- Volume 2, Issue 7A, 2020
Volume 2, Issue 7A, 2020
- Abstracts from Annual Conference 2020
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- Oral Abstract
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Prevalence of Agr phase variants in Staphylococcus aureus
Staphylococcus aureus is an important human pathogen whose success is largely attributed to its vast arsenal of virulence factors that facilitate its invasion into, and survival within, the human host. The expression of these virulence factors is controlled by the quorum sensing Accessory Gene Regulator (Agr) system. However, a large proportion of clinical S. aureus isolates are consistently found to have a mutationally inactivated Agr system. These mutants have a survival advantage in the host but are considered irreversible mutants. Here we show, for the first time, that a fraction of Agr-negative mutants can revert their Agr activity. By serially passaging Agr negative strains and screening for phenotypic reversion of haemolysis and subsequent sequencing, we identified two mutational events responsible for reversion: a genetic duplication plus inversion event and a poly(A) tract alteration. Additionally, we demonstrate that one clinical Agr-negative MRSA isolate could reproducibly generate Agr-revertant colonies with a poly(A) tract genetic mechanism. We also show that these revertants activate their Agr system upon phagocytosis. To assess the significance of our findings we screened a series of primary clinical isolates, which had undergone minimal handling post-isolation, and successfully identified a fraction which were Agr phase variants. Taken together, we propose a model where some Agr-negative S. aureus strains are phase variants who can revert their Agr activity and may act as a cryptic insurance strategy against host-mediated stress.
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The SKI complex is a broad-spectrum antiviral drug target
More LessStarting from a yeast suppressor screening platform, we have identified the SKI complex as a potential broad-spectrum antiviral target. We found that the NS1 protein of influenza A virus (IAV) and the ORF4a protein of Middle East respiratory syndrome coronavirus (MERS-CoV), which both function to bind double-strand RNA and inhibit cellular interferon responses, cause a slow growth phenotype when expressed in yeast. Knockout of the components of the yeast SKI complex caused a loss of this slow growth phenotype, suggesting a functional link between the viral proteins and the SKI complex. The SKI complex is a helicase that unwinds double-strand RNA and sends it to the RNA exosome for degradation. We next investigated whether the highly conserved human SKI complex was important for replication of IAV and MERS-CoV. RNAi based experiments showed that both viruses were inhibited when the SKI complex was removed, suggesting the complex has a proviral role in replication. Through in silico modelling using the published crystal structure of the SKI complex, we looked for potential binding pockets for chemical compounds. We screened a selection of these compounds for antiviral activity and have found four different chemicals capable of inhibiting IAV infection. Our most studied of these also inhibits not only MERS-CoV, but also Ebolavirus Makona. Our data suggests the SKI complex may be a target for broad-spectrum antiviral therapy and we have multiple chemical structures from which to work to develop therapeutic approaches.
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Resolving a clinical tuberculosis outbreak using palaeogenomic genome reconstruction methodologies
More LessThis study describes the analysis of DNA from heat-killed (boilate) isolates of Mycobacterium tuberculosis from two UK outbreaks where DNA was of sub-optimal quality for the standard methodologies routinely used in microbial genomics. A blunt-end Ligation based DNA library construction method, developed for sequencing ancient degraded DNA, was successfully used to obtain whole genome sequences from the sub-optimal samples. Thus, allowing analysis of the outbreak by WGS gene-by-gene MLST, SNP mapping and phylogenetics to be achieved. All cases were spoligotyped to the same Haarlem H1 sub-lineage. This is the first described application of ancient DNA library construction protocols to allow whole genome sequencing of a clinical tuberculosis outbreak. Using this method it is possible to obtain epidemiologically meaningful data even when DNA is of insufficient quality for standard methods.
Keywords: Mycobacterium tuberculosis, whole genome sequencing, outbreak investigation, ancient DNA, library construction, palaeogenomics.
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Deep splicing plasticity of the human adenovirus type 5 transcriptome as a driver of virus evolution
More LessViral genomes are characterised by having high gene density and complex transcription strategies. One of the most complex is adenovirus which has a double stranded DNA genome and is the archetypal viral system in which splicing was first discovered. Understanding the transcriptional landscape using conventional mRNA cloning or more recent Illumina-based deep sequencing methods offers insight but also has limitations, including the potential for reverse transcription or PCR amplification artefacts and bias. Here we used direct RNA long read length sequencing on an Oxford Nanopore MinION device to gain a quantitative system-wide overview of transcription and splicing as it dynamically changes during a human adenovirus type 5 infection. This global overview revealed an extensive and hitherto unappreciated complexity of alternative splicing and secondary initiating codon usage. Allied to this, analysis of viral polyadenylation patterns over time showed that most viral transcripts tended to shorter polyadenylation lengths as the infection progressed. Moreover, development and use of an ORF-centric bioinformatics pipeline for analysis of sequenced mRNA, provided both a quantitative and deeper qualitative understanding of the genetic potential of this virus. The data strikingly illustrated that across the viral genome adenovirus made multiple distinctly spliced transcripts that coded for the same ORF. Indeed, as many as 11,000 different splicing patterns were recorded across the viral genome over the three time points analysed. This constitutive low level use of alternative splicing patterns and secondary ORFs potentially enables the virus to maximise its coding potential over evolutionary timescales.
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Incidence of other bacterial pathogens among the patients suspected with pulmonary tuberculosis attending Hasiya Bayero Paediatric hospital Kano, Nigeria
More LessTuberculosis co-infection with other bacterial pathogens is one of the major health problems especially in tuberculosis endemic region. Bacterial pathogens that localized in the lower respiratory tract are could sometimes preclude Mycobacterium tuberculosis or manifest as pulmonary symptoms that could mislead clinicians. Therefore this study was planned to examine suspected TB patients in Kano, Nigeria for other pathogenic bacteria with view to establishing frequency of occurrence towards facilitating better outcome of therapeutic cover. A total of 170 non duplicated sputum samples were collected from patients with suspected pulmonary tuberculosis. All the samples were processed according to standard bacteriological procedures including macroscopic examination, culture and microscopic examination using both Acid Fast Bacilli (AFB) staining and Gram staining methods. Isolated organisms were subjected to appropriate biochemical testes. High incidences were observed among age group 10 – 29 and 30 – 49 years with male and urban people been the predominant patients. Overall 20.6% of samples were AFB positive indicated TB co-infection while 62.4% were positive for other bacterial growth. Nalidixic acid and Nitrofurantoin found to have highest resistant among Gram negative organisms isolated following by Levofloxacin, Ceftriaxone and Ceftazidime while the Ofloxacin and Ciprofloxacin have the least resistant. Among Gram positive Ceftazidime shown to has the highest resistant and Ofloxacin found to has the least resistant. The study indicated predominance of Streptococcus pneumoniae (47.2%) and Staphylococcus species (32.1%) with an evidence of 15.1% and 2.8% co-infection with TB respectively. x
Keywords: Mycobacterium tuberculosis, Co-infection, Bacterial pathogens, pulmonary tuberculosis.
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Viruses in multiple sclerosis pathogenesis
Multiple sclerosis (MS) is an extremely debilitating auto-immune disease of people characterised by demyelination of the central nervous system and progressive neurological dysfunction. The etiology of the disease is complex (including factors such as genetics, sex hormones and vitamin D levels). The involvement of viral infections as a risk factor in triggering disease has long been suspected and two classes of viruses in particular, the herpesvirus Epstein-Barr virus (EBV) and the Human endogenous retrovirus “W” family (HERV-W) have been the focus of much recent research. Despite near ubiquitious infection (EBV), or integration into the genome as a repetitive element no longer able to function as a virus (HERV-W) a picture is gradually emerging of how these are involved in MS pathogenesis. In the case of EBV, having had infectious mononucleosis (clinical disease in patients infected post-puberty) increases the risk of developing MS. For HERV-W our recent work has confirmed that RNA is over-expressed in MS patients compared with healthy controls (though basal levels vary with ethnic background) and that EBV infection of B cells triggers expression of HERV-W RNA and proteins. There is a growing body of evidence that expression of HERV-W can trigger innate immune system inflammatory responses. There is also increasing evidence for molecular mimicry between epitopes of the HERV-W env protein, the EBV EBNA1 protein and peptides from brain proteins implicated in MS pathogenesis. Crucially these peptides are also able to bind the HLA-DR2b locus that is the strongest genetic risk factor for MS development.
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Selfless viruses - the biology and regulation of gene transfer agents
More LessGene transfer agents (GTAs) are genetic elements that have the potential to carry out high frequency horizontal gene transfer. GTAs are similar to small bacteriophages in many ways but, instead of prioritizing the spread of their own genes, they package and disseminate the entire genome of their bacterial host. Indiscriminate transfer of bacterial genes could clearly have a major impact on bacterial evolution, fitness and antimicrobial resistance. Over the past decade, numerous pleiotropic systems have been shown to influence GTA production (e.g. nutrient stress, quorum sensing, SOS response etc.) but all act indirectly and so the mechanism of activation has remained elusive. I will present recent work that identified the missing link that couples GTA production to host regulatory pathways. I will also offer insights into the mechanism of random DNA packaging, the evolutionary role of GTAs in the environment and their potential ecological niche.
