- Volume 1, Issue 1A, 2019

Streptomyces species are well known for harbouring a large number of extracytoplasmic function (ECF) RNA polymerase sigma (σ) factors; nearly all of regulated genes required for morphological differentiation and/or response to environmental stress. The activity of ECF σ factors is typically modulated by a cognate anti-σ factor protein co-encoded at the same locus. In previous work, we identified σAntA as a cluster-situated regulator of starter unit biosynthesis in the production of antimycin, an anticancer compound. Unlike a canonical ECF σ factor, whose activity is controlled by a cognate anti-σ factor, σAntA is an orphan, which raises intriguing questions about how its activity is controlled. Inspection of the σAntA amino acid sequence revealed a carboxyterminal di-alanine, which is the only motif of the Clp-protease recognition signal obligately required for proteolysis. Here, we show by immunoblotting that the abundance of 3xFLAG-σAntA in vivo is enhanced by alteration of the C-terminal Ala-Ala to Asp-Asp and that abundance of both variants is elevated in the absence of genes encoding the Clp-protease and its regulatory subunits ClpX and ClpA. We also show that (His)6-SUMO-σAntA, but not a variant lacking the C-terminal di-alanine motif, is degraded by the ClpXP protease in vitro. These data unambiguously establish direct proteolysis as an alternative control strategy for ECF RNA polymerase σ factors and expands the paradigmatic understanding microbial signal transduction regulation.

Non-tuberculous mycobacteria (NTMs), such as Mycobacterium abscessus and chimaera, can cause high mortality and morbidity amongst patients who are immunocompromised or have chronic lung diseases, such as cystic fibrosis. Mycobacterial biofilms can form in the alveolar walls of such patients following inhalation from environmental reservoirs. Recently, pathogