- Volume 1, Issue 1A, 2019
Volume 1, Issue 1A, 2019
- Oral Abstract
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- Infection Forum
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An uncharacterised protein mediates motility, biofilm formation, and host colonisation in adherent invasive Escherichia coli
More LessAdherent Invasive Escherichia coli (AIEC) is a non-diarrhoeagenic intestinal E. coli patho type with a putativea etiological role in Crohn’s Disease (CD). AIEC pathogenes is ischaracterised by adhesion to, invasion of, and replication within intestinal epithelial cells and macrophages, and biofilm formation. Using a heterologous expression screen, we identified a gene in the AIEC LF82 genome encoding a protein which self-assembles into filaments in HeLa cells, which we hypothesised was a novel pilin or biofilm matrix component, designated bcmA (biofilm coupled to motility in AIEC). Using a crystal violet-based assay, we found LF82ΔbcmA have defective biofilm formation, which can be fully complemented by episomal bcmA expression. Microscopic analysis of LF82 biofilms demonstrated LF82ΔbcmA form patchy, sparse biofilms, and revealed an intracellular localisation for GFP-tagged bcmA, suggesting the protein is not a surface-exposed adhesin or biofilm matrix component. We therefore assessed the role of bcmA in flagellar-mediated motility and found that – despite displaying wild-type flagellar morphologies – LF82ΔbcmA have profound swimming and swarming defects. Work in a Caenorhabdit is elegans infection model suggests bcmA is not required for full virulence; however, preliminary evidence suggests LF82ΔbcmA have defective C. elegans gut colonisation. Taken together, our data demonstrates roles for bcmA in AIEC host colonisation via an undefined role in motility. bcmA is highly conserved among pathogenic gammaproteobacteria, including Salmonella Typhi, Shigella boydii and Klebsiella pneumoniae. This suggests bcmA may not only be significant for AIEC pathogenesis, but may also represent an important virulence determinant in several major gammaproteobacterial pathogens.
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The macrophage intracellular niche and its role in cryptococcosis
More LessCryptococcus neoformans is an opportunistic fungal infection that causes cryptococcal meningitis in immunocompromised individuals. Macrophages play a critical role in determining the outcome of infection, and can either phagocytose and kill the cryptococcal cells, or disseminate infection. While it is known that macrophages impact the progression of cryptococcal disease, it is not known how the macrophage intracellular niche contributes to complex infection outcomes. Clinical and experimental studies have identified potential genetic differences, in both host and pathogen, but statistical robustness has been difficult to achieve due the large variability in the outcome of infections. Therefore, in attempt to quantitatively explain this variability, we have used a zebrafish model of cryptococcal infection where we can directly relate the initial level of fungal infection with final infection outcome. We find that at low levels of initial fungal burden the outcome of infection is stochastic, while over high ranges of initial infection the outcome is linearly related to the initial burden, but with a further stochastic component that contributes to increased variability. Using these experimental data and data from clinical trials we have generated a computational simulation of in vivo cryptococcal infections which allows us to consider different infection variables and how they alter the progression of cryptococcosis. By combining such computational simulations with our experimental models we demonstrate that the macrophage intracellular niche determines the unknown stochastic component, independent of fungal burden. Using this knowledge, we can better identify the molecular, population genetic, and clinical parameters associated with the outcome of cryptococcosis.
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Global mapping of protein subcellular location in apicomplexans: the parasite as we’ve never seen it before
Apicomplexans are human and animal protozoan pathogens responsible for diseases including malaria, cryptosporidiosis and toxoplasmosis. As obligate intracellular parasites they are highly organised cells with numerous novel and specialised sub-compartments that form the basis of their invasion biology, host defence evasion, and novel metabolic traits. However, our understanding of these cells is highly constrained by our limited knowledge of the locations and functions of most of the cell’s proteome. Even in the best-studied apicomplexans (Plasmodium spp. and Toxoplasma gondii) only a small fraction of proteins’ locations have been experimentally determined, with most assignments based on predictions from orthologues in distant relatives. Moreover, many parasite proteins are annotated as ‘hypotheticals’, for example 4113 of 8121 Toxoplasma proteins, and many are unique to parasites stymying even predictions of location or function by comparative biology. To address this deficit in our basic understanding of the compositional organisation of the apicomplexan cell, we have used a spatial proteomics method called hyper LOPIT to simultaneously capture the steady-state subcellular association of thousands of proteins in the apicomplexan Toxoplasma. These protein atlases reveal: extensive protein association networks throughout the cell providing testable hypotheses of their function; conservation and novelty of compartment proteomes between apicomplexans; differential selective pressures across the different cell compartments; and clear instances of protein relocation from one organelle to a different one during apicomplexan speciation. This new, global view of the organisation of the apicomplexan cell proteome provides a much more complete framework for understanding the mechanisms of function and biology of these cells.
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Investigating the role of the bacterial mechanosensitive channel YnaI in Salmonella pathogenesis
More LessMechanosensitive channels are required for bacteria cells to survive hypoosmotic shock (transition from high to low osmolarity environment). YnaI is one of the mechanosensitive channels found amongst many bacterial species including Salmonella Typhimurium. Previous studies have suggested that S. Typhimurium YnaI may be implicated in host colonization during infection of farmed animals. Disruption of ynaI impaired intestinal colonization in pigs, cattle and chicken. To investigate S. Typhimurium YnaI structure and function, the S.Typhimurium ynaI was cloned into a plasmid, followed by physiological characterization of the S. Typhimurium YnaI constructs expressed in an E. coli channel-less mutant strain. To further understand the role of YnaI in S. Typhimurium pathogenesis, S. Typhimurium ynaI was deleted and the ability of mutant to survive and replicate in host cells was investigated. The S. Typhimurium YnaI channel has unique characteristics when expressed in E. coli: S. Typhimurium YnaI channel conferred almost complete protection against 0.3 M NaCl hypoosmotic shock when overexpressed, but interestingly, high level expression of S. Typhimurium YnaI inhibited growth in two different complex media and in minimal media. Deletion of ynaI from S. Typhimurium led to increased internalization in macrophages and epithelial cells. Data derived from this study reveals novel characteristics of S. Typhimurium YnaI which may provide insights into other functions of the S. Typhimurium YnaI channel.
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Global gene expression profiling of a virulent Klebsiella pneumoniae strain during pulmonary infection
BackgroundKlebsiella pneumoniae (Kpn) is an important respiratory pathogen associated with significant mortality, fierce inflammatory responses and high rates of antimicrobial resistance. The increasing incidence of multidrug resistant Kpn has significantly narrowed the therapeutic options available; as such, there is an urgent need to better understand Kpn pathophysiology to identify novel therapeutic targets. Here we performed an in vivo transcriptomic analysis of Kpn isolated from a mammalian host with pulmonary infection.
MethodsC57BL/6 mice were intranasally inoculated with the virulent Kp52.145 strain (serotype O1:K2); with lungs extracted, homogenised and pooled (n=3; in duplicate) at 32 h post-infection for bacterial RNA purification and RNA-Seq (Illumina). Differential gene expression (analysed using Degust [Voom/Limma]; FDR cut-off =0.01, abs log-FC=2) was assessed in comparison with mid-log phase growth in Lennox broth.
ResultsOverall, we identified >900 differentially expressed genes (DEGs), comprising ∼17 % of the combined chromosomal and plasmid coding sequence repertoire. 52 % of the DEGs were upregulated during infection, including several siderophore-independent iron-, manganese- and zinc-uptake systems (e.g. hmuRSTUV, sitABCD, mntH and znuACB). We also observed a marked in vivo oxidative stress signal, with several Kpn oxidative stress response genes (e.g. oxyR, katE, katG) upregulated during infection. In contrast, expression of mgrB, a negative-regulator of the PhoPQ two-component system associated with resistance to host antimicrobial peptides was downregulated.
ConclusionThis study provides a novel insight into Kpn gene expression during pulmonary infection. Overall, our data suggest that adaptation to metal starvation, oxidative stress and innate immune defenses are critical for the success of Kpn lung infection.
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The biocide triclosan triggers multiple regulatory systems in Staphylococcus aureus to induce antibiotic tolerance
More LessThe biocide triclosan is used extensively in household and hospital settings, resulting in chronic exposure to the biocide in individuals that use triclosan-containing products. Triclosan is thought to induce antibiotic tolerance and alter biofilm formation, although the underlying mechanisms causing these changes are yet to be elucidated. If true, the widely used biocide may contribute to antibiotic treatment failures, and therefore requires investigation. To determine how triclosan induces antibiotic tolerance, Staphylococcus aureus was pre-treated with triclosan prior to treatment with the clinically relevant antibiotics ciprofloxacin, rifampicin, and vancomycin. Planktonic S. aureus cultures pre-treated with triclosan had 1000 fold higher viable counts compared to non triclosan pre-treated cultures. Inspection of biofilms by live/dead staining found that triclosan pre-treatment protected S. aureus biofilms from treatment with otherwise lethal doses of ciprofloxacin, rifampicin, or vancomycin. Biofilms of mutant strains with a defective stringent response were not protected from antibiotic treatment, even in the presence of triclosan. Interestingly, stringent response mutants still exhibited triclosan-induced antibiotic tolerance in planktonic culture, but mutants with a defective agr quorum sensing system did not. Confocal laser scanning microscopy revealed that incubation of S. aureus with triclosan altered biofilm structure, resulting in increased proportions of polysaccharide in the biofilm matrix that could potentially mediate protection against antibiotics. Neither the stringent response mutants nor agr mutants influenced triclosan-induced biofilm changes, suggesting another, currently uncharacterised response. We suggest that triclosan triggers multiple global regulatory systems in S. aureus, subsequently inducing tolerance to multiple antibiotic classes and altering biofilm structure.