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Plasmids and temperate phages influence each other’s transfer rates
More LessAccessory genes, such as antibiotic resistance genes (ARGs) can spread horizontally by mobile genetic elements, including plasmids and (temperate) bacteriophages leading to increased bacterial fitness in particular environments. In contrast to plasmids, temperate phages i.e. viruses that can incorporate their own genetic material into their host bacteria (then called lysogen) can additionally increase their hosts’ fitness through their ability to kill phage-susceptible competitors. However, in contrast to ARG-transfer by plasmids (conjugation), which has been extensively studied, ARG-transfer by phages (lysogenization) has received far less attention. Here we combined experiments using E. coli, phage lambda and the Rp4 plasmid (phage and plasmid both with an ampicillin resistance gene) and mathematical models to test which mechanism (lysogenization or conjugation) dominates under which conditions.
In the absence of selection and in two-species experiments (Donor & Recipient), conjugation was significantly more common than lysogenization. However, in three-species experiments (DonorPlasmid, DonorPhage& Recipient) lysogenization rates increased by several orders of magnitude. By using phage-friendly environments that favour phage adsorption we were able to shift the ratio between lysogens and transconjugants even further towards lysogens and in extreme-events to the extinction of transconjugants. Mathematical models additionally allowed us to investigate how starting population sizes of both donors and the recipient influence conjugation and lysogenization dynamics. Taken together, our results suggest that plasmids and temperate phages can influence each other’s transfer rates which, in the present study system, seems to be largely driven by population size effects.
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A two-lane mechanism for selective biological ammonium transport
The transport of charged molecules across biological membranes faces the dual problem of accommodating charges in a highly hydrophobic environment while maintaining selective substrate translocation. A particular controversy has existed around the mechanism of ammonium exchange by the ubiquitous Amt/Mep/Rh transporter family, an essential process in all kingdoms of life. Here, using a combination of SSME electrophysiology, yeast functional complementation, and extended molecular dynamics simulations, we reveal a unique two-lane pathway for electrogenic NH4+transport in two archetypal members of the family. The pathway underpins a mechanism by which charged H+and neutral NH3 are carried separately across the membrane after NH4+deprotonation. This mechanism defines a new principle of achieving transport selectivity against competing ions in a biological transport process.
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Engineered ureolytic Bacillus subtilis and its future in Microbial Induced Calcium Carbonate Precipitation (MICCP)
More LessAs the global population grows there is an urgent need for increased, yet sustainable civil infrastructure. The ability to harness biological processes in order to improve ground stability; as well as creating construction materials without adding to climate damage is necessary. In almost every environment on earth, microorganisms and microbially mediated mineralisation (biomineralisation) processes are active. It is well documented that microbes present in soil can induce the precipitation of calcium carbonate (CaCO3) in both the laboratory and the natural setting through microbial induced calcium carbonate precipitation (MICCP). MICCP utilises microorganisms as a result of their active metabolism, to precipitate CaCO3, strengthening the surrounding matrix. MICCP is used in a variety of different applications such as carbon sequestration, environmental remediation and improving construction materials.
The enzyme urease catalyzes the hydrolysis of urea to ammonia and CO2, and is acknowledged to be instrumental in MICCP. Bacillus subtilis is a model, gram positive, spore-forming soil bacterium that produces a functionally active urease, but with low efficiency, and the activation is not completely understood. Sporosarcina pasteurii is one of the most commonly used MICCP microbes as its urease operon has been well studied and the bacterium has proven to produce ecologically stable bioconstruction materials. The ability to clone the urease operon of S. pasteurii into the model B. subtilis would create an engineered ureolytic organism whose urease activity could be controlled. This control would enable the CaCO3 morphology and material properties to be tailored and would create a truly responsive biomaterial.
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Unveiling the CAZyme repertoire of a sponge-derived Pseudoalteromonas sp. strain for the degradation of marine polysaccharides
Despite their well‐known importance to the holobiont homeostasis, carbohydrate‐active enzymes (CAZymes) from the sponge microbiome have to date been largely understudied from an industrial perspective. Enzymes that degrade marine polysaccharides (MPs) are receiving increasing attention as alternatives in the biorefinery sector and for use in the generation of high‐value‐added hydrolysis products. Screening of the agarolytic Pseudoalteromonas sp. strain PA2MD11, isolated from the Brazilian sponge Plakina cyanorosea, on selective media indicated that the strain degrades agarose, κ‐carrageenan and sodium alginate. Subsequently in silico analysis of the strain's genome indicated that around 3 % of the total genome encoded for CAZymes. These consisted mostly of glycoside hydrolases (GH) and glycosyltransferases (GT). Four agarase genes were also present, three from the family GH50 and one from the family GH16, with sequences from other free‐living and psychrophile Pseudoalteromonas genomes as its closest relatives. These agarases are structurally related to exo‐(GH50) and endo‐β‐agarases (GH16), and appear to encode for a complete agarolytic pathway. The two alginate lyases were classified as polysaccharide lyases (PL) from the families 6 and 17, with chondroitin‐ and heparin‐like domains, respectively, and shared common homologues sequences with an estuarine Pseudoalteromonas lipolytica strain. Data on the cloning and heterologous expression of these genes will be presented. To our knowledge, this is the first time that a “genome mining” based approach has been undertaken to identify these groups of MPs‐degrading CAZymes from cultivable members of the sponge microbiome.
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Cell-to-cell ATP differences can modulate cellular decision-making
More LessCells generate phenotypic diversity both during development and in response to stressful and changing environments, aiding survival. Functionally vital cell fate decisions from a range of phenotypic choices are made by regulatory networks, the dynamics of which rely on gene expression and hence depend on the cellular energy budget (and particularly ATP levels). However, despite pronounced cell-to-cell ATP differences observed across biological systems, the influence of energy availability on regulatory network dynamics is often overlooked as a cellular decision-making modulator, limiting our knowledge of how energy budgets affect cell behaviour. Here, we consider a mathematical model of a highly generalisable, ATP-dependent, decision-making regulatory network, and show that cell-to-cell ATP variability changes the sets of decisions a cell can make. Our model shows that increasing intracellular energy levels can increase the number of supported stable phenotypes, corresponding to increased decision-making capacity. Model cells with sub-threshold intracellular energy are limited to a singular phenotype, forcing the adoption of a specific cell fate. We suggest that energetic differences between cells may be an important consideration to help explain observed variability in cellular decision-making across a broad range of biological systems, including bacteria and the blood stem cell system.
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‘Seq & Destroy’: the full genome sequencing of archived wild type and vaccine rinderpest virus isolates prior to their destruction
More LessRinderpest, a once much feared livestock disease, was declared eradicated in 2011, however virus-containing material is still held in laboratories worldwide. Prior to the destruction of our institute’s stocks, we determined the full genome sequence of the distinct samples of rinderpest virus (RPV) in our repository. This data would decipher the historical epidemiology of RPV and allow for recovery of the virus should the need arise.
For each sample (n=123), sequencing libraries were prepared using either transposon-based fragmentation of cDNA (Nextera XT DNA Library Prep kit) or single primer isothermal amplification (Trio RNA-Seq kit) and sequenced on the Illumina MiSeq. Regions of low or no coverage were re-sequenced using a Sanger sequencing approach.
Examination of the sequences of RPV isolates has shown that the African isolates form a single disparate clade, rather than two separate clades as was previously believed. We have also identified two groups of goat-passaged viruses which have acquired an extra 6 bases in the long untranslated region between the matrix and fusion protein coding sequences, and a group of African isolates where translation of the fusion protein begins from a non-standard start codon (AUA). In addition, the viruses that were force-passaged through alternate hosts such as rabbits or goats, appear to diverge from the clades that represent viruses which were maintained in the wild.
Our unique set of sequence data will be invaluable for forensic epidemiology investigations in the event of an unforeseen outbreak and aid in the understanding of the evolution of related morbilliviruses.
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Identification of dengue virus immunodeficiency (IMD) pathway antagonists in Aedes Aegypti cells
More LessThe flavivirus dengue virus (DENV) is the most significant arthropod borne virus (arbovirus) of humans, causing serious morbidity and mortality, with nearly half of the world’s population at risk of infection. Due to a lack of antivirals and limited vaccine options, vector control remains a vital defence against dengue disease. The mosquito Aedes aegypti is the major vector for DENV, and understanding mosquito immune responses and how DENV may evade them is critical. We have shown that DENV-2 can inhibit the exogenous induction of immune deficiency (IMD) signalling by classical immune stimuli. Therefore, we aimed to identify DENV antagonists of the IMD pathway, and define the molecular virus and host determinants of IMD antagonism in a well characterised Ae. aegypti derived cell line, Aag2. Each DENV protein was expressed individually in Aag2 cells and tested for their ability to block IMD signalling induced by exogenous stimuli. This screen identified NS4A as a potential antagonist of the IMD pathway. Further, we have found that the N-terminus of NS4A is responsible for this inhibition. The antagonism of IMD signalling is specific to flaviviruses transmitted by a mosquito vector, illustrating the importance of both the IMD pathway for mosquito immunity and the antagonism of this pathway by DENV. By enhancing our understanding of how DENV evades the mosquito immune response at a molecular level, we will gain insight into virus-host interactions constraining arbovirus transmission and emergence, which may be exploited for developing transmission-incompetent vectors to reduce the burden of dengue disease.
