- Volume 2, Issue 7A, 2020

Starting 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.

This 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.

Viral 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.

Tuberculosis 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.

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.

Gene 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.

Accessory 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.

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.

As 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.

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.

Cells 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.

Rinderpest, 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.

The 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.

The 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.

Myxobacteria 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.

Several 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.

The 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.

Varroa 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.

Alphaviruses 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.