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The role of SARM in the control of immune response driven by Klebsiella pneumonia infection
More LessIntroductionKlebsiella pneumonia is a Gram-negative, capsulated bacteria, which is an important cause of community-acquired and nosocomial pneumonia. Klebsiella co-opts cellular functions dedicated to control immune balance to limit the activation of inflammatory responses. SARM (Sterile α-and armadillo-motif containing protein), the fifth identified member of the TIR (Toll-interleukin 1 receptor (1LR)) adaptor family, negatively regulates IRF and NF-kB activation by affecting TLR4 and TLR3 TRIF-dependent signalling. It is currently unclear the role, if any, of SARM in bacterial infections. Here, we aim to dissect the contribution of SARM in Klebsiella infections, and, specifically, to investigate whether Klebsiella may exploit SARM as part of the pathogen’s portfolio immune evasion strategies.
ResultsSARM contributed to Klebsiella anti-inflammation strategies in macrophages through by increasing AKT phosphorylation (to limit phagosome-lysosome fusion), and preventing the activation of NF-kB (to control inflammatory responses). SARM also negatively regulated type I IFN regulatory factor (IRF3) by decreasing IRF3 phosphorylation. Notably, SARM played a role as inflammasome inhibitor, as observed by increased IL-1β secretion in the supernatants of infected sarm-/-. SARM also inhibited ASC oligomerization in Klebsiella-infected macrophages as seen by the increased ASC monomers release by sarm-/- BMDMs. SARM was also required for pyroptosis following Klebsiella infection. Interestingly, Klebsiella induced the expression of SARM in a TLR4-TRAM-TRIF-IRF3-IFNAR dependent manner, demonstrating that Klebsiella exploits type I IFN to trigger SARM to control inflammasome activation, and the activation of inflammatory responses.
ConclusionsThese findings have uncovered how Klebsiella manipulates the TLR adaptor SARM to dampen the activation of host defences.
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PorA-Loop4 derived peptides of Neisseria meningitidis cause a G1 cell cycle arrest through the Akt signalling pathway in human brain microvascular endothelial cells
More LessNeisseria meningitidis (meningococcus) is a major meningitis-causing bacteria and is known for its ability to breach blood-brain barrier (BBB). Meningococcus binds to Laminin receptor (LAMR) on the surface of endothelium, which is part of the BBB. The meningococcal surface proteins PorA and PilQ were previously identified as bacterial ligands responsible for binding and, subsequently, the LAMR-binding moiety of PorA was localised to its fourth extracellular loop (PorA-Loop4). Using a circularised peptide corresponding to PorA-Loop 4 from N. meningitidis MC58, the PorA-LAMR interaction induced specific cellular responses in human brain microvascular endothelial cells (HBMECs) including G1 cell cycle arrest. Flow cytometric analysis indicated that the treatment of HBMECs with PorA-Loop4 for 24 h caused a significant reduction of cells (20 %) at S-phase and a corresponding increase (23 %) in G1 population. Immunoblotting and quantitative real time PCR (qRT-PCR) analysis suggested that a blockade in Akt signalling (key proteins including Akt, GSK-3β, CyclinD1, and CDK4) contributes to the G1 arrest. Immunoblotting showed that the expression of phosphorylated GSK-3β and CDK4 were significantly increased in treated HBMECs. In contrast, the expression of phosphorylated Akt and Cyclin D1 were decreased following treatment. Transcriptome analysis using qRT-PCR confirmed that treatment of HBMECs with PorA-Loop4 peptide for 2, 4, 8, or 24 h increased gene expression of CDK4, and decreased expression of Cyclin D1. Immunofluorescent imaging of Akt, GSK-3β, CyclinD1, and CDK4 in Loop4-treated HBMECs are consistent with qRT PCR and immunoblot results. The data confirm that PorA-Loop4 induce G1 arrest through the Akt signalling pathway via Akt/GSK-3β/CyclinD1/CDK4.
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Rational design of TFDs as novel oligonucleotide antimicrobials to treat Gram-negative infections
More LessWe have designed a new Gram-negative antimicrobial- it is an oligonucleotide Transcription Factor Decoy (TFD) that binds to and inhibits bacterial transcription factors controlling genes essential for growth and pathogenicity. The TFD is highly effective in vitro and in vivo, tested in Galleria mellonella survival models and in a mouse model of intra-abdominal infection. TFDs are formulated as nanoparticles, composed of a proprietary lipidic molecule (CM2), that binds to essential prokaryotic phospholipids, such as Cardiolipin, to deliver the oligonucleotide across the bacterial membrane. Studies with models of bacterial membranes showed that translocation was dependent on the presence of Cardiolipin but occurred in the absence of ATP or a pH gradient. Flow Cytometry studies found that the efficiency of TFD delivery to bacterial cells was high, and Live/Dead staining confirmed that cells were not being lysed by the nanoparticles. Further investigation of the mechanism of delivery used a proteomics and metabolomics study of the response of E. coli to nanoparticulate delivery. A number of highly induced transcription factors were identified consistent with stress induced by disruption of respiratory centres bound by Cardiolipin within the membrane. A TFD was designed to inhibit one of the induced transcription factors. This TFD was shown to bind tightly to its cognate transcription factor, have a potent MIC in vitro and be efficacious in murine models of infection. Hence, the work demonstrates that TFDs can be rationally designed to create new antimicrobials to efficiently target bacterial transcription factors.
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Investigating the role of low-oxygen-activated (lxa) encoded proteins in the pathogenesis of Burkholderia cepacia complex and their contribution to chronic infection in cystic fibrosis
More LessBurkholderia cepacia complex (Bcc) is a group of 22 closely related species of Gram-negative bacteria that cause chronic infections in people with cystic fibrosis (CF) that are rarely eradicated. The high-level antibiotic resistance and poorly understood mechanisms by which Bcc survive and persist during chronic infection mean that combatting these chronic infections is particularly challenging. We have previously found that a group of 20 low-oxygen-activated (lxa) encoded proteins were consistently upregulated in sequential Bcc isolates in two chronically infected CF patients over time of infection. Many of these proteins have not previously been studied in Bcc but the consistent upregulation over time of infection suggests that they play an important role during chronic infection. Two particular proteins of interest within the lxalocus that were consistently upregulated were a universal stress protein (USP) and a phospholipid binding protein (PBP). Single gene deletion mutants (Δusp and Δpbp) in the wildtype Bcc strain K56-2 (WT) both showed a 90 % reduction in attachment to CFBE41o- cells compared to WT. There was also a 5-fold reduction in the virulence of Δpbp in the acute infection model Galleria mellonella (P<0.005) and an increased sensitivity of Δusp to peroxide-induced oxidative stress (P<0.0001) and low pH (P<0.05) relative to WT. A reduction in both uptake and survival of Δusp in U937 macrophage-like cell line compared to WT, suggests the USP plays a role in the intra-macrophage survival of Bcc. Overall, these proteins, previously associated with low-oxygen conditions may play a considerable role in Bcc pathogenesis and its adaption during chronic infection.
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Identification of niche-specific virulence factors via experimental evolution of Streptococcus pneumoniae
More LessStreptococcus pneumoniae (the pneumococcus) is an important human pathogen, adept at colonising various ecological niches within the host. Colonisation of the nasopharynx, followed by asymptomatic carriage and non-inflammatory clearance is the predominant outcome of infection, but diverse disease manifestations including pneumonia, septicaemia and meningitis occur in a minority of individuals. Through experimental evolution of pneumococci in mouse disease models, we are investigating the genetic basis of the niche adaptations that enable pneumococci to switch from a commensal lifestyle in the nasopharynx, to a pathogenic phenotype in the lungs, brain or blood. Experimental evolution was carried out via serial passage of pneumococci separately through pneumonia and nasopharyngeal carriage mouse models, to generate lineages adapted to the lung and nasopharyngeal environments, respectively. Starting from a non-passaged (lab adapted) isolate, ten independently-evolved lineages of lung-adapted pneumococci have been generated, each having been passaged 20-times through a mouse pneumonia model. Pneumococci recovered from the infected lungs were used to inoculate further mice for the next passage round. We will present the results from whole genome sequencing and phenotypic analysis of lung-passaged bacterial isolates including growth characteristics, toxin production, adherence and invasion with lung epithelial cells. We will also describe how the acquired pneumococcal adaptations, which facilitate survival in the lung environment, can influence bacterial gene expression during exponential growth. These studies will provide insight into genetic changes associated with pneumococcal commensal to pathogen switch. Identifying such genetic determinants of virulence will be valuable for the development of vaccine candidates and targets for therapeutic intervention.
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Investigating evolution of the paediatric cystic fibrosis lung microbiota using induced sputum sampling and culture-independent techniques
More LessBackgroundChildren with cystic fibrosis are frequently non-productive of sputum even during exacerbation. Current routine sampling methods are either invasive (Bronchoalveolar lavage; BAL) or insensitive (cough swab) which makes pathogen surveillance challenging. We investigated induced sputum (IS) as a promising complementary sampling technique, looking at both its comparison to BAL and the evolution of the lung microbiota across children aged 0.5–18 years.
MethodsBAL and IS samples were collected as part of the CF-SpIT study (UKCRN14615; ISRCTNR12473810). DNA was extracted from samples and Illumina NextSeq sequencing of the 16S rRNA gene V4 region was performed. Bioinformatics data processing was carried out using Mothur. Microsoft Excel and R statistical software were used for downstream analyses.
ResultsComparison of the microbiota of 30 BAL-IS matched patient samples indicated that at a presence/absence level, IS captured >80 % of the pathogens observed in BAL samples. These findings validated the expansion of the study to 136 un-matched IS sputum samples, and we found that as patient age increased, bacterial diversity decreased, and changes in the abundance of key genera occurred over time. Both Neisseria and Haemophilus decreased with age, whilst Pseudomonas and a sub-group of Prevotella increased.
ConclusionsHere we uniquely demonstrate using culture-independent techniques that IS captures the majority of the bacterial diversity observed in BAL samples. The age associated decline in lung microbiota diversity, previously documented using BAL samples, can also be captured using IS samples. This suggests that surveillance of microbiota evolution may be possible using this method.
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Induction of inflammasome-dependent signalling in the human monocytic cell line THP-1 by Campylobacter lipooligosaccharides
More LessThe Campylobacter lipooligosaccharides (LOS) can stimulate membrane-bound innate immune receptors in human macrophages. However, the association of Campylobacter LOS in the stimulation of cytosolic receptors or the inflammasome remains poorly characterised. Therefore, the aim of this study was to determine the role of Campylobacter LOS in the activation of NLRP3 inflammasome-dependent signalling in a human monocytic cell line. The induction of NLRP3 inflammasome-mediated IL-1β and Caspase-1 secretion in THP-1 supernatants was quantified using ELISA following co-culture of THP-1 cells with LOS extracts from wild type C. jejuni 11168, mutant C. jejuni 11 168 with reduced LOS and two wild type C. coli strains (RM1875 and 76339). Our results demonstrate that LOS purified from both C. jejuni and C. coli can induce Caspase-1 and IL-1β production in human macrophages. However, C. jejuni 11 168 mutant LOS with modified lipid A and lack of core oligosaccharides stimulated significantly reduced Caspase-1 and IL-1β. This result was also replicated in co-culture of live wild type and mutant C. jejuni with THP-1 cells. This study provides new insight into the interaction of Campylobacter with human macrophages and suggests that variation in LOS structure may alter NLRP3 inflammasome activation.
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- Intra- and Interspecies Metabolic Networks: You Are What You Eat
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Understanding the killing mechanism of action by virus-infected yeasts
More LessKiller yeasts are microorganisms, which can produce and secrete proteinaceous toxins, a characteristic gained via viral infection. These toxins are able to kill sensitive cells of the same or a related species. From a biotechnological perspective, killer yeasts have been considered as beneficial due to their antifungal/antimicrobial activity, but also regarded as problematic for large-scale fermentation processes, whereby those yeasts would kill species off starter cultures and lead to stuck fermentations. Here, we propose a mechanistic model of the toxin-binding kinetics pertaining to the killer population coupled with the toxin-induced death kinetics of the sensitive population to study toxic action in silico. Our deterministic model explains how killer Saccharomyces cerevisiae cells distress and consequently kill the sensitive members of the species, accounting for the K1, K2 and K28 toxin mode of action at high or low concentrations. The dynamic model captured the transient toxic activity starting from the introduction of killer cells into the culture at the time of inoculation through to induced cell death, and allowed us to gain novel insight on these mechanisms. The kinetics of K1/K2 activity via its primary pathway of toxicity was 5.5 times faster than its activity at low concentration inducing the apoptotic pathway in sensitive cells. Conversely, we showed that the primary pathway for K28 was approximately 3 times slower than its equivalent apoptotic pathway, indicating the particular relevance of K28 in biotechnological applications where the toxin concentration is rarely above those limits to trigger the primary pathway of killer activity.
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When the metabolic model says NO: untangling the Gordian knot of TB’s intracellular metabolism
The causative agent of TB, Mycobacterium tuberculosis (Mtb) is once again the world’s number one infectious killer. M. tuberculosis resides primarily within macrophages and metabolic reprogramming within this intracellular niche is a crucial determinant of virulence. We previously applied the metabolic modelling-based tool 13C-flux spectral analysis (13C-FSA) to show that intracellular M. tuberculosis co-metabolises multiple gluconeogenic and glycolytic carbon substrates by utilizing the reactions of the phosphoenolpyruvate (PEP)-pyruvate-oxaloacetate (OAA) or anaplerotic (ANA) node. However, predicting the metabolic mode of operation required for intracellular survival is chellenging using a metabolic network as the ANA node consists of several apparently functionally redundant bidirectional reactions. Here we use multiple techniques including 13C isotopomer profiling, lipid analysis and fluorescent reporter strains to dissect the role of the ANA node. We show that this node has unexpected roles in the life cycle of M. tuberculosis including lipid biosynthesis, protection from known toxic intracellular carbon sources and redox regulation. Inhibiting enzymes at this node with novel therapeutic compounds restricts the growth of M. tuberculosis and limits the ability of this formidable pathogen to survive within the human host cell identifying the ANA node as a potential druggable pathway for controlling TB.
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The effect of antibiotic and nutrient limitation to antibiotic resistant bacteria in single-cell level
More LessHeterogeneity in bacterial populations can manifest in various ways, such as resistant cells, which can be observed in harsh environments after the use of antibiotics. Many studies have looked at the evolution of resistance and the effect of inhibitory and sub-inhibitory concentrations of antibiotics by batch culture measurements without considering the heterogeneity of bacterial populations. But antibiotic susceptibility and fitness costs of resistance mutations or plasmids are affected by the growth rate and physiology of individual cells. Single-cell analysis in microfluidic systems has opened up new possibilities enabling us to investigate the various putative mechanisms behind the persistence phenomenon required direct observation under the microscope. In this study, we use a gradient mixer and a novel micro-chemostat, to create concentration gradients of growth substrates and/or antibiotics to study the effect of nutrient and antibiotic concentration on individual cells growing under constant and defined conditions in cell-sized channels. The single-cell elongation, morphology and growth rate of ribosome-targeting antibiotics resistant E. coli was tracked by combining the microfluidics, microscope phase contrast imaging and fluorescent tag in high throughput mode. A mechanistic cellular model was used to describe the reaction between antibiotics and ribosome and the resulting effects on bacterial growth; then we linked the intracellular chemical-reaction kinetics processes to the population level and predicted the behaviour of population responses. Our approach has enabled the investigation of single-cell individuality and predictions of population dynamics under different environment.
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Analysis of bacterial competition using imaging mass spectrometry
More LessThe bacterial order actinomycetales are responsible for the production of 65–70 % of microbially produced specialised metabolites with diverse biological activities, with some actinomycetale strains containing over 30 Biosynthetic Gene Clusters encoding for these metabolites. However, only approximately 10 % of these genes are typically transcribed in a mono-culture setting. Furthermore, it has been observed that microbial interactions may induce these cryptic gene clusters providing an ecological advantage to the producer strains. To understand the chemical exchange between strains isolated from the marine environment, microbial interactions were assessed using 49 actinomycetale strains, two Pseudomonas and one Bacillus strain. In total, 72 tri-cultures (three strains) were analysed resulting in 29 strains that showed an altered phenotypes as a result of the interaction. These were then evaluated in a one-to-one culture (two strains) followed by bioactivity screening. Using this data, nine tri-cultures and 27 one-to-one cultures were evaluated using tandem Mass Spectometry, enabling chemically interesting interactions to be prioritized for Time of Flight Secondary Ionisation Mass Spectometry (ToF-SIMS) analysis. ToF-SIMS enables the spatial distribution of parent ions within a sample, in this case, two bacterial strains interacting in a Petri dish. The results that will be presented demonstrate that microbial interactions induce the production of metabolites and ToF-SIMS represents an exciting strategy to study bacterial chemical ecology.