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Inter and intra species social exploitation of antimicrobial resistance alters antibiotic efficacy
More LessThe widespread use and misuse of antibiotics has led to the global spread of antimicrobial resistance. It is increasingly evident that very low concentrations of antibiotics, well below the MIC of sensitive strains, can select for antimicrobial resistance. However, it is less clear how social interactions within bacterial communities can alter sub-MIC selection dynamics, potentially confounding the outcomes of antibiotic treatments. Here we explore how antimicrobial resistances that inactivate antibiotics can be socially exploited by sensitive members of the microbial community at the inter and intra species level. We first show that a beta-lactamase encoded multi-drug resistance plasmid provides high levels of protection to plasmid-free antibiotic-sensitive cells within single-species populations in a frequency-dependent manner. Second, a similar protection can also occur between different species during polymicrobial infections. Using model Cystic Fibrosis lung communities, we demonstrate that the focal pathogen Pseudomonas aeruginosa can socially exploit antibiotic resistance in the presence of Stenotrophomonas maltophilia bacterium that can hydrolyse imipenem antibiotic. In contrast, the presence of Staphylococcus aureus, another commonly co-occurring CF pathogen, provided P. aeruginosa with no protection, but instead, made P. aeruginosa more susceptible to antibiotic due to intensified competition. These findings reveal that social exploitation of pre-occurring antimicrobial resistance, and inter-specific competition, can have a large effect on the efficacy of antibiotic treatments, highlighting the importance of microbial ecology for understanding antibiotic resistance evolution.
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Uncovering the molecular mechanisms of the Pseudomonas aeruginosa PA14 response to myxobacterial predation
More LessMyxobacteria are Gram-negative bacteria, notable for their predatory and antimicrobial activities, which dictate the outcomes of their interactions with neighbouring organisms. They are abundant and widespread in nature, and can significantly affect the microbiome of an environment.
We hypothesise that there are underlying molecular mechanisms in prey specieswhich govern the prey’s susceptibility/resistance to the antimicrobial activity of myxobacteria. In this work we attempt to define the mechanisms by which Pseudomonas aeruginosa PA14 resists predation bythe model myxobacterium Myxococcus xanthus.
Pseudomonas aeruginosa is an opportunistic pathogen of humans and plants. With the rise in antibiotic resistant organisms, Pseudomonas spp. are categorised as World Health Organisation priority 1 antibiotic-resistant bacteria and are our prey of choice in this study.
In collaboration with Dr N. Tucker (Strathclyde), and using a strain of M. xanthus expressing mCherry (courtesy of E. Hoiczyk, Sheffield), we developed 96-well plate assays of predation which measured the optical density of both predator and prey and the florescence of predator at different point intervals. Predation was assayed againsta library of approximately 5700 PA14 mutants to identify strains with increased/decreased susceptibility to predation. Responses of PA14 mutants varied with time and between mutants, allowing us to create a shortlist of candidate genes involved in the prey response to predation.
We are currently performing a preliminary analysis of the data using the Integrated Genomic Viewer and Circos plots to assess the genomic organisation of the prey genes that influence susceptibility and resistance to predation.
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Explosive intron expansion and fickle rDNA copies within plastid genomes of Euglenophyta
More LessSeveral eukaryotic lineages gained the ability of photosynthesis by acquiring plastids in the events of primary endosymbiosis with cyanobacteria, or secondary endosymbiosis with plastid-bearing eukaryotes. Plastids possess genomes (ptDNA) with genetic contents considerably reduced as a result of gene losses and transfers to the host’s nucleus. Still, ptDNA encodes components of various metabolic processes, including photosynthesis. Plastid genomes usually retain quadripartite structure with two rDNA-bearing inverted repeats, but the reason for its conservation, and the consequences of its decline, have not been fully understood.
As the model group to study plastid genome evolution, we chose euglenids (Euglenophyta), whose ancestor acquired the secondary plastid by endosymbiosis with a green alga. The organization of ptDNA in this lineage is rather diverse: we have shown that loss of one repeat occurred at least three times, while some species in the genus Euglena possess tandemly repeated rDNA copies. The ptDNA of euglenids is also intron-rich, but we did not confirm the previously proposed strong correlation between the prevalence of introns and quantity of maturases.
Although euglenophytes are predominantly photosynthetic, a few of them lost their photosynthetic capabilities independently. Thus far, only Euglena longa has been shown to possess vestigial plastids with reduced genome; we observed that another strain lost its plastid genome completely. Currently, we are investigating the loss and retention of metabolic functions in the plastids of other non-photosynthetic euglenophytes. This, along with investigation of ptDNA structure, will bring new insights into the evolutionary processes shaping the diversity of eukaryotic plastids.
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In vitro reconstitution of the polymicrobial community associated with CF airway infections
More LessThe airways of persons with cystic fibrosis (CF) provide a nutritionally rich environment that is prone to colonisation by a diverse and dynamic community of microbes (including both bacteria and fungi). Traditionally, Pseudomonas aeruginosa (PA) was thought to be the dominant pathogen associated with CF airway infections. However, it is becoming increasingly clear that interspecies interactions play a crucial role in modulating the physiology and gene expression profile(s) of the protagonists, and also impact on the response to therapeutic intervention. However, there are currently no suitable experimental models that permit long-term successful co-cultivation of PA with other CF-associated pathogens. Simply mixing bacterial or fungal species together and hoping for the best is not a recipe for success, since PA rapidly outcompetes most other species when grown in batch culture. In this work, we rectify this by describing a “3Rs-compliant” continuous-flow in vitro co-culture model. Using our model, it is possible to maintain remarkably stable steady-state co-cultures of major CF-associated pathogens (PA, Staphylococcus aureus and Candida albicans). Our findings reveal that even numerically minor (0.1%, by cell number) species can have a profound impact on quorum sensing and virulence factor production by PA. Furthermore, we show that complete polymicrobial communities derived from CF sputum can be inoculated directly into the model, thus enabling the recapitulation of the entire microbiome associated with CF airway infections to be studied under physiologically relevant conditions.
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Healthy Honey Bees – analysis of the Deformed Wing Virus population to assess rational Varroa control on a Scottish island
More LessVarroa destructor is an ectoparasitic mite associated with significant losses of honey bee colonies globally. The mite vectors a range of pathogenic viruses, most important of which is Deformed Wing Virus (DWV, (+)ssRNA). Overwintering colony losses, accounting for the death of ∼25% of all colonies each year, are associated with high levels of Varroa-DWV infestation. Effective miticide treatments are available to control Varroa. However, the absence of coordinated treatment means environmental transmission of mites continued unchecked. We aim to determine whether rational, coordinated treatment is beneficial, using features of the DWV population as an indicator of colony health. This study uses coordinated treatment of Varroa in a geographically isolated environment (Isle of Arran, Scotland). It is reported that a high level of a near-clonal virus population is associated with Varroa infestation and colony losses, whereas Varroa-free healthy colonies carry only low levels of a diverse population of DWV. The study area contains 50-85 colonies and 25 beekeepers. Sampling and virus analysis – strain diversity and viral loads – have been conducted before and after treatment. Changes in virus diversity are quantified by next generation sequencing analysis to determine population diversity. In the first two years we have observed a geographic decrease in Varroa and changes in the composition of the virus population. This study will inform our development of rational Varroa control strategies for beekeepers in temperate regions and could be used to inform policy changes regarding treatment regimes in Scotland and elsewhere for this global pathogen.
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Identifying and characterizing a novel Sindbis virus capsid and IRAK1 interaction
More LessAlphaviruses are arthropod-borne positive-sense RNA viruses that have the capacity to cause large scale outbreaks of severe disease. As of now, there are no effective therapeutic strategies with which alphaviral disease may be treated. Thus, there is a need for research that defines the mechanism(s) by which the alphaviruses establish infection and cause pathogenesis. Our previous work identified non-assembly interactions between the SINV Capsid protein and the viral genomic RNA that were important for viral RNA stabilization early during infection. These efforts led us to evaluate the Protein:Protein interactions of the SINV Capsid protein using the BioID2 discovery approach to define the mechanism of action underlying the SINV Capsid-mediated genome stability.
To our surprise, these efforts indicated that the SINV CP protein interacts with the host IRAK1 protein in tissue culture models of infection. To validate the interface we utilized a Bimolecular Fluorescence Complementation approach to confirm the SINV Capsid-IRAK1 interaction. After confirming the novel Capsid Protein:Protein interaction we hypothesized that the SINV Capsid protein may interfere with IRAK1-dependent signaling during infection. To this end, we assessed the dose-responsiveness of several IRAK1-dependent signaling pathways, including TLRs 4 and 7 in the presence of the SINV Capsid protein. We found that the TLR-agonist response, was significantly decreased in the presence of the SINV Capsid protein. Collectively, these data are highly suggestive that the SINV Capsid protein interferes with TLR signaling during viral infection contributing to the evasion of the host innate immune response.
