and metatranscriptomic technologies is hindered by high costs and skills barrier necessary to generate and interpret the data. To address this, a tool for Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) was developed for inferring the functional potential of an observed microbiome profile, based on 16S data. This allows functional inferences to be made from metataxonomic 16S rDNA studies with little extra work or cost, but its accuracy relies on the availability of completely sequenced genomes of representative organisms from the community being investigated. The rumen microbiome is an example of a community traditionally underrepresented in genome and sequence databases, but recent efforts by projects such as the Global Rumen Census and Hungate 1000 have resulted in a wide sampling of 16S rDNA profiles and almost 500 fully sequenced microbial genomes from this environment. Using this information, we have developed "CowPI," a focused version of the PICRUSt tool provided for use by the wider scientific community in the study of the rumen microbiome. We evaluated the accuracy of CowPI and PICRUSt using two 16S datasets from the rumen microbiome: one generated from rDNA and the other from rRNA where corresponding metagenomic and metatranscriptomic data was also available. We show that the functional profiles predicted by CowPI better match estimates for both the meta-genomic and transcriptomic datasets than PICRUSt, and capture the higher degree of genetic variation and larger pangenomes of rumen organisms. Nonetheless, whilst being closer in terms of predictive power for the rumen microbiome, there were differences when compared to both the metagenomic and metatranscriptome data and so we recommend, where possible, functional inferences from 16S data should not replace metagenomic and metatranscriptomic approaches. The tool can be accessed at http://www.cowpi.org and is provided to the wider scientific community for use in the study of the rumen microbiome.

The anti-fecundity effect of 5-azacytidine (5-AzaC) on Schistosoma mansoni is linked to dis-regulated transcription, translation and stem cell activities.

Uncontrolled host immunological reactions directed against tissue-trapped eggs precipitate a potentially lethal, pathological cascade responsible for schistosomiasis. Blocking schistosome egg production, therefore, presents a strategy for simultaneously reducing immunopathology as well as limiting disease transmission in endemic or emerging areas. We recently demonstrated that the ribonucleoside analogue 5-azacytidine (5-AzaC) inhibited Schistosoma mansoni oviposition, egg maturation and ovarian development. While these anti-fecundity effects were associated with a loss of DNA methylation, other molecular processes affected by 5-AzaC were not examined at the time. By comparing the transcriptomes of 5-AzaC-treated females to controls, we provide evidence that this ribonucleoside analogue also modulates other crucial aspects of schistosome egg-laying biology. For example, S. mansoni gene products associated with amino acid-, carbohydrate-, fatty acid-, nucleotide- and tricarboxylic acid (TCA)- homeostasis are all dysregulated in 5-AzaC treated females. To validate the metabolic pathway most significantly affected by 5-AzaC, amino acid metabolism, nascent protein synthesis was subsequently quantified in adult schistosomes. Here, 5-AzaC inhibited this process by 68% ±16.7% (SEM) in male- and 81% ±4.8% (SEM) in female-schistosomes. Furthermore, the transcriptome data indicated that adult female stem cells were also affected by 5-AzaC. For instance, 40% of transcripts associated with proliferating schistosome cells were significantly down-regulated by 5-AzaC. This finding correlated with a considerable reduction (95%) in the number of 5-ethynyl-2'-deoxyuridine (EdU) positive cells found in 5-AzaC-treated females. In addition to protein coding genes, the effect that 5-AzaC had on repetitive element expression was also assessed. Here, 46 repeats were found differentially transcribed between 5-AzaC-treated and control females with long terminal repeat (LTR) and DNA transposon classes being amongst the most significant. This study demonstrates that the anti-fecundity activity of 5-AzaC affects more than just DNA methylation in schistosome parasites. Further characterisation of these processes may reveal novel targets for schistosomiasis control.

Accumulation and exchange of parasites during adaptive radiation in an ancient lake.

In the ancient Lake Baikal, Russia, amphipod crustaceans have undergone a spectacular adaptive radiation, resulting in a diverse community of species. A survey of microsporidian parasites inhabiting endemic and non-endemic amphipod host species at the margins of Lake Baikal indicates that the endemic amphipods harbour many microsporidian parasite groups associated with amphipods elsewhere in Eurasia. While these parasites may have undergone a degree of adaptive radiation within the lake, there is little evidence of host specificity. Furthermore, a lack of reciprocal monophyly indicates that exchanges of microsporidia between Baikalian and non-Baikalian hosts have occurred frequently in the past and may be ongoing. Conversely, limitations to parasite exchange between Baikalian and non-Baikalian host populations at the margins of the lake are implied by differences in parasite prevalence and lack of shared microsporidian haplotypes between the two host communities. While amphipod hosts have speciated sympatrically within Lake Baikal, the parasites appear instead to have accumulated, moving into the lake from external amphipod populations on multiple occasions to exploit the large and diverse community of endemic amphipods in Lake Baikal.