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Decaying Ascophyllum nodosum as a source of algal cell wall degrading enzymes with potential utility in enzyme-assisted extraction technologies
Seaweeds are of huge interest in the food, pharmaceutical and agricultural industries due to their high nutritional content and the prevalence of useful bioactive compounds. Current extraction methods of macroalgal-derived metabolites are however problematic due to the complexity of the algal cell wall which hinders extraction efficiencies. The use of advanced extraction methods such as enzyme-assisted extraction (EAE), which involve the application of commercial algal cell wall degrading enzymes to hydrolyze the cell wall carbohydrate network, are becoming more popular as they allow the development of more efficient and eco-friendly processes. Ascophyllum nodosum samples were collected from the Irish coast and incubated in artificial seawater for six weeks at three different temperatures (18 °C, 25°C and 30 °C) to induce decay. Microbial communities associated with the intact and decaying macroalga were examined using Illumina Miseq sequencing and culture-dependent approaches, including the novel iChip device. The bacterial populations associated with the seaweed were observed to change markedly upon decay with a substantial decrease in the relative abundances of certain phyla including Planctomycetes and Verrucomicrobia observed during the decay period. Over 800 bacterial isolates cultured from the macroalga were screened for the production of algal cell wall polysaccharidases and a range of species from the phylum Bacteroidetes together with a number of Vibrio species which displayed multiple hydrolytic enzyme activities were identified. Extracts from these enzyme-active bacterial isolates were then used in EAE of phenolics from Fucus vesiculous and were shown to be equally efficient as commercial enzymes in their extraction efficiencies.
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Understanding metabolic processes shaping adaptation of E. coli to the gut
More LessMany microbes colonise the gut establishing interactions with their host and their nutritional environment. Studying genetics and metabolism brought about the drive and potential to engineer communities to promote health and improve industrial processes. However, structuring artificial communities in predictable ways is underdeveloped. We studied Escherichia coli’s genetic targets and physiological mechanisms during gut colonisation and adaptation and how metabolic environment/microbiota complexity shape these processes. We introduced a tractable E. coli K-12 in mice Germ-free or with polymicrobial communities. Whole Genome Sequencing identified potential adaptive targets. Here, we established phenotypic assays as well characterising effects of key mutations and metabolomics was performed with 1H-NMR of intestinal contents. Genes for sugar alcohol metabolism (gat) was the only target common to both mouse models, evidencing specificity. Facing complex microbiota E. coli targeted use of sugar alcohols (srlR, kdgR) and anaerobic respiration (dcuB, focA) [1] whereas alone, we observed instead mutations pointing to increased ability for amino acid use (lrp, dtpB, alaA). Mutations selected correlated dinamically with metabolomics: our results fit the model whereby other microbiota members scavenge oxygen and breakdown complex sugars, limiting E. coli to anaerobically respire simple by-product carbon sources. In the opposing scenario (functional absence) improved amino acid use are favoured colonisation factors. Through experimental evolution we gained insight on shaping E. coli’s metabolic traits through genetic engineering to colonise specific host environments. This work also highlights the versatility of E. coli as potential biotic sensor. [1] Barroso-Batista, J. et al. The first steps of adaptation of Escherichia coli to the gut are dominated by soft sweeps, 2014.
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The Candida albicans arginase family encodes enzymes with diverse catabolic activities that differentially influence host–fungus interactions
More LessIn the blood stream, arginine is an essential amino acid that is required by phagocytes to synthesize iNOS. Previously we showed that the fungus Candida albicans induces host arginase production that diverts arginine from the pathway that leads to the production of nitrite oxide. We therefore investigated whether C. albicans arginase activity also contributed to the protection of the fungus by competing for arginine during infections. Three C. albicans genes had been annotated as putative arginase encoding genes. Heterologous expression of these genes suggested all three had some arginase activity and one gene product (Car1) encoded a bone fide arginase that was required for growth on arginine. However, single and double mutations in the two other genes (AGM1 and GBU1) did not affect growth on arginine as a single nitrogen source and were found instead to encode agmatinase and guanidinobutyrase respectively that participate in two other pathways related to arginine metabolism. This family of three enzymes therefore exhibits mixed biochemical activities and collectively participate in the catabolism of exogenous and endogenous sources of arginine. Virulence of the triple mutant lacking all three genes was reduced in a Galleria infection model, but single or double mutants were fully virulent. None of the single or multiple mutants affected host NO production suggesting they do not influence the oxidative burst of phagocytes. In addition, CAR1 expression was required for hyphal growth. This family of enzymes therefore represent a novel enzyme set that is essential for growth in vivo and indirectly for fungal virulence.
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In vitro reconstitution of the polymicrobial community associated with cystic fibrosis airway infections
More LessThe airways of cystic fibrosis (CF) patients provide a rich and unique environmental niche, prone to lifelong chronic infection by a diverse and dynamic polymicrobial community. Such dense microbial ecosystems have a network of interspecies communication between each member of the community, serving to modulate virulence, impact metabolism and contribute towards antimicrobial resistance (AMR). Currently no models exist which enable the long-term culture of a true polymicrobial community. Most existing animal models are only suitable for short term infection studies, often utilising relatively healthy hosts and which use axenically cultured clonal strains, providing little parallel to the complex biochemical interactions occurring within chronic CF infections. Here we describe a simple in vitro model utilising artificial sputum medium to allow the successful coculture of major CF-associated pathogens and begin to recapitulate and maintain the CF microbiome within a relatively steady-state. An in vitro model confers several advantages for studying widespread community changes and pathogenic interactions. Perhaps most importantly, in vitro models can be easily perturbed through the addition of antibiotics or introduction of new species/strain variants, allowing the impact of external stressors upon the emergence and changes in lifestyles of key pathogens to be effectively studied. A simple, robust and physiologically relevant CF model could be applied to address any number of fundamental biological questions surrounding interspecies interactions occurring within polymicrobial infections.
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- Irish Fungal Society Clinical Case Studies
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Cross-sectional study of respiratory Aspergillus spp. colonization or infection in patients with various stages of chronic obstructive pulmonary disease (COPD) using culture vs non-culture based technique
BackgroundCOPD patients are now recognized to be at increased risk of colonization by Aspergillus spp. which may progress to invasive pulmonary aspergillosis (IA). Published data on the frequency of Aspergillus detection in COPD are limited.
MethodsA cross-sectional study was undertaken to determine Aspergillus colonization or infection in COPD patients undergoing bronchoscopy for any indication. Culture as well as galactomannan antigen (GM) and Aspergillus nucleic acid detection (PCR) were performed on bronchoalveolar lavage fluid (BAL).
ResultsOne hundred and fifty patients were included (44.7 % female, mean age 68.2 years). 21.3 % were inpatients, 74.7 % outpatients and 4 % were ICU patients. Investigation of lung masses was the most common indication (43.3 %) for bronchoscopy. Most patients (81.3 %) were either GOLD stage 1 or 2 COPD. Cancer was the most frequent co-morbidity (60.48 %). 12 % and 48.7 % were on systemic and inhaled steroids respectively. Lung mass was the most common (28.43 %) CT imaging finding. Seventeen patients (11.3 %) had a positive result for Aspergillus (Culture+Galactomannan+PCR). 76.4 % out of these seventeen were in the early stages (GOLD stage 1 or 2) of COPD.
ConclusionAspergillus sp. was detected in 3.3 % of patients by culture, which increased to 11.3 % if culture was combined with either a positive GM or PCR result. Overall the frequency of Aspergillus detection in this population of COPD patients was low which may reflect the predominance of Gold stages 1 and 2 among the study population.
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Repurposing histone deacetylase inhibitors (HDACi) to treat Candida glabrata infections
More LessCandida glabrata currently accounts for 25 % of all fungal cases in UK hospitals, second only to C. albicans. This number is expected to rise given the intrinsic anti-fungal resistance of this species and the difficulty in treating it. In an effort to identify novel-anti fungal targets in C. glabrata, we used comparative genomics within Saccharomycotina yeast to predict which genes are under positive selection in this species specifically. Such genes are predicted to have influenced the adaptation of C. glabrata from a free-living microbe to a human pathogen, potentially due to functional shift(s) of the proteins they encode. Our analysis predicts that histone acetylation pathways are under positive selection in C. glabrata. Thereforewe hypothesised that we could use histone deacetylase inhibitors (HDACi) to interfere with histone acetylation levels and impact C. glabrata virulence. By treating C. glabrata withbroad spectrum HDACis we show it has a reduced capacity to form biofilms, it is less well adapted to high salt conditions typically found within a human host, and most importantly, it reverts to a more anti-fungal sensitive state. RNAseq analysis indicates that HDACi treatment interferes with the C. glabrata transcriptional response to anti-fungal treatment, rendering it incapable of combating against these drugs. Furthermore, using an in vivo worm model of candidiasis, we show that HDACi treatment in conjunction with the anti-fungal fluconazole, can increase the survival rate of individuals with C. glabrata infections. Taken together our data suggest that the health threat posed by C. glabrata might be addressed by repurposing HDACi to treat this infection.
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- Marine Protists as Emerging Models for Functional Genomics and Cell Biology
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Transformation of the dinoflagellate chloroplast
More LessDinoflagellate algae are ecologically and environmentally important, as symbionts of corals and many other aquatic organisms, and the causative agents of red tides. However, attempts over the last twenty years to establish genetic manipulation systems for dinoflagellates have met with little success. We have exploited the unusual chloroplast genome of dinoflagellates to establish a system for transformation of this organelle. The chloroplast genome of peridinin-containing (the ancestral state) dinoflagellates is highly reduced and composed of a number of small, plasmid-like molecules, referred to as ‘minicircles’. We have constructed shuttle vectors that are fusions of minicircles and Escherichia coli plasmids and carry selectable markers. We used biolistic transformation to introduce these into the model dinoflagellate Amphidinium carterae. We found that the plasmids confer the expected phenotype on the dinoflagellate cells, and we can detect the plasmid DNA and associated transcripts following selection, indicating successful transformation. This opens up the possibility of studying many aspects of dinoflagellate chloroplast biology, including the maintenance and expression of the minicircles, and the role of the chloroplast in phenomena such as coral bleaching.