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Biocoatings: Painting bacteria on surfaces
More LessBackground: Biocoatings are nanoporous polymer materials which encapsulate bacterial cells with carbohydrates as osmoprotectants. Here, we optimised biocoatings to offer a favourable environment for the metabolic activity of bacteria.
Methods: E. coli were used as a model organism and mixed with the colloidal polymer particles (i.e. synthetic latex), inorganic nanoparticles and different carbohydrates. Films were casted and dried to create a coalesced latex film and finally rehydrated to re-establish bacterial metabolism. The toxicity of the sterile latices to the bacteria was tested by using the colourimetric redox indicator resazurin. Visualisation of the bacteria inside the biocoatings was performed by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM).
Results: We introduced halloysite (clay nanotubes) to create nanoporosity, which created voids in the structure that will permit gas exchange. The biocoatings were tested in liquid and rehydrated states with resazurin to find the most promising composition ensuring bacterial viability. Rehydrated biocoatings were visualised by CLSM by tracking the constitutively expressed yellow-fluorescent protein (YFP) for viable cells and the membrane exclusion dye propidium iodide for dead cells. The structure of the biocoatings appeared to be unaffected by freeze-drying compared to chemical fixation. Following this fixation, SEM allowed the observation of the organisation of the latex polymers, halloysite and bacteria.
Conclusions: The biocoatings were highly porous thanks to halloysite. E. coli survived the film formation process. Next, we will use E. coli and cyanobacteria to achieve higher efficiency for a variety of applications e.g. pollutant degradation, solar energy harvesting and carbon recycling.
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Host-dependent differences in replication and virion release strategy of the Sulfolobus Spindle-shaped Virus strain SSV9 (a.k.a., SSVK1): Lytic replication in hosts of the family Sulfolobaceae
The Sulfolobus Spindle-shaped Virus (SSV) system is a model for studying thermophilic archaeal virus biology. Several factors make the SSV system amenable to studying archaeal genetics and virus-host interactions in extreme environments. It has been shown that populations of Sulfolobus, the natural host, exhibit biogeographic structure. The acidic (pH<4.5) high temperature (65-88°C) habitats have low biodiversity, which diminishes prospects for host switch. SSVs and their hosts are readily cultured in liquid media and on plates. Given the wide geographic separation between various SSV-Sulfolobus habitats, the system is also amenable to studying allopatric versus sympatric virus-host interactions. We previously reported that SSVs exhibit differential infectivity on allopatric and sympatric hosts. We discovered a strikingly broad host-range for strain SSV9 (a.k.a., SSVK1). For decades, SSVs have been described as “non-lytic” dsDNA viruses that infect species of Sulfolobus and release virus particles via blebbing as a preferred strategy over host lysis (in reported laboratory infections). Here, we show, that SSVs infect more than one genus of the family Sulfolobaceae and, in allopatric hosts, SSV9 does not release virions via blebbing. Instead, SSV9 appears to lyse all susceptible allopatric hosts, while exhibiting canonical non-lytic virion release (historically reported for SSVs) on a single sympatric host. Lytic versus non-lytic virus release does not appear to be driven by multiplicity of infection. Data suggest that SSV9 is more stable than other SSVs in suspension; however, genetic substrates (e.g., CRISPR profiles) underlying non-lytic versus lytic virion release remain unresolved and are the subject of ongoing investigation.
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Tropism and neutralisation studies on bat influenza H17N10
The diversity of subtypes within Influenza A recently expanded with identification of H17N10 and H18N11 from bats. To study the tropism and zoonotic potential of these viruses, we successfully produced lentiviral pseudotypes bearing haemagglutinin H17 and neuraminidase N10. We investigated a range of cell lines from different species for their susceptibility to infection by these pseudotypes and show that a number of human haematopoietic cancer cell lines and the canine kidney MDCK II (but not MDCK I) cells are susceptible. Using microarrays and qRT-PCR we show that the dog leukocyte antigen DLA-DRA mRNA is over expressed in late passaged parental MDCK and commercial MDCK II cells, compared to early passaged parental MDCK and MDCK I cells, respectively. The human orthologue HLA-DRA encodes the alpha subunit of the MHC class II HLA-DR antigen-binding heterodimer. Small interfering RNA- or neutralizing antibody-targeting HLA-DRA, drastically reduced the susceptibility of Raji B cells to H17-PV. Conversely, over expression of HLA-DRA and its paralogue HLA-DRB1 on the surface of unsusceptible HEK293T/17 cells conferred susceptibility to H17-PV. The identification of HLA-DR as an H17N10 entry mediator will contribute to understanding the tropism of the virus and help to elucidate its zoonotic transmission. We also show that H17 pseudotypes can be efficiently neutralised by the broadly-neutralizing HA2 stalk monoclonal antibodies CR9114 and FI6. The lentiviral pseudotype system is a useful research tool, amenable for investigation of bat influenza tropism, restriction and pandemic preparedness, without safety issues of producing a replication-competent virus, to which the human population is naïve.
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DAAs treatment is associated with up-regulation of CD81 on peripheral B-lymphocytes
Cellular surface expression of CD81 (an essential co-receptor for HCV) is critical for successful HCV infection. Furthermore, CD81 cross-linking with HCV-E2 protein impedes activation signaling pathways in different lymphocytes (T-cells, B -cells and NK cells). The expression of CD81 on peripheral lymphocytes is known to be downregulated following successful dual anti-HCV therapy. On the other hands, no reports are yet available regarding its expression levels following the newly used treatment regimen in Egypt; direct-acting antivirals (DAAs): Sofosubvir & daclatsvir for three months. Thus, the aim of the current study was to evaluate the expression levels of CD81 on T and B lymphocytes in HCV-infected patients before and after successful treatment with DAAs. Cellular CD81 expression was measured on CD3+ (T lymphocytes) and CD19+ (B Lymphocytes) lymphocytes by flow cytometry from 19 patients with chronic HCV infection.
All the HCV viruses were of genotype 4. We found no correlation between CD81 expression on either CD3+ or CD19+ lymphocytes and viral load. The expression of CD81 on CD19+ lymphocytes was markedly reduced at the end of the treatment. On the contrary, CD81 was significantly increased on CD3+ lymphocytes following successful treatment.
Our data indicate that successful treatment of HCV infection is associated with a reduction in surface CD81 expression on B lymphocytes with a concomitant increase on T lymphocytes.
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Investigating the effect of Herpes Simplex Virus 1 Latency-Associated Non-Coding RNAs on the Human Neuronal Transcriptome
More LessHerpes simplex virus 1 (HSV-1) is a prevalent neurotropic virus that persists for the host’s lifetime due to HSV-1 establishing latency in sensory neurons. During latency, the only abundantly transcribed HSV-1 gene is the latency-associated transcript (LAT), which is processed into the 1.5kb or 2.0kb major LAT intron and several microRNAs. These latency-associated non-coding RNAs (ncRNAs) have been reported to impact the establishment, maintenance and reactivation from latency. However, the molecular mechanisms of these ncRNAs are not fully characterised, especially in the context of human neurons.
This study investigated how the latency-associated ncRNAs affect the human neuronal transcriptome. We developed an experimental system to deliver the latency-associated ncRNAs to human neurons, differentiated from SH-SY5Y neuroblastoma cells. The cells were infected with a replication-defective HSV-1 mutant, in1382, that establishes a quiescent infection in which LAT is strongly expressed. Alternatively, we utilised lentiviruses engineered to express the first 3.1kb of LAT, without or with mutations in splice sites that prevents splicing of the major LAT intron, or five HSV-1 microRNAs, shown to be abundant in latently infected human ganglia. Following RNA-Seq of uninfected versus infected or transduced SH-SY5Y cells, we identified 178 host genes that had significant differential expression in response to in1382 quiescent infection and lentivirus delivery of LAT or the latency-associated microRNAs. A subset of these were validated by PCR. This work provides insight into possible roles of the latency-associated ncRNAs in neuronal cell biology and latency that could aid future investigations examining how HSV-1 latency affects human neurons.
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Characterisation of Burkholderia prophages and discovery of a novel inducible phage from B. vietnamiensis G4 that is widely distributed across the species
More LessBurkholderia species have environmental, industrial and medical significance, and are important opportunistic pathogens in individuals with cystic fibrosis (CF). Approximately 10% of Burkholderia genomes (6-9 Mb) are horizontally acquired material, representing a rich source of mobile genetic elements including prophages. There is limited research on Burkholderia bacteriophages, their contributions to genome evolution, virulence and antimicrobial resistance, or biotechnological and therapeutic applications. We investigated prophage carriage in Burkholderia and aimed to isolate and characterise inducible bacteriophages from B. vietnamiensis.
Burkholderia genomes were screened for prophages using PHASTER. Prophage genomes were compared using MASH and visualised with ProgressiveMauve. Phylogenomics was used to assess the distribution of prophages across B. vietnamiensis strains. Spontaneously induced phages were characterised to determine linkage between prophage regions and isolated phages, bacteriophage morphology and host range.