The rumen microbiome: an underexplored resource for novel antimicrobial discovery.

Antimicrobial peptides (AMPs) are promising drug candidates to target multi-drug resistant bacteria. The rumen microbiome presents an underexplored resource for the discovery of novel microbial enzymes and metabolites, including AMPs. Using functional screening and computational approaches, we identified 181 potentially novel AMPs from a rumen bacterial metagenome. Here, we show that three of the selected AMPs (Lynronne-1, Lynronne-2 and Lynronne-3) were effective against numerous bacterial pathogens, including methicillin-resistant Staphylococcus aureus (MRSA). No decrease in MRSA susceptibility was observed after 25 days of sub-lethal exposure to these AMPs. The AMPs bound preferentially to bacterial membrane lipids and induced membrane permeability leading to cytoplasmic leakage. Topical administration of Lynronne-1 (10% w/v) to a mouse model of MRSA wound infection elicited a significant reduction in bacterial counts, which was comparable to treatment with 2% mupirocin ointment. Our findings indicate that the rumen microbiome may provide viable alternative antimicrobials for future therapeutic application.

Characterization of the Microbiome along the Gastrointestinal Tract of Growing Turkeys.

The turkey microbiome is largely understudied, despite its relationship with bird health and growth, and the prevalence of human pathogens such as Campylobacter spp. In this study we investigated the microbiome within the small intestine (SI), caeca (C), large intestine (LI), and cloaca (CL) of turkeys at 6, 10, and 16 weeks of age. Eight turkeys were dissected within each age category and the contents of the SI, C, LI, and CL were harvested. 16S rDNA based QPCR was performed on all samples and samples for the four locations within three birds/age group were sequenced using ion torrent-based sequencing of the 16S rDNA. Sequencing data showed on a genus level, an abundance of Lactobacillus, Streptococcus, and Clostridium XI (38.2, 28.1, and 13.0% respectively) irrespective of location and age. The caeca exhibited the greatest microbiome diversity throughout the development of the turkey. PICRUSt data predicted an array of bacterial function, with most differences being apparent in the caeca of the turkeys as they matured. QPCR revealed that the caeca within 10 week old birds, contained the most Campylobacter spp. Understanding the microbial ecology of the turkey gastrointestinal tract is essential in terms of understanding production efficiency and in order to develop novel strategies for targeting Campylobacter spp.

Temporal Metagenomic and Metabolomic Characterization of Fresh Perennial Ryegrass Degradation by Rumen Bacteria.

Understanding the relationship between ingested plant material and the attached microbiome is essential for developing methodologies to improve ruminant nutrient use efficiency. We have previously shown that perennial ryegrass (PRG) rumen bacterial colonization events follow a primary (up to 4 h) and secondary (after 4 h) pattern based on the differences in diversity of the attached bacteria. In this study, we investigated temporal niche specialization of primary and secondary populations of attached rumen microbiota using metagenomic shotgun sequencing as well as monitoring changes in the plant chemistry using mid-infrared spectroscopy (FT-IR). Metagenomic Rapid Annotation using Subsystem Technology (MG-RAST) taxonomical analysis of shotgun metagenomic sequences showed that the genera Butyrivibrio, Clostridium, Eubacterium, Prevotella, and Selenomonas dominated the attached microbiome irrespective of time. MG-RAST also showed that Acidaminococcus, Bacillus, Butyrivibrio, and Prevotella rDNA increased in read abundance during secondary colonization, whilst Blautia decreased in read abundance. MG-RAST Clusters of Orthologous Groups (COG) functional analysis also showed that the primary function of the attached microbiome was categorized broadly within "metabolism;" predominantly amino acid, carbohydrate, and lipid metabolism and transport. Most sequence read abundances (51.6, 43.8, and 50.0% of COG families pertaining to amino acid, carbohydrate and lipid metabolism, respectively) within these categories were higher in abundance during secondary colonization. Kyoto encyclopedia of genes and genomes (KEGG) pathways analysis confirmed that the PRG-attached microbiota present at 1 and 4 h of rumen incubation possess a similar functional capacity, with only a few pathways being uniquely found in only one incubation time point only. FT-IR data for the plant residues also showed that the main changes in plant chemistry between primary and secondary colonization was due to increased carbohydrate, amino acid, and lipid metabolism. This study confirmed primary and secondary colonization events and supported the hypothesis that functional changes occurred as a consequence of taxonomical changes. Sequences within the carbohydrate metabolism COG families contained only 3.2% of cellulose activities, on average across both incubation times (1 and 4 h), suggesting that degradation of the plant cell walls may be a key rate-limiting factor in ensuring the bioavailability of intra-plant nutrients in a timely manner to the microbes and ultimately the animal. This suggests that a future focus for improving ruminant nutrient use efficiency should be altering the recalcitrant plant cell wall components and/or improving the cellulolytic capacity of the rumen microbiota.