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Why does a heterotrophic marine protist produce carotenoids? Genetic approaches to investigate the ecophysiology of the thraustochytrid Aurantiochytrium limacinum
More LessThraustochytrids are abundant and ubiquitous osmoheterotrophic marine protists (labyrinthulomycetes, stramenopiles) thought to function ecologically as fungus-like decomposers. Some thraustochytrids have the ability to synthesize carotenoids, including carotenes (e.g. beta-carotene) and xanthophylls (e.g. astaxanthin), which is uncommon among heterotrophic eukaryotes. Carotenogenic thraustochytrids appear to have acquired carotenoid biosynthetic enzymes by horizontal gene transfer from bacteria. Heterotrophic production of carotenoids is typically associated with protection against oxidative stress, and in thraustochytrids may be particularly associated with protecting large amounts of essential omega-3 polyunsaturated fatty acids stored in lipid droplets. To gain better understanding of carotenoid function in thraustochytrids, and thus new insight into the ecophysiology of these organisms, we have produced mutants of the thraustochytrid Aurantiochytrium limacinum in which the trifunctional gene Aurli_150841, encoding the first three carotenogenesis-specific reactions (phytoene synthase, phytoene desaturase, lycopene cyclase), has been interrupted by double homologous recombination with a construct containing a zeocin resistance (BleoR, shble) expression cassette. As predicted, the Aurli_150841 knockout mutants lack the carotenoid pigmentation found in the wild-type. Complementation with the wild-type Aurli_150841 to confirm that this phenotype is due to the knockout is in progress. Differences between the wild-type and Aurli_150841 knockout mutants in features such as growth rate and biomass yield, lipid content, survival in stationary phase and response to oxidative stress are being evaluated under growth conditions that induce different amounts of carotenoid accumulation in the wild-type.
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Developing genetic manipulation platforms for Naegleria gruberi
More LessNaegleria gruberi, is a free-living microbial eukaryote, which belongs to the group of Excavates. The organism is widely distributed especially in aquatic environments and it is famous for its ability to transform from an amoeba to flagellate and cyst forms depending on its surroundings. In silico examination of the published Naegleria gruberi genome opened up the possibility of functional exploration of the organism by molecular cell biology. Despite this, several attempts to genetically transfect or genetically manipulate the organism have been unsuccessful so far due to the unique morphological and cellular adaptations of the organisms, but also due to its resistance to certain basic antibiotics. Using a series of protocols and combination of cell biological tools, we attempted to genetic manipulate Naegleria using both CRISPR/Cas9 and traditional genetic transformation protocols. Preliminarily data from these investigations will be discussed. This work is going to provide traits found in the last eukaryotic common ancestor and provide a model for investigating the cell biology of other free-living eukaryotes.
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Discovering the biology behind the organism while developing genetic tools for Corallochytrium limacisporum
More LessTo address biological questions that cannot be answered by current model organisms, we need to develop genetic tools in the specific taxa that can provide the best answers. Such is the case of the origin of animals in which, genetic tools need to be developed among the closest unicellular relatives of animals. To fill this gap, we are developing genetic tools in Corallochytrium limacisporum, a close unicellular relative of animals that also has a fascinating biology. Corallochytrium is a marine free-living walled saprotroph that develops through a choenocyte. Moreover, because of the basal phylogenetic position of Corallochrytria together with Ichthyosporea, this lineage is especially informative to fill the void of information between yeast and metazoans. We have successfully developed transient and a stably transfection protocols by introducing the resistance gene to puromycin, allowing us to select individual transformants. Deep characterization of the established transformed lines has revealed important biological features of this organism such as the plasticity of its genome, the mode of plasmid integration, and some differences between the two known strains (Hawaii and India). Currently we are better characterizing these features and, in parallel, developing genome-editing technologies. Progress and the potential implications of our research will be presented and further discussed.
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- Microbial Dark Matter
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Shining new lights on chytrid cell biology: quantitative live cell imaging of rhizoid development in an early-diverging fungus
More LessChytridiomycota (Chytrids) are the most basal lineage within the true fungi, however they have largely remained in the dark in terms of their fundamental cell biology. In aquatic ecosystems, chytrids can dominate ‘dark matter’ surveys and are important saprotrophs of recalcitrant organic carbon. They therefore play an integral biogeochemical role in carbon cycling. Unlike ‘higher’ dikaryan fungi that feed via multicellular hyphae, chytrids are unicellular and develop an anucleate rhizoid system that acts as the trophic interface of the cell. Understanding the functions of the rhizoid has the potential to shed light on the trophic biology of ‘dark matter’ chytrids. We applied 3D and 4D live-cell confocal microscopy to morphometrically quantify rhizoid development in the model saprotrophic chytrid Rhizoclosmatium globosum under different nutrient treatments. Rhizoid branching was highest under carbon-rich conditions, whereas under carbon-starved treatments, rhizoids grew significantly longer and were less branched, in what we interpret to be a ‘search strategy’ for nutrient sources. F-actin and the cell wall were identified throughout the rhizoid system. Chemical inhibition of actin and cell wall glucan synthesis induced the development of hyperbranched paramorphs, suggesting that these components underpin rhizoid branching and organising cell polarity at the rhizoid tip. Previous studies have shown that inhibition of these components induces an identical phenotype in dikaryan fungi. These findings represent an important step in understanding the trophic biology of a biogeochemically important microbe and unveil striking similarities in cell development between early-diverging and ‘higher’ dikaryan fungi.
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Redefining a new genomic blueprint of the human gut microbiota
The human gut microbiota composition is linked to both health and disease, but knowledge of individual microbial species is needed to decipher their biological role. Despite extensive culturing and sequencing efforts, the complete bacterial repertoire of the human gut microbiota remains undefined. Here we identify 1952 uncultured candidate bacterial species by reconstructing 92 143 metagenome-assembled genomes from 11 850 human gut microbiomes. These uncultured genomes substantially expand the known species repertoire of the collective human gut microbiota, with a 281 % increase in phylogenetic diversity. Whilst the newly identified species are less prevalent in well-studied populations compared to reference isolate genomes, they improve classification of understudied African and South American samples by over 200 %. These candidate species encode hundreds of novel biosynthetic gene clusters and possess a distinctive functional capacity that might explain their elusive nature. We also highlight newly identified species overrepresented in patients with gastrointestinal diseases, suggesting an underappreciated role in human health and disease. Our work uncovers the uncultured gut bacterial diversity, providing unprecedented resolution for taxonomic and functional characterization of the intestinal microbiota.
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Marine fungal dark matter in the global ocean
More LessMarine fungi are a major part of ‘microbial dark matter’, with most organisms known from sequence data and currently not in culture. Interest in marine fungi has substantially increased over the past decade, and studies using culture independent methods have indicated that fungal diversity in the oceans may be greater than previously estimates based on cultivation alone. There remains much to learn about the true diversity of marine fungi in the global oceans and the ecological roles that they could play. The Tara Oceans expedition has allowed for significant advancements in our understanding of the global diversity of planktonic microorganisms. Interrogation of the Tara Oceans 18S rRNA gene dataset for fungal sequences shows that fungi are found throughout the global oceans, appearing in all marine regions covered by the Tara Oceans expedition. In the survey, Ascomycota and Basidiomycota were common, while some locations had increased abundances of Chytridiomycota. Differences in community composition were observed between oceanic regions and, although clear signals were not apparent due to the nature of the sampling, there was some indication of community variation between upwelling, coastal, shelf and gyral provinces. Different size sampling fractions appeared to capture different portions of the pelagic fungal community. These findings highlight a number of ecological questions: How important are oceanic currents in determining fungal biogeography? What is the relationship between marine fungi and biogeochemical processes? What is actually there? By targeting these questions directly, we will be able to bring the dark matter of marine fungi into the light.
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The Sodalis system and flux balance analysis as a tool for investigating insect-microbe interactions and the evolution of symbioses
The development of new microbial growth and analytical techniques is becoming increasingly relevant in relation to ‘unculturable’ organisms. This may involve the modification of existing methods or the development of new, custom procedures. One important application of this is in the symbiotic bacteria of insects. Symbionts, due to adaptations to their host, are often difficult to culture in vitro. With the growing interest in the use of modified microbiomes to control vector-borne diseases, improved culture techniques that further the understanding of an insect’s microbiome are becoming increasingly important. The tsetse fly, genus Glossina, is the insect vector for Trypanosoma brucei. This parasite is responsible for human African trypanosomiasis (HAT), endemic in sub-Saharan Africa, as well as the wasting disease nagana in cattle. The tsetse’s secondary symbiont, Sodalis glossinidius, provides a unique potential target for reducing the spread of T. brucei. Here, we describe the use of metabolic modelling to design an entirely defined growth medium for S. glossinidius. This medium was used to verify predictions about carbon and nitrogen usage in the symbiont, including amino acid and vitamin auxotrophies. Furthermore, we discuss the use of multiobjective evolutionary algorithms combined with flux balance analysis to investigate computationally the evolution of symbioses, with S. glossinidius and its free-living relative S. praecaptivus as an exemplar. This work not only improves our understanding of the metabolic interactions within the tsetse microbiome, but serves also as a template for future investigations into symbiont evolution.
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Identification of viral transcripts in RNA-seq datasets from bees, ants, wasps and mites
More LessMany honey bee colonies suffer large losses due to colony collapse disorder. This phenomenon, which has dramatically increased in frequency since 2006, has led to widespread efforts in sequencing honey bee pathogens, including RNA viruses such as deformed wing virus. However, honey bees coexist with a number of other arthropods, whose viruses are less thoroughly characterised. Many viruses currently classified as honey bee pathogens may therefore have a much wider host range. In particular, ants, which like bees are members of the Hymenoptera order, often coexist with bees and the two groups have previously been shown to exchange viruses. Parasitism by Varroamites, known to act as effective vectors for a number of RNA viruses, is also almost ubiquitous amongst honey bees, but little is known about viruses endemic to mites. We have previously demonstrated that it is possible to detect and characterise viral RNA in publicly available RNA-seq datasets. There are over 3000 such datasets for diverse Hymenoptera and mite species. We have developed a computational pipeline to identify viral transcripts in these datasets. This pipeline performs quality control, removes low complexity reads and reads generated from host RNA and various known contaminants, assembles the remaining reads into transcripts and detects the presence of regions with homology to known RNA viruses. Viral fragments identified with this pipeline will be examined phylogenetically to identify novel pathogens, clarify host range and specificity, and characterise transmission patterns.
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- Microbial Physiology, Metabolism and Molecular Biology Forum
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The HamE scaffold positively regulates MpkB phosphorylation to promote development and secondary metabolism in Aspergillus nidulans
More LessMitogen-activated protein kinase (MAPK) pathways are conserved signalling cascades in eukaryotes which regulate a myriad of processes in fungi from sexual reproduction to stress responses. These pathways rely on recruitment of three kinases on a scaffold protein to facilitate efficient kinase phosphorylation and subsequent downstream signalling to the nucleus. The model filamentous fungus Aspergillus nidulans utilises a MAPK pathway termed the pheromone module to regulate both development and secondary metabolism. This complex consists of the MAP3K (SteC), MAP2K (MkkB), MAPK (MpkB) and adaptor protein SteD. To date, there has been no scaffold protein identified for this MAPK pathway. In this study, we characterised a protein termed HamE, which we propose as a scaffold that regulates kinase phosphorylation and signalling in the pheromone module. Mass spectrometry analysis and BIFC experiments revealed that HamE physically interacts with both MkkB and MpkB and transiently interacts with SteC. Deletion of hamE or any of the pheromone module kinases results in reduced sporulation and complete abolishment of cleistothecia production. Mutants also exhibited reductions in expression of secondary metabolite gene clusters, including the velvet complex and sterigmatocystin genes. HamE acts as a positive regulator of MpkB phosphorylation, allowing for HamE to subsequently regulate development and secondary metabolism.
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Mechanistic analysis of the minimalistic twin-arginine translocation system found in Bacillus subtilis
More LessThe twin-arginine translocation (Tat) machinery mediates the transport of folded proteins across the cytoplasmic membranes of prokaryotes and thylakoid membranes of chloroplasts. In Gram-negative bacteria three integral membrane components, TatA, TatB and TatC, are essential for generating an active complex. Most Gram-positive bacteria however, have a minimalist Tat complex formed solely from TatA and TatC subunits. Bacillus subtilis encodes two TatAC systems; TatAdCd and TatAyCy. These complexes operate in parallel but have differing substrate specificities. To date, little is known about how the B. subtilis TatAC-complexes assemble, however, the Escherichia coli TatABC complex is well studied and a substrate-triggered positional exchange of TatA and TatB has been identified as the first step in the assembly of an active complex. In this study, we aim to identify site specific interactions taking place between the B. subtilis Tat components and how these interactions differ whilst the complex is engaged in transport of a substrate. Molecular modelling was used to identify likely interaction interfaces between TatA and TatC, and site-directed mutagenesis was used to generate single cysteine variants of both proteins. Subsequently in vivo disulfide crosslinking was undertaken in both E. coli and B.subtilis, in the presence and absence of overproduced Tat substrates, to determine changes that occur between resting state and activated complexes. Overall, this work will enable us to outline a possible mechanism of assembly in TatAC-complexes of Gram-positive bacteria and identify whether the two distinct translocases assemble in a similar fashion.
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Coupling of subunit availability to activation of PMF-driven flagellar type III secretion
More LessBacterial flagella are assembled from thousands of protein subunits that are unfolded and exported via a specialized type III secretion system. Subunit export is fuelled by the proton motive force (PMF) facilitated by a cytoplasmic ATPase complex comprising FliH, FliI and FliJ, which are evolutionarily related to components of the F1 ATPase. The FliJ stalk component of the ATPase binds the export gate protein FlhA, allowing it to utilise ΔΨ to drive highly efficient subunit export. What is unclear is how FliJ activation of FlhA is regulated to prevent constitutive proton influx when there are no subunits available. FliJ-mediated export gate activation could be regulated by other proteins that bind FliJ. We have shown that FliJ recruits unladen export chaperones, transferring them to their cognate subunits to create a local cycle of chaperone-subunit binding. To investigate whether chaperones also regulate FliJ activation of FlhA, we sought to isolate chaperone variants that were defective in FliJ binding but retained their ability to bind subunits and other export components. Disruption of chaperone-FliJ binding attenuated motility and cognate subunit export. To test whether chaperones blocked the FlhA-FliJ interaction, we developed in vitro and in vivo competition assays. Our data showed that chaperones and FlhA compete for a common binding site on FliJ, and that unladen chaperones, which would be present in the cell when subunit levels are low, disrupt the FliJ-FlhA interaction, preventing activation of the export gate. This provides a mechanism whereby the export gate is only activated when subunits are available.
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A large-scale investigation of stress response mechanisms in the industrial yeast Kluyveromyces marxianus
Microbial production strains need to operate under sub- optimal growth conditions such as low pH, high osmolarity and thermal stress. The capacity to carry out industrial fermentations at higher temperatures reduces the risk of bacterial contamination and lowers cooling costs. We want to understand the basis of thermotolerance in the industrial yeast Kluyveromyces marxianus. As part of the EU-funded project, CHASSY, K. marxianus was grown in chemostat cultures under different stress conditions and a multi-omics analysis performed to study a range of stress responses, including elevated temperature (40 °C). Transcriptomes were generated from steady state cultures growing at identical growth rates under different stress conditions and gene set enrichment analysis (GSEA) performed. A range of functions were identified as being specifically expressed at higher temperatures and these are now being further investigated. One example is the temperature-specific expression of two putative hexose transport genes. Subsequent mutational inactivation using CRISPR and heterologous complementation established that at least one of these two genes is required for growth at (40 °C). We are now trying to determine the substrates for, and the precise function of, these genes. We also developed a ribosome profiling pipeline for K. marxianus and are using this to investigate the translational response to temperature stress. The combined study of both transcription and translation at steady state and as a culture responds to a temperature shift will give a comprehensive view of the basis of thermotolerance in K. marxianus and should identify strategies to exploit this in biotechnological processes.
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Effects of an RNA chaperone on mutation tolerance
More LessDue to their intrinsic thermodynamic properties, RNA can misfold easily in cells. One way to mitigate RNA misfolding is through the actions of RNA chaperones, which bind and unwind structured RNA molecules and thereby offer opportunities for these misfolded species to refold properly. Such rescue activity has implications for the fitness effects of individual mutations-- at least mutations that compromise RNA folding or structure might be buffered by RNA chaperones. However, little is known about the rules governing such mutation buffering. Here, we describe how a model RNA chaperone, the DEAD-box RNA helicase CYT-19, affects the fitness effects of mutations in a model structured RNA, the Tetrahymenagroup I intron, whose self-splicing activity is dependent on its structure. We performed deep mutational scanning on the P1ex region of the intron which is critical for its self-splicing activity, and assayed differential splicing activity of all possible P1ex mutants in the presence and absence of CYT-19 to identify mutations that are buffered by RNA chaperone activity. I will discuss the properties of the chaperone-dependent and chaperone-independent mutation pools. Our results highlight that, to understand RNA robustness in vivo, we need to consider how mutational fitness effects are modulated by RNA chaperones and other trans-acting factors.