Prophage carriage across 456 Burkholderia strains (spanning 43 species) was high; 716 intact prophages were discovered and polylysogeny was common. In B. vietnamiensis alone, 115 prophages were identified from 81 strains, with evidence of shared prophage carriage between related and diverse strains. Three novel inducible phages were isolated from B. vietnamiensis strain G4 and their genomic origins localised putatively. One phage (vB_BvM-G4P1; family Myoviridae) had inhibitory activity against multiple strains of 5 B. cepacia complex species, including species prevalent in CF infections.
Prophages are numerous in Burkholderia genomes and contribute to strain diversity. There is huge potential for further investigation into the functional implications of prophage carriage and its impact on genome evolution, in addition to the isolation of novel bacteriophages.
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Identification of isoprene-degrading bacteria in phyllosphere and soil communities from high isoprene-emitting oil palm trees by DNA-stable isotope probing
Isoprene is the most abundant biogenic volatile organic compound (BVOC) on Earth, with annual global emissions almost equal to those from methane. Due to its volatile nature and high reactivity, isoprene plays a complex role in atmospheric chemistry and hence, climate. However, very little is known about its biological degradation in the environment. The vast majority of isoprene (500 Tg ·y-1) is produced by terrestrial plants and oil palm is considered one of the highest isoprene-producing trees, with estimated emissions of 175 μg·g-1 dry leaves ·h-1. Oil palm is also a heavily cultivated crop since it is the source of 30% of the vegetable oil in the world and in countries such as Malaysia represents >85% of total agricultural land. The vast expansion of a single crop that emits such high amounts of isoprene have raised serious concerns about its impact on air quality and climate change. We performed DNA Stable Isotope Probing (DNA-SIP) to study the isoprene-degrading community of oil palm trees in a Malaysian plantation and identified novel genera of isoprene-utilising bacteria in both oil palm soils and leaves. isoA amplicon sequencing data also confirmed that oil palm trees harbour a novel diversity of isoA genes, which encode the alpha subunit of the isoprene monooxygenase, a key enzyme in isoprene metabolism. In addition, metagenome assembled genomes (MAGs) were reconstructed from metagenomes from oil palm soil and leaf incubations and analysed to identify isoprene degradation gene clusters in these microorganisms. Finally, analysis of unenriched metagenomes showed that isoA-containing bacteria are more abundant in soils than in the oil palm phyllosphere.
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Peroxynitrite is essential for the initiation of cytomegalovirus replication in vitro and in vivo
Human cytomegalovirus (HCMV) is a pathogenic beta-herpesvirus that establishes a lifelong infection in hosts. It causes significant morbidity and mortality in the immunocompromised and is associated with a range of birth defects following congenital infection. Current therapeutic approaches that target key viral proteins are toxic and antiviral drug resistance is common. Thus, targeting host genes and cellular pathways essential for HCMV infection offers an alternative strategy for the development of antivirals. Here we show that host oxidative/nitrosative stress responses to CMV are critical for virus replication. Oxidative/nitrosative stress occurs due to accumulation of reactive oxygen/nitrogen species (ROS/RNS). Using a range of ROS/RNS scavengers, we identified that peroxynitrite, a powerful oxidant and nitrating agent, dramatically promoted virus replication in both in vitro and in vivo models of CMV infection. HCMV rapidly induced production of intracellular peroxynitrite upon infection. Inhibition of peroxynitrite within the first 24 hours alleviates efficient HCMV infection in both cell-free and cell-associated infection systems, indicating that peroxynitrite may influence pathways necessary for HCMV entry and/or replication. Furthermore, peroxynitrite inhibition also inhibited HCMV reactivation from latency. Interestingly, the neurotransmitter and naturally-occurring peroxynitrite antagonist 5-hydroxytryptamine, commonly known as serotonin, also impinged on HCMV-induced peroxynitrite production and exhibited anti-HCMV activity. Thus, overall, our study demonstrates a novel role for intracellular peroxynitrite in CMV pathogenesis and implies that peroxynitrite could be targeted as a novel approach to inhibiting CMV infection.
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Characterisation and identification of STEC O157 pathogenicity using machine learning
More LessShiga toxin-producing Escherichia coli O157: H7 (STEC) is a zoonotic pathogen that is globally dispersed, causing severe gastroenteritis when transmitted from ruminants to humans through direct or indirect contact with animals, their environment or contaminated food. Symptoms are varied in severity; from mild to bloody diarrhoea with more serious sequalae including hemolytic uremic syndrome (HUS) which can be fatal. Although there is compelling evidence that the Shiga toxin sub-type is a key predictor of disease severity, differences in virulence potential of strains with the same Shiga toxin profile are often observed. In this study, we employ machine learning algorithms to explore the relationship between the STEC genome with clinical outcome.
Kmer-counts of variable length (9-100 base pair) from 1148 isolates of STEC O157:H7, representing two years of routine surveillance in England, were matched to their respective clinical outcome data. A Random Forest classifier was developed and validated with the objective of inferring the clinical symptoms associated with a given STEC genome. Clinical outcomes were categorised into asymptomatic, diarrhoea, bloody diarrhoea and HUS. The model correctly classified 160 out of 190 cases of bloody diarrhoea, 81 out of 128 cases of diarrhoea and 7 out of 12 cases of HUS, with average AUC ROC score of 90%. Kmers deemed important for distinct classification were characterised and matches related to Shiga toxin 2a phage integration and excision genes and adhesion and transporter proteins were identified. This is consistent with reported virulence factors in the literature, supporting this approach of de novo pathogen characterisation.
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Evidence that Chlamydia abortus vaccine strain 1B produces indistinguishable placental lesions to wild type strains
Chlamydia abortus is the most commonly diagnosed cause of abortion in small ruminants around the world [1]. Control of chlamydial abortion is achieved in several European countries using an attenuated live 1B C. abortus vaccine strain, which can be distinguished from virulent wild-type strains by PCR-RFLP analysis [2]. Application of this method has provided molecular evidence that the 1B strain can cause abortion in ewes [3, 4]. The objective of this study was to define the distribution of lesions and bacterial load in cotyledons from ewes vaccinated with the 1B strain compared to normal wild-type infections.
A Chlamydia-free flock of 75 multiparous adult ewes were vaccinated twice, two years apart, each prior to mating, with the commercial 1B vaccine. In the second lambing season following the last vaccination, placentae (n=116) were collected and analysed by C. abortus real-time qPCR [3]. Only two of the placentae, both from the same ewe, were found to be positive. Viable organisms were isolated from these placentae and confirmed by RFLP-PCR [3] to be vaccine-type. All cotyledons from these placentae were analysed by histopathology and immunohistochemistry [5], and compared with those from wild-type infected placentae. The lesions in the vaccine-type infected placentae were indistinguishable from the wild-type infected placentae in terms of their severity, load and distribution.
These results suggest that the 1B vaccine strain of C. abortus can cause chlamydial abortion in ewes producing typical placental lesions to wild-type infected animals, and could be circulating with the potential to cause natural infection and disease.
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3D co-cultures of epithelial cells with immune cells as a model of HSV and oncolytic HSV infections
More LessVirus infectivity is commonly investigated with in vitro monolayer cell cultures or in vivo animal models. Ease of growth and manipulation and low cost characterise standard cell culture. Animal models allow investigation of infectivity in the context of tissue structure and environment but are costly and can be limited by species variations. Neither approach can recapitulate the human context, the tissue microenvironment and human immune components. Three-dimensional organotypic raft tissue models can provide most of the advantages of in vitro and in vivo models.
We successfully established HSV and oncolytic HSV (HSV1716) infection in 3D raft cultures of epithelial non-tumour (HaCaT) and tumour (SiHa, OVCAR3 and TOV21G) cell lines. Our 3D models allowed the evaluation and quantification of virus replication and the recovery of the virus both in culture media and tissues.
We developed a complex 3D co-culture of epithelial cells with human immune cells in order to mimic the tissue microenvironment. This innovation allowed us to study the effect of immune cells in cell killing by HSV1716 in the in vitro tissues. In HSV1716-infected co-culture tissues, immune cells were identified throughout the tissue and some migrated to the areas of infection. The immune activity was identified through increased IL-8 release. Moreover, combining infection with immune cell infiltration increased tumour cell killing in the 3D co-culture model. This new co-culture model could be further developed to identify the role of immune cells in oncolytic viroimmunotherapy and to dissect the involvement of specific single immune cell subpopulations.
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Artificial Intelligence and deep learning turns to antibiotic discovery
More LessDeep learning (DL) is a subset of Artificial Intelligence employing neural networks that require the use of a training set and are modelled on circuit pathways in the human brain. Whilst AI is a burgeoning field today, its roots are in the 1950s. DL algorithms use multiple layers to progressively extract higher level features from the raw input. Many different architectures of neural network exist, and for the most part are involved in applications with image recognition. Some recent uses of DL include self-driving cars; there are also creative projects using DL to create fake faces or human poses for use as models, composing music, creating novel art from different styles of art and writing fake news.