Exploring and Expanding the Fatty-Acid-Binding Protein Superfamily in Fasciola Species.

The liver flukes Fasciola hepatica and F. gigantica infect livestock worldwide and threaten food security with climate change and problematic control measures spreading disease. Fascioliasis is also a foodborne disease with up to 17 million humans infected. In the absence of vaccines, treatment depends on triclabendazole (TCBZ), and overuse has led to widespread resistance, compromising future TCBZ control. Reductionist biology from many laboratories has predicted new therapeutic targets. To this end, the fatty-acid-binding protein (FABP) superfamily has proposed multifunctional roles, including functions intersecting vaccine and drug therapy, such as immune modulation and anthelmintic sequestration. Research is hindered by a lack of understanding of the full FABP superfamily complement. Although discovery studies predicted FABPs as promising vaccine candidates, it is unclear if uncharacterized FABPs are more relevant for vaccine formulations. We have coupled genome, transcriptome, and EST data mining with proteomics and phylogenetics to reveal a liver fluke FABP superfamily of seven clades: previously identified clades I-III and newly identified clades IV-VII. All new clade FABPs were analyzed using bioinformatics and cloned from both liver flukes. The extended FABP data set will provide new study tools to research the role of FABPs in parasite biology and as therapy targets.

Proteomics and in silico approaches to extend understanding of the glutathione transferase superfamily of the tropical liver fluke Fasciola gigantica.

Fasciolosis is an important foodborne, zoonotic disease of livestock and humans, with global annual health and economic losses estimated at several billion US$. Fasciola hepatica is the major species in temperate regions, while F. gigantica dominates in the tropics. In the absence of commercially available vaccines to control fasciolosis, increasing reports of resistance to current chemotherapeutic strategies and the spread of fasciolosis into new areas, new functional genomics approaches are being used to identify potential new drug targets and vaccine candidates. The glutathione transferase (GST) superfamily is both a candidate drug and vaccine target. This study reports the identification of a putatively novel Sigma class GST, present in a water-soluble cytosol extract from the tropical liver fluke F. gigantica. The GST was cloned and expressed as an enzymically active recombinant protein. This GST shares a greater identity with the human schistosomiasis GST vaccine currently at Phase II clinical trials than previously discovered F. gigantica GSTs, stimulating interest in its immuno-protective properties. In addition, in silico analysis of the GST superfamily of both F. gigantica and F. hepatica has revealed an additional Mu class GST, Omega class GSTs, and for the first time, a Zeta class member.

Genetic diversity of the feminising microsporidian parasite Dictyocoela: new insights into host-specificity, sex and phylogeography.

Microsporidia of the genus Dictyocoela are parasites of gammarid amphipod Crustacea. They typically exhibit low virulence and efficient vertical transmission and at least some strains are capable of feminising their hosts. Sequencing of a region of the 16S rDNA of Dictyocoela spp. from various gammarid host species and localities in Europe and northern Asia indicates that Dictyocoela is genetically diverse and that different strains predominate in different host species. However, the presence of intermediate sequences casts doubt upon previous attempts to describe Dictyocoela spp. on the basis of rDNA divergence alone. Phylogenetic analysis provides little support for coevolution between gammarids and Dictyocoela. Furthermore, observations of heavily infected individuals, together with genetic evidence of recombination, suggest that some strains of Dictyocoela may be horizontally transmitted and are sexually reproducing. These findings suggest that Dictyocoela may be phenotypically, as well as genotypically, diverse, with the potential to exhibit a range of different interactions with its host populations.

Distribution and host range of the microsporidian Pleistophora mulleri.

Microsporidia of the genus Pleistophora are important parasites of fish and crustacea. Pleistophora mulleri has been described previously as a parasite of the gammarid amphipod crustacean Gammarus duebeni celticus in Irish freshwater habitats. Through a survey of European G. duebeni populations, P. mulleri was found to be widely distributed in the western British Isles (Wales, Scotland, and the Isle of Man), and populations of the subspecies Gammarus duebeni duebeni as well as G. d. celticus were infected. Pleistophora infections were also detected in G. d. duebeni sampled from the Bay of Gdansk on Poland's Baltic coast, indicating a wide distribution of Pleistophora in European G. duebeni. Sequencing and phylogenetic analysis of the 16S rRNA, 18S rRNA, and Rpb1 genes of P. mulleri suggest that this species may be synonymous with P. typicalis, a parasite of fish. These findings suggest that amphipod crustaceans may act as intermediate or reservoir hosts for microsporidian parasites of fish.