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Of microscopes and microbes; novel applications of optical microscopy to microbiology
More LessThe development of optical microscopy has been traditionally driven by the needs of eukaryotic cell biology. Therefore, there are a number of unmet requirements for the application of advanced microscopy techniques to the field of microbiology. Conventional techniques, such as widefield epi-fluorescence and confocal laser scanning microscopy, are common-place in most laboratories, however these methods have trade-offs in terms of their attainable resolution and limited imaging volume. Here we present the application of several optical microscopy methods with the aim addressing the unmet needs of the field; increasing spatial resolution and sampling volume. We demonstrate the use of Interference Reflection Microscopy (IRM) for investigating the morphology and gliding motility of Myxococcus xanthus. This label-free technique provides super-resolution in the axial plane where changes in cell shape on the order of 100 nm can be detected in live cells. Using IRM we show novel insights into the gliding behaviour of these bacteria. We also present the application of the Mesolens to microbiology. The Mesolens is a large optical microscope with the unique combination of a low magnification and a high numerical aperture which results in an imaging volume >100 mm 3 with isotropic sub-cellular resolution. We demonstrate the use of the Mesolens to image live bacterial communities at multiple spatial scales simultaneously and offer new insights for bacterial community dynamics and biofilm architecture. Our work details novel applications of advanced microscopy to the field, and in doing so fills the technology gap which has previously restricted the study of complex microbial behaviours.
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The chitin attenuator: the Ca2+/calcineurin pathway maintains the viability of Candida albicans cells with supra-normal chitin levels
More LessChitin is an essential structural polysaccharide component of the cell walls and septa of fungi. Recent reports have suggested that Candida cells can resist killing by echinocandins by up-regulation of chitin synthesis thereby sustaining cell wall integrity both in vitro and in vivo (Lee et al. 2012). This increase in chitin content seen in C. albicans cells that are less susceptible to caspofungin is coordinated simultaneously by the PKC, Ca2+/calcineurin and HOG pathways (Munro et al. 2007, Walker et al. 2008). However, when echinocandins are removed, the chitin content quickly returns to basal levels, suggesting that elevated chitin cell wall content represents a fitness cost. We show here that those cells that die in the presence of caspofungin often have supra-normal chitin levels rather than low chitin levels, and therefore that having too much chitin in the cell wall may be detrimental for viability. Chitin content may therefore need to be clamped at levels that enable cells to survive cell wall stresses but are not so high that they negatively affect cell viability.
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A CRISPR-associated Rossmann fold (CARF) domain regulates transcription of an RNA repair system in Escherichia coli
More LessCRISPR-associated Rossmann fold (CARF) domain signalling underpins the modulation of CRISPR-Cas systems. Unlike the majority of known CARF domains associated with nuclease activity in CRISPR-Cas systems, the CARF domain of the transcriptional regulator RtcR modulates the opposite function by activating an RNA end sealing system. The Rtc RNA repair system in Escherichia coli consists ofthe universally conserved RNA cyclase RtcA and RNA ligase RtcB, and is known to be induced by antibiotics and oxidative stress. We aim to investigate the CARF domain mediated transcriptional regulation of the Rtc system in vivo and in vitro. A reporter based assay confirmed that the RtcR CARF domain has a negative regulatory effect on RtcR activity and subsequent induction of the rtcBA operon. Both predictive modelling and site-directed mutagenesis suggest the Rtc induction is not due to oxidation of cysteines present in the RtcR regulatory CARF domain. Interestingly, the enzymes RtcA and RtcB, products of the actions of RtcR as a transcription regulator, are required for RtcR to stimulate expression of the rtcBA operon and directly bind to the RtcR CARF domain as shown in vivo by two-hybrid and in vitro by gel filtration. Given that CARF domains are known to be activated by RNA binding, a range of RNA molecules linked to the Rtc system or CARF domains in general are currently being investigated as potential inducers in an in vitro transcription system. Together, our data indicate an expanded range for CARF domain signalling, including via protein-protein interactions.
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Deciphering the secretion mechanism and novel protein-protein interactions of TecA, a Burkholderia cenocepacia toxin
More LessBurkholderia cenocepacia (Bc) is an environmental opportunistic pathogen that causes persistent, often severe, lung infections in individuals with cystic fibrosis and other underlying diseases. Rho GTPases are central molecular switches that regulate cytoskeletal dynamics, trafficking, immune responses and cell proliferation in eukaryotic cells. Many microbes produce proteins that target Rho GTPase signalling. Bc employs a type VI secretion system (T6SS) to survive in macrophages by disarming Rho GTPases and causing actin cytoskeletal defects. Bc protein TecA is a non-VgrG T6SS effector that is responsible for actin disruption. TecA and other bacterial homologs bear a cysteine protease-like catalytic triad, which inactivates Rho GTPases by deamidating a conserved asparagine in the GTPase switch-I region. RhoA deamidation induces Pyrin inflammasome activation1. Our goal is to determine the detailed TecA secretion mechanism and the interacting partners inside the bacterial cytoplasm and inside the macrophages. In this study, we found by Co-IP/MS analysis that TecA interacts with the T6SS tube protein HcP, the membrane anchored TssM and with the elongation factor Tu. We also found that TecA is secreted in the absence of HcP and the secretion mechanism is discussed. 1. Aubert, D.F., X. Hao, J. Yang, X. Shi, W. Gao, L. Li, F. Bisaro, S. Chen, M.A. Valvano, and F. Shao. 2016. A Burkholderia Type VI Effector Deamidates Rho GTPases to Activate the Pyrin Inflammasome. Cell Host and Microbe 19 : 664–674.
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- Missing Microbes and the Hygiene Hypothesis: New Challenges and Perspectives
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Prevalence of microbial parasites in captive animals across wildlife parks
More LessMicrobial eukaryotes (parasites/protists) are widely distributed and are common inhabitants of the gastro-intestinal (GI) tract of humans and animals. Some species, including Giardia, Entamoeba and Cryptosporidium are associated with symptomatic gastro-intestinal illness. However, others, for example Blastocystis, have questionable pathogenicity as they can be found in symptomatic and asymptomatic individuals. The aim of this study is to investigate selected protists which present health concerns to humans or animals. To date, approximately 180 faecal samples from 33 mammalian species, four bird species and one reptile across two wildlife parks in the Southeast England have been collected. A combination of cell culturing techniques, microscopy and molecular biology have been carried out to positively identify different protists including Blastocystis, Cryptosporidium, Eimeria, Entamoeba, Giardia and Isospora. Preliminary data show over fifty percent of the animals are sequence positive for at least one species, with approximately thirty percent exhibiting co-habitation with two or more different species. This study provides one of the first thorough investigations into distribution and prevalence of GI tract protists in wildlife parks in the UK. As a result, it has enhanced our awareness regarding what may constitute a normal eukaryotic component of the gut microbiome, in addition to aiding conservation efforts by examining the impact captivity has on an animal’s microbiome and potential implications this may have on their release.
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The ‘missing’ gastric microbe; the impact of gastric cancer-associated microbiota on Helicobacter pylori growth in vitro and its implications in gastric carcinogenesis
More LessIn recent years, multiple studies have examined the bacterial communities present in the gastric microbiota during the progression to gastric cancer (GC). Although Helicobacter pylori is the biggest risk factor for GC, the microbiota of GC comprises a decreased load of H. pylori and an enrichment of bacteria, such as Prevotella spp., Veillonella spp., Actinomyces spp. However, interactions between H. pylori and these cancer-associated bacteria is hugely understudied. Here, we have used hypoxic growth conditions (5 % O2) to investigate polymicrobial interactions between cancer-associated bacteria and H. pylori in vitro. We found that whilst the co-culture of H. pylori with Prevotella spp. and Veillonella spp. had no effect on growth of either bacteria, Actinomyces oris completely inhibited the growth of H. pylori. Moreover, A. oris did not inhibit the growth of other Gram-negative pathogens such as Salmonella Typhimurium and E. coli, whilst there was a slight growth inhibition of Campylobacter jejuni. Furthermore, ultrafiltration of A. oris culture supernatants revealed that inhibition is mediated by a secreted factor larger than 5 kDa, which can be heat inactivated. Interestingly, Actinomyces viscosus can also specifically kill H. pylori suggesting that this inhibition could be conserved across the Actinomyces genus. We are currently identifying the inhibitory factor responsible for inhibiting H. pylori growth. Furthermore, we are investigating whether A. oris can clear gastric H. pylori infection in a mouse model of infection and the implications of this on gastric carcinogenesis. In conclusion, whilst data-rich microbiota studies continue to thrive, it is imperative that we understand the mechanisms underpinning changes to the gastric microbiota and whether these bacteria are drivers or ‘passengers’ of gastric carcinogenesis.
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- Non-human Pathogens
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Epidemiology of tick borne pathogens of dogs in Nigerian communities
More LessTick borne diseases (TBDs) have significant impact on the health and welfare of domestic animals and humans. There is extremely little data on the prevalence of tick species, TBDs or their impact in Nigeria. Nigeria’s scenario is further worsened by lack of basic diagnostic facilities and treatment, compared with the average person’s income. The multiple climate zones and animal husbandry practices in Nigeria also make it difficult to extrapolate studies from one zone to the other five geopolitical zones. TBDs reported in Nigerian dogs include Anaplasma species. (A. platys A. omatnenne), Babesia species (B. rossi, B. canis and B. gibsoni), Theileria species. (T. equi, T. sable), Ehrlichia sp. (E. canis, E. ruminantum), Hepatozoon species (H. canis) and Candidatus Neoehrlichia mikurensis. The prevalence of zoonotic pathogens of dogs such as Borrelia sp., in humans in West Africa also indicates that these are likely to present in dogs in Nigeria. This main study aim is to identify ticks taken from dogs in Nigeria using morphological and molecular methods, determine which host species the ticks have fed on, and identify pathogens that they are carrying. It also aims to compare molecular and point of care diagnostics for TBDs in blood from Nigerian dogs. The overall objective is to provide robust data on which tick species and TBDs are present in Nigerian dogs and the potential zoonotic or epizootic risk.