Streptomyces spp. are well-known as important producers of bioactive compounds such as antibiotics. These bioactive compounds are often encoded as secondary metabolites in the organisms by large gene clusters such as non-ribosomal peptide synthases (NRPS) and polyketide synthases (PKS). The NRPS and PKS assemble peptides using enzymatic units arranged in modules that can function in an iterative or sequential fashion independent of messenger RNA. Each NRPS or PKS is capable of assembling one type of peptide. Fusions of the two also exist.
Here we have trained deep learning neural networks to provide us with simulated “fake” secondary metabolite sequences. We examine the characteristics of these sequences and how they could be used to guide us with antibiotic discovery.
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BK polyomavirus genotypes and progression of BKV disease in renal transplant recipients
More LessBackground. BK virus (BKV) reactivation is a significant cause of BK Polyomavirus associated nephropathy (BKPyVN) resulting in acute graft rejection in 1-10% of post-renal transplant recipients. However, the association of BKV genotypes with development of BKPyVN is poorly understood. Here we aimed to determine the prevalence of BKV genotypes in post-renal transplant recipients, and its association with BKPyVN disease progression.
Method. Two methods were utilised to genotype BKV. A 800bp fragment of virus VP1 antigen region was amplified using nested (PCR) followed by sequencing. The genotypes were determined according to a previously developed algorithm based on analysing 100bp region of the VP1 gene. Furthermore, the logarithm results were validated with the constructed phylogenetic tree. The results were correlated with patient viral loads and development of BKPyVN.
Results. BK virus DNA was detected in 32 (69%) of 46 post-renal transplant recipients with BK viremia, while BKPyVN was only reported in two (4.3%)patients. 30 out of 32 samples were successfully genotyped (93.7%) with 23 (76.6%) belonging to the BKV Ib-2 subtype and seven (23.3%) belonging to the BKV Ib-1 subtype and with no cases representing genotype II, III and IV. All cases with confirmed BKPyVN matched to the BKV Ib-2 genotype. Additionally, no significance differences were observed between BKV genotypes in regards to viral loads, development of viremia, HLA mismatch, age or sex.
Conclusion. The results indicate no correlations between BKV genotypes and the development of BKPyVN. Furthermore,a high distribution of BKV genotype Ib-2 was found among BKV infected patients within this cohort.
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Safe water for all: A nature-based approach for cyanotoxin elimination from potable water
Cyanobacterial blooms are a serious threat to public health and water quality due to the production of cyanotoxins as a result of nutrient pollution from industry, agriculture, domestic waste as well as global warming. The microcystins (MCs) are the most abundant cyanotoxins consisting of >200 analogues causing both acute and chronic toxicity, sometimes resulting in death. In Asian countries, such as Sri Lanka, reports of kidney disease are constantly increasing. Although no direct link between metal and pesticide contamination in water and kidney disease has been found, high concentration of cyanobacteria cells in drinking water wells implies that the nephrotoxic effects of cyanotoxins might play a key factor in the reports of Chronic Kidney Disease of unknown aetiology (CKDu) in Sri Lanka. Therefore, we propose a nature-based approach for water treatment which will study the hypotheses that cyanotoxins can cause CKDu. Sri Lankan bacterial isolates (Alcaligens sp., Roseateles sp., Bacillus sp., and Micrococcus sp.) known to degrade microcystins, were used to form biofilm on biochar from Sri Lankan crop residues, such as coconut shells. The immobilisation of the microbes was assessed via a high-throughput colourimetric assay, followed by monitoring the biodegradation rate of microcystins when added to the immobilised cultures. Biodegradation products were analysed and identified through molecular networking and quantified via LC-MS/MS. Ultimately, this project will provide safe water in line with UN Sustainable Development Goal 6.1 as well contributing in sustainable goals 7 (Affordable and Clean Energy), 11 (Sustainable Cities and Communities) and 12 (Responsible Production and Consumption).
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How the colonic environment influences Enterohaemorrhagic E. coli outer membrane vesicle production, and the interaction between outer membrane vesicles with human host cells
More LessEnterohaemorrhagic E. coli (EHEC) may instigate bloody diarrhoea and haemolytic uraemic syndrome (HUS) due to Shiga toxin (Stx) production. Stx has been detected within outer membrane vesicles (OMVs), which are membrane-derived nanosized proteoliposomes. During colonisation, EHEC encounters many environmental surroundings such as the presence of bile salts and carbon dioxide (CO2). Here, the influence of different intestinal cues on EHEC OMV production was studied. OMV yield was quantified by densitometric analysis of outer membrane proteins F/C and A, following OMV protein separation by SDS-PAGE. Compared to cultures in Luria broth, higher OMV yields were attained following culture in human cell growth medium and simulated colonic environmental medium, with further increases in the presence of bile salts. Interestingly, lower yields were attained in the presence of T84 cells and CO2. The interaction between OMVs and different human cells was also examined by fluorescence microscopy. Here, OMVs incubated with cells showed internalisation by semi confluent but not fully confluent T84 cell monolayers. OMVs were internalised into the lysosomes in confluent Vero and Caco-2 cells, with Stx being transported to the Golgi and then the Endoplasmic reticulum. OMVs were detected within polarised Caco-2 cells, with no impact on the transepithelial electrical resistance by 24 hours. These results suggest that the colonic environmental factors influences OMV production in vivo. Additionally, results highlight the discrepancies which arise when using different cells lines to examine the intestine. Nevertheless, coupled with Stx, OMVs may serve as tools of EHEC which are involved in HUS development.
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The molecular interaction between ML336 and the VEEV non-structural proteins
More LessVenezuelan Equine Encephalitis Virus (VEEV) is a positive sense RNA virus in the family Togaviridae. VEEV circulates in the Americas, causing occasional large scale epidemics. Our group has previously discovered and described the anti-VEEV compound ML336. We found that ML336 inhibits viral RNA synthesis during infection. This RNA synthesis inhibition is highly specific for VEEV, and ML336 has no effect on the closely related chikungunya virus, or on cellular RNA synthesis. We also found that this activity was maintained in a cell-free viral RNA synthesis system, supporting our hypothesis that ML336 is a direct acting antiviral compound. We recently discovered that treatment with ML336 reduces the amount of double stranded RNA present in infected cells. This reduction supports that ML336 is interfering with the synthesis of viral RNA. This was measured qualitatively with microscopy, and quantitatively with flow cytometry. We have also reported that resistant viral mutants emerge when grown in the presence of inhibitory compounds and these mutations mapped to the N terminal domains of both nsP2 and nsP4. This region of nsP2 has recently been shown to be a helical region which serves as an accessory domain to the viral helicase. However, the region of nsP4 in question currently lacks known function. Based on this genetic data we hypothesized that ML336 and related compounds interact with these two domains to interfere with the activity of the replicase complex. We are currently examining this interaction using ectopically expressed protein.
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The Type VI Secretion System of Pseudomonas aeruginosa: a gun loaded with antimicrobial bullets
Pseudomonas aeruginosa is an opportunistic pathogen that can cause severe respiratory infections in people who are immunocompromised. P. aeruginosa possesses the Type VI Secretion System (T6SS), a bacterial weapon that injects effectors into neighbouring prokaryotes and eukaryotes. The T6SS is crucial for bacterial warfare, allowing P. aeruginosa to kill its competitors, which promotes its dominance in mixed microbial environments. P. aeruginosa has three T6SSs, H1/2/3-T6SS, these are structural homologs but deliver unique effectors. Effectors are delivered via the secreted component, a Hcp tube topped with a VgrG and PAAR spike. Only the first three identified effectors are delivered by Hcp1. Since then, there has been a bias in identification of VgrG or PAAR delivered effectors, mostly as these are encoded next to the spike proteins. Some P. aeruginosa effectors not only kill bacteria but have a dual role in pathogenesis. Our aim was to identify a comprehensive set of Hcp-delivered effectors for all three systems. Using Hcp1/2/3, systematic pull-down screens were performed to identify novel interaction partners. After confirming interaction, antibacterial toxicity was evaluated, identifying new Hcp delivered T6SS effectors for Hcp2 and Hcp3, which are toxic in the bacterial cytoplasm. These new anti-bacterial effectors may kill bacteria in novel ways, which could lead to novel antibiotics. Additionally, a toxin fusion proved too large for secretion and blocked the T6SS, revealing a Hcp-delivered effector size limit. Future work will focus on fully characterising these new toxins, as well as to look into the potential eukaryotic role of other interaction partners.
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Unravelling the requirement for host cell chloride channels during HRSV infection
More LessIon channels are a diverse class of transmembrane proteins that selectively allow ions across membranes, influencing a multitude of cellular processes. Modulation of these channels by viruses is emerging as an important host-pathogen interaction that regulates critical stages of the virus multiplication cycle including entry, replication and egress.
Human respiratory syncytial virus (HRSV) causes severe respiratory tract infections globally and is one of the most lethal respiratory pathogens for infants in developing countries, with frequent development of bronchiolitis. Furthermore, it is the most significant cause of hospitalisation of infants in the UK. Evidence also indicates that severe childhood HRSV infection contributes towards the increased incidence of adult asthma. No HRSV vaccine is available, and currently the only treatment is immunoprophylaxis which is prohibitively expensive and only moderately effective; thus new treatment options are required.
Utilising GFP-expressing HRSV in combination with an extensive panel of channel specific pharmacological inhibitors, we have identified an important role of cellular chloride (Cl-) channels during HRSV infection. Interestingly, pharmacological inhibition of specific Cl- channel families has ruled out involvement of the CFTR and instead highlighted a critical requirement for calcium-activated Cl- channels (CaCCs). Time of addition studies using CaCC blockers have indicated that these channels play a post-entry role during HRSV infection. Using genetic knockdown techniques we have isolated a single channel of interest and are now further investigating its role in facilitating HRSV multiplication, as well as assessing the importance of Cl- channels in replication cycles of other negative sense RNA viruses.
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Recombination between African and Asian lineages of Zika virus in vitro and its consequences for viral phenotype
More LessRecombination is a process of extensive genetic exchange that is known to contribute to virus evolution and has been frequently observed in positive-sense RNA viruses. Zika virus (ZIKV) is an emerging arbovirus of the family Flaviviridae with two distinct lineages – African and Asian. While some phylogenetic evidence suggests that recombination in the envelope-encoding region of the ZIKV genome has occurred during evolution, there has been no experimental evidence for ZIKV recombination to date. We conducted co-infections of mammalian and insect cells, using the prototype African ZIKV strain (MR766) and an Asian isolate from the 2015-16 ZIKV outbreak in Brazil (BeH819015), and used a recombinant-specific PCR assay to detect recombinant sequences from total cell RNA extracts. In brief, a 564bp fragment spanning the boundary between the structural and the non-structural genes of the viral genome was amplified using a primer pair consisting of an Asian-specific and an African-specific primer. A total of 24 individual sequences were screened. All were in-frame recombinants and they formed 10 unique junctions. Several of the detected recombinant sequences were chosen for construction of full-length infectious clones to test the viability and phenotype of the recombinant viruses. This study represents the first isolation of recombinant ZIKV sequences from co-infected cultured cells and demonstrates the capacity of ZIKV to recombine in an experimental system. Further investigation is required to better understand the evolutionary potential of this mechanism and its putative role in the emergence of ZIKV.
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H5N8 highly pathogenic avian influenza virus introduction risk routes in a high biosecurity floor reared poultry setting
The European H5N8 highly pathogenic avian influenza virus (HPAIV) epizootic during 2016-2017 resulted in both wild bird and poultry deaths throughout the EU. This in vivo study investigated the potential for indirect infection of naïve birds through contaminated drinking water or feed, to assess potential disease incursion into a high biosecurity commercial floor housed setting.
Three-week-old Ross 308 chickens were exposed to H5N8 A/wigeon/Wales/52833/2016 (H5N8-2016) virus at a high (1 x 106 EID50/ml) or low (1 x 104 EID50/ml) dose, in either drinking water or feed for a 24 hour period. Chickens directly-infected with a high dose of H5N8-2016 (intra-nasal) acted as positive controls. Viral shedding, environmental contamination and clinical signs were monitored for ten days post infection (dpi).
All directly-infected birds shed virus and were humanely terminated at 3 dpi. Immunohistochemical analysis of nasal epithelium and caecal tonsil lymphoid tissue, obtained at post mortem from directly-infected chickens (2 dpi), showed the presence of influenza antigen in both tissues. Only birds exposed to high dose virus in drinking water, shed virus and showed clinical disease presentation (67% mortality). Interestingly low levels of antigen were detected in the nasal epithelium, whereas higher levels were detected in the caecal tonsil.
All surviving chickens from each group, remained uninfected and did not seroconvert. Our findings suggest virus bio availability in different substrates is variable (feed and water) and possible routes of viral contamination leading to disease ingress at poultry premises may have different outcomes including disease presentation.
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Investigation of Pseudomonas aeruginosa biofilm formation within washing machines using novel nanosensor technology
More LessBiofilms are communities of microorganisms that attach to various surfaces and are widely associated with infections. Our investigation is focussed on a current and growing concern: the formation of biofilms in washing machines. Many countries wash clothes at reduced temperatures (30°C to 40°C) rather than at higher temperatures above 60°C that would kill the bacteria. Survival of the bacteria is associated with biofouling, malodour and an increased infection risk due to the distribution of human pathogens such as Pseudomonas aeruginosa, one of the predominant bacteria found in washing machines.
Little is known about environmental microniches present in biofilms. Here, we focus on the pH variation throughout P. aeruginosa biofilms knowing that the pH can influence biofilm formation and could be an important aspect for the prevention of biofilms. We use novel pH-sensitive optical nanosensors that penetrate P. aeruginosa biofilms and emit fluorescence in response to pH variation. Using time lapse imaging, pH changes were tracked in real time at a single cell level which will ultimately facilitate monitoring of environmental changes induced as biocides penetrate biofilms. We also look at the isolation and identification of P. aeruginosa from household washing machines. Whole genome analysis was performed to identify different genomic features relevant to antimicrobial resistance (AMR) and biofilm formation. Furthermore, testing of different washing detergent formulations revealed a range of abilities to disrupt biofilm formation or kill P. aeruginosa, which will facilitate the development of more effective washing agents to limit the emergence of AMR within biofilms.
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Revisiting the role of PML protein targeting and disruption of PML bodies in human cytomegalovirus infection
More LessPromyelocytic leukaemia (PML) bodies are nuclear organelles implicated in post-translational modification by small ubiquitin-like modifier (SUMO) proteins and in the antiviral host cell response to infection. The 72-kDa immediate-early protein 1 (IE1) is considered the principal antagonist of PML bodies encoded by the human cytomegalovirus, one of eight human herpesviruses. Previous work has suggested that the interaction between IE1 and PML proteins, the central organisers of PML bodies, and the subsequent disruption of these organelles serve a critical role in viral replication by counteracting intrinsic antiviral immunity and the induction of interferon (IFN)-stimulated genes. However, this picture has emerged largely from studying mutant IE1 proteins known or predicted to be globally misfolded und metabolically unstable. We systematically screened for stable IE1 mutants by clustered charge-to-alanine scanning. We identified a mutant protein (IE1cc172-176) selectively defective for PML interaction. Functional comparisons between the mutant and wild-type protein revealed that IE1 can undergo modification by mixed polymeric SUMO chains and that it targets PML and Sp100, the two main constituents of PML bodies, via distinct mechanisms. Unexpectedly, IE1cc172-176 supported viral replication almost as efficiently as wild-type IE1. Moreover, lower instead of higher (as expected) levels of tumor necrosis factor alpha, IFN-beta, IFN-lambda and IFN-stimulated gene expression were observed with the mutant compared to the wild-type protein and virus. These results suggest that the disruption of PML bodies is linked to induction rather than inhibition of antiviral gene expression. Our findings challenge current views regarding the role of PML bodies in viral infection.
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Air pollution induces an adaptive response in Staphylococcus aureus and alters host-pathogen interaction
Air pollution is a major global health problem, with around 91% of the world’s population living in areas that exceed the WHO air pollution guidelines. This complex mix of pollutants almost always includes particulate matter (PM), and this has the greatest impact on human health. PM exposure contributes to a range of diseases such as COPD, heart disease and respiratory infections. Our recent publication was the first to document that as well as damaging the host, PM has a direct impact on bacteria that can cause respiratory infections. We showed that Black Carbon (BC) exposure results in species-specific alterations in biofilm structure in both Streptococcus pneumoniae and Staphylococcus aureus, altered biofilm protectivity against antibiotic exposure, and S. pneumoniae bacterial colonisation in vivo.
Following on from this ground-breaking work, our current data show that the bacterial response to BC occurs at the genetic level, altering the transcription of key genes involved in biofilm formation, colonisation and virulence. Bacterial adhesion to and invasion of human epithelial cells is significantly increased when S. aureus are pre-exposed to BC prior to infection compared to naïve S. aureus cells. In a murine respiratory colonisation model, both S. aureus co-infected alongside BC, and crucially S. aureus pre-exposed to BC, show increased colonisation of the nasopharynx and lungs. These data suggest that the bacteria are responding and adapting to exposure to air pollution, and this has an impact on how the bacteria infect the host.
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Differences in acid-base regulation of haploid and diploid life-cycle stages of Coccolithus braarudii and their consequences for the sensitivity towards ocean acidification
More LessCoccolithophores are calcifying microalgae that carry characteristic calcite platelets (coccoliths) on their surfaces. Most coccolithophore species exhibit diploid and haploid life cycle stages, each adjusted to different environmental conditions. The diploid life cycle stage of the coccolithophore C. braarudii is heavily calcifying with calcification rates that exceed the rates of photosynthesis. Haploid life-cycle stages are often weakly calcifying, generating significantly less H+ from the intracellular calcification reaction. We show how these different cellular “H+ burdens” require substantially different physiological molecular strategies to regulate intracellular pH under changing environmental conditions. Voltage-gated H+ channels (Hv) have been shown to play a role in the release of H+ in the diploid life cycle previously (Taylor et al. 2011). Combining scanning electron microscopy, electrophysiology, gene expression approaches and physiological measurements, we here show a direct link between the function of proton channels and coccolith formation of the diploid but not the haploid life-cycle stage. Our data also indicate how the different mechanisms for acid-base regulation of the diploid and haploid life-cycle stages may result in different sensitivities towards ocean acidification.