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Evolutionary genomics of the host-restricted pathogen Staphylococcus aureus subsp. anaerobius reveals extensive genome decay and signatures of adaptation
Staphylococcus aureus subsp. anaerobius is a microaerophilic, catalase-negative bacterium responsible for abscess pathology (Morel’s disease) in small ruminants. We performed whole-genome sequencing to a collection of isolates taken in Europe and Africa over the last 30 years, and carried out an evolutionary genomic analysis to understand the molecular bases of its host adaptation and restricted metabolism. Phylodynamic analyses showed that S. anaerobius emerged from a Staphylococcus aureus progenitor about 1000 years ago (716–1184), with an evolutionary rate of ∼1.2 SNPs/year (approximately 10-fold slower than S. aureus clones), before differentiating into two distinct lineages separating African and European isolates. The S. anaerobius genome displays signatures of extreme adaptation to a highly specific niche, with 205 pseudogenes that together represent over 10 % of the genome and affect many metabolic and pathogenic pathways. In addition, S. anaerobius contains 87 highly similar insertion sequences (IS) located in intergenic regions. Our functional analysis suggests that the IS transcription could affect the expression of their flanking genes, using at least two complementary strategies: antisense RNA or modification of promoter regions. We propose that the IS-mediated control of gene expression underpins an orchestrated mechanism of host adaptation. Remarkably, we also identified 6 large genomic transversions (size range: 70–346 kb) flanked by IS, presumably the result of homologous recombination. In summary, S. anaerobius evolved from S. aureus undergoing restrictive host specialisation, which shaped its genome through widespread pseudogenisation, accumulation of IS that modulate gene expression, and large chromosomal rearrangements.
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Genome epidemiology of Mycobacterium bovis infection in contemporaneous, sympatric badger and cattle populations in Northern Ireland
IntroductionBovine tuberculosis (bTB) is an epidemiologically complex disease affecting both cattle and badgers in the UK and Ireland. Traditional molecular typing schemes have been used to characterise the spatial structure of the pathogen and relationship of M. bovis derived from sympatric animals. However, these methods lack the resolution to describe transmission dynamics at the farm level or to inform on the extent to which the hosts contribute. Whole genome sequencing can improve resolution of molecular epidemiology investigations in this epi-system.
MethodsWe collected 598 M. bovis isolates from contemporaneous badgers (n=119) and cattle (n =479), located in a 100 km2 area of Northern Ireland. Cultures were DNA extracted and genome sequenced. Bioinformatic analysis was undertaken using the reddog pipeline and maximum likelihood phylogenetic analyses were conducted using RAxML, with the major endemic clade in the region subjected to phylodynamic analysis using Bayesian Evolutionary Analysis Sampling Trees (BEAST) software.
Results and DiscussionAll 598 isolates produced reads of good quality, aligning to >90 % of the M. bovis reference genome with coverage of at least x10. A total of 1598 SNPs were detected. Phylogenetic analysis indicated the presence of nine major lineages circulating in the region. Eight exhibited long branch-lengths suggesting they were not endemic in the area. One lineage was endemic, comprising isolates from 60 badgers and 363 cattle. From the substitution rate of 0.36 SNPs per annum, this lineage arose in the study area in the mid-1980s. Data were consistent with ongoing transmission within and between both hosts.
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Mastitis and animal husbandry – high-throughput sequencing as a support tool
More LessMastitis is a disease of the mammary gland which affects most mammals and is one of the costliest ongoing challenges in modern dairy farming. The most significant challenges in mastitis management are the speed and accuracy of diagnosis and the use of antibiotics. Current mastitis diagnosis in veterinary practices in the United Kingdom utilises culture-based techniques. However, this approach has some limitations including; processing time, species selection biases and culture failure. This ongoing PhD project seeks to assess the utility of high-throughput sequencing technology in addressing the current challenges in mastitis diagnosis. Samples were obtained from cattle from farms in North Yorkshire, United Kingdom. Cattle were selected from monthly somatic cell count records as mastitis positive cases with a cell count between 250 000 and 550 000 cells ml−1 or control animals with a count below 200 000 cells. Mastitis positive cases were further split into bacteriology positive and negative groups. During sampling a cow-side somatic cell test (California Milk Test) was performed to confirm the monthly database readings. All farms sampled were conventional dairy farms with comparable management systems. Samples were collected by trained veterinarians following a pre-defined protocol designed to limit sample contamination. Microbial community diversity, richness and composition will be compared between sample groups to survey for variations which may explain why culture fails in culture negative subclinical cases. 131 sample animals have been assigned to study groups with samples collected, DNA extracted and prepared for sequencing on the Illumina MiSeq platform with results expected in January 2019.
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Characterisation of bovine dendritic cells following FMDV infection
More LessDendritic cells (DCs) are the sentinels of the immune system, responsible for recognising invading pathogens and priming the adaptive immune system for appropriate responses. Hence they are considered potential targets for vaccines against pathogens such as foot-and-mouth disease virus (FMDV). Little is known of the events of FMDV replication in bovine moDCs. Present work therefore sought to characterize FMDV and its immune complex (IC) replication in bovine moDCs in vitro. A chimeric heparin sulphate FMDV (O1M) was used in this study. Immuno-fluorescence microscopy (IFM) and quantitative RT-PCR was used to analyse viral replication at 0–6, 8, 16 and 24 hpi. Plaque assays were used to investigate the yields of live virus produced in moDCs at 0, 4, 8 and 24 hpi. FMDV and IC FMDV could infect moDC. In moDC infected with FMDV alone, or with immune-complexed (IC) FMDV, replication was observed by IFM between 2–4 and 1–16 hpi. In contrast, for both FMDV and FMDV IC infections RT-PCR analyses showed viral replication peaked at 4 hpi and then decreased between 8 to 24 hpi. Plaque assays using supernatants of the infected moDC showed no evidence of an increase in viral titre at 24 hpi. The detection of viral nsp (3AB and derivatives) suggests replication of FMDV persists for longer in moDCs when entry is mediated by IC. However, the lack of increase in virus yield suggests replication is abortive. One possible explanation for this difference could be that bovine moDCs are able to recognise non-immune complexed FMDV more rapidly.
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Phylogenetics and vector competence of a bovine ephemeral fever virus strain from Israel
Bovine ephemeral fever virus (BEFV) [Family Rhabdoviridae: genus Ephemerovirus] causes a transient febrile illness in cattle that results in economic losses and morbidity, with occasional mortality. Epidemics of the disease have been particularly costly in countries of the Middle East, including Israel. The virus is considered a vector-borne pathogen although the exact relationship with a number of blood-feeding arthropods has not been established. In order to improve developments in diagnostic detection, phylogeographic investigations and virus-vector relationships of BEFV we have derived the first genome of this virus from an isolate from Israel and used this to assess recent outbreaks of disease. We have also investigated the vector competence of BEFV with a number of target arthropod species. The complete genome sequence of BEFV (Israel strain 1) is 14 850 base pairs in length and shows over 95 % identity with the only other Middle Eastern genome for BEFV from Turkey. A wider phylogeny shows that these viruses form a clade, previously described as cluster II, which is clearly distinct from BEFV strains derived from China, Japan and Australia. However, there is a distinct separation of those viruses from Israel to others in the Middle East. Preliminary vector competence studies suggest that at least three species of mosquito that are potential transmission vectors of the virus are incapable of infection with, or transmission of, BEFV. Current studies are ongoing to assess other blood-feeding insect species as potential vectors.
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SNP based transmission study of badgers infected with Mycobacterium bovis in the edge risk area of England
More LessMycobacterium bovisis the causative agent of bovine tuberculosis (bTB), one of the most costly and persistent agricultural infectious diseases still widespread in England today. However, not all parts of England are created equally with respect to the incidence of bTB. In 2012 the Department for the Environment, Food and Rural Affairs (DEFRA) divided the country into three risk areas; the high, low and edge risk areas. The perpetuation of the bTB epidemic is often blamed on the known wildlife host of M. bovis, the European badger (Meles meles). Despite this, no large scale studies examining both the prevalence and transmission patterns of M. bovis in this species has been undertaken. Here we describe the results of a major survey of 600 road-kill badgers from 6 counties within the edge risk area of England. Mycobacterium like colonies were isolated from over 80 carcasses of which, 65 were confirmed to be tuberculosis complex positive by PCR. These isolates were spoligotyped, VNTR typed and then subsequently whole-genome sequenced. We describe a SNP based transmission analysis of the sequenced isolates that provides a higher degree of resolution between the badgers compared to the described molecular typing methods.
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