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RNA-seq provides molecular insights into interaction modification in simplified microbial community
More LessA major challenge in microbial ecology is to understand the stability of interspecies interactions when progressing from pairs of interacting species to multispecies interaction networks. A lack of direct evidence, and a conceptual framework to explore how direct and indirect effects shape cellular responses in species-rich networks has hindered progress in our understanding of these combined effects. Here we aimed to investigate whether higher-order interactions shape community dynamics and transcriptional profiles of all interacting partners in a simplified microbial community that includes a primary producer (Nannochloropsis oceanica CCAP849/10) and two heterotrophic bacteria (Marinobacter sp. FDB33 and Alteromonas sp. FDB36). By combining co-cultivation assays, quantification of absolute abundances, nutrient analysis, and simultaneous RNA-sequencing, we reveal genome-wide transcriptional responses in all binary co-cultivation partners and show that the third partner can profoundly alter binary interactions at the phenotypic and transcription level. Our study demonstrates the context-dependency of binary interactions, whereby environmental conditions and the presence of specific organisms can affect the cellular physiology of the interacting partners and ultimately the stability of the community. Furthermore, our approach provides a powerful tool for probing the molecular basis of emergent properties in more complex systems.
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Investigating the mechanism of the Tup1-Cyc8 (Ssn6) Co-Repressor complex in the yeast Saccharomyces cerevisiae
More LessThe Tup1-Cyc8 (Ssn6) complex is a powerful epigenetic repressor of genes in the yeast Saccharomyces cerevisiae. The highly conserved complex brings about a repressive chromatin structure at regulatory regions of its target genes or prevents the recruitment of factors needed for activation of transcription. A gap in the current understanding is whether each of the subunits contribute differently to repression. The FLO family of genes are repressed by the Tup1-Cyc8 complex, these genes encode the proteins required for flocculation, a stress response in yeast where the cells aggregate, or form flocs, to protect cells within the floc. Interestingly, each mutant strain has a distinct flocculant phenotype. The tup1Δ strain displays large, dense flocs compared to smaller, more dispersed flocs associated with the cyc8Δ strain. RT-qPCR showed that FLO1 is highly de-repressed in the tup1Δ strain whereas it is de-repressed to a significantly lower level in the cyc8Δ strain. Using the Anchor Away (AA) technique, which allows for a nuclear protein to be conditionally sequestered to the cytoplasm, I am investigating differences in the sequence of events at the FLO1 promoter when Tup1p or when Cyc8p is removed from the nucleus. Six hours after Cyc8p is removed from the nucleus transcription of FLO1 almost reaches the maximum transcription seen in the cyc8Δstrain. However, six hours after removing Tup1p the level of transcription of FLO1 is still over ten times lower than the maximum transcription in tup1Δ. This difference indicates that each of the subunits have independent functions within the complex.
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From waste to bioplastics: bio-based conversion of volatile fatty acids to polyhydroxyalkanoates
More LessProduction of polyhydroxyalkanoates (PHAs) as bio-alternative to petroleum-based plastics is an important field in the biorefinery to move forward in the development of the circular economy. PHAs are bioplastics stored inside microbial cells as carbon reservoirs and can be produced from a broad range of renewable resources such as waste streams. One important waste stream is food waste that can be converted into volatile fatty acids (VFAs) by anaerobic digestion. The produced effluent from food waste is not only rich in VFAs but also, other nutrients such as nitrogen and phosphorus that can be used by the microorganisms to produce PHAs. The aim of this research is to convert VFAs produced from food waste into PHAs, in which two approaches have been studied. The first approach was to use microbial mixed cultures (MMCs) while the second used microbial pure cultures.
The MMCs were enriched in sequencing batch bioreactor cultivations, where nitrogen and carbon starvation were combined to enhance the selection phase. PHA accumulation of the selected cultures was studied in nitrogen-limited fed-batch cultivations. The second approach studied five different PHA producing bacteria: Cupriavidus necator, Burkholderia cepacea, Bacillus megaterium, Bacillus cereus and Bacillus cereus. To select the most promising bacteria, synthetic medium with the same VFAs composition as in MMCs study was used for pre-screening experiments. Both, pure and mixed culture studies, resulted in the production of PHAs containing (R)-3-hydroxybutyrate, (R)-3-hydroxyvalerate and (R)-3-hydroxyhexanoate as monomers and VFAs were consumed with a high rate by the microorganisms.
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Synthrophic interactions between Bacteroides and Bifidobacterium on yeast beta-glucan
More LessThe Human Gut Microbiota (HGM) comprises two major phyla, the Bacteroidetes and Firmicutes, although important members of the Actinobacteria (Bifidobacterium) and Verrucomicrobia (Akkemansia) also make an important contribution to this ecosystem.
Accumulating datasupport the notionthat the HGM can be modulated by probiotics and prebiotics to prevent or revert common diseases of the gastrointestinal tract (GIT) such as Inflammatory Bowel Disease. Because it is believed that these GIT diseases are linked to the fact that current Western populations follow a more fat-based diet, significant efforts have been made to search for novel prebiotics/probiotics in order to restore and improve gut health.
So far, no publications have described probiotic properties of Bacteroidetes. Nonetheless, a case can be made that certain Bacteroides species present primary glycan degraders that interact in a syntrophic manner with other members of the microbiota, such as bifidobacteria, which are considered beneficial members of the microbiota.
In this study, we present the simbiotic interactions between Bacteroides and Bifidobacterium spp. acting on yeast beta-glucan (1,3/1,6 mixed linkage beta-glucan). Bacteroides cellulosilyticus and Bacteroides ovatus act as keystone organism to share beta-1,3/1,6-glucooligosaccharides with other members of the HGM, including Bifidobacterium breve UCC2003 and Bifidobacterium bifidum. We show in these Bifidobacterium spp. a specific beta-1,3-glucosidase, which degrade some of these sharing oligosaccharides. Also, we have identified the specific sugar symporter, which incorporate these oligosaccharides into the cytoplasm of B. breve UCC2003. With the help of RT-qPCR, we have quantified and monitored how these two members of the HGM are able to symbioticly use this dietary glycan.
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Elucidation of environment dependent antibiotic resistance mechanisms
The propensity of pathogens to evolve resistance to antibiotics used in clinical infectious disease therapeutics has been an increasing concern in recent decades. Acquisition of resistance often translates into treatment failure and puts patients at risk of serious adverse outcomes. Current laboratory testing of antibiotic susceptibility does not account for the different microenvironments that bacteria encounter within the human body, providing results that often do not translate into the clinic. Our goal is to better understand evolutionary strategies employed by Staphylococcus aureus in development of resistance in distinct environments.
We used adaptive laboratory evolution (ALE) to generate isogenic strains resistant to several antibiotics. Different media were used to mimic distinct environments and multi-omics approaches applied in the understanding of resistance mechanisms.
Evolved strains presented phenotypes similar to those observed in clinical resistant isolates. Mutational analysis indicated that resistance was specific and condition-dependent. Distinct mutations led to resistance phenotypes under a particular environmental condition, but these mutations did not necessarily translate into resistance under a different environmental condition. Furthermore, resistant strains possessed distinct transcriptional landscapes, even when the same systems were mutated, suggesting that similar evolutionary paths translate into distinct resistance mechanisms.
We identified several resistance mechanisms employed by S. aureus that were not only environment-dependent, but also environment specific. Additionally, we showed that ALE can be applied in pathogens of interest to study antibiotic resistance evolution and prediction of clinical resistance mechanisms, as supported by the significant overlap of mutations identified via ALE and those reported in clinical isolates.
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M1CR0B1AL1Z3R—a user-friendly web server for the analysis of large-scale microbial genomics data
More LessLarge-scale mining and analysis of bacterial datasets contribute to the comprehensive characterization of complex microbial dynamics within a microbiome and among different bacterial strains, e.g., during disease outbreaks. The study of large-scale bacterial evolutionary dynamics poses many challenges. These include data-mining steps, such as gene annotation, ortholog detection, sequence alignment, and phylogeny reconstruction. These steps require the use of multiple bioinformatics tools and ad-hoc programming scripts, making the entire process cumbersome, tedious and error-prone due to manual handling. This motivated us to develop the M1CR0B1AL1Z3R web server, a ‘one-stop shop’ for conducting microbial genomics data analyses via a simple graphical user interface (Avram, et al., Nucleic Acids Res., 2019). Some of the features implemented in M1CR0B1AL1Z3R are: (i) extracting putative open reading frames and comparative genomics analysis of gene content; (ii) extracting orthologous sets and analyzing their size distribution; (iii) analyzing gene presence-absence patterns; (iv) reconstructing a phylogenetic tree based on the extracted orthologous set; (v) inferring GC-content variation among lineages. M1CR0B1AL1Z3R facilitates the mining and analysis of dozens of bacterial genomes using advanced techniques, with the click of a button. M1CR0B1AL1Z3R is freely available at https://microbializer.tau.ac.il/ [https://microbializer.tau.ac.il/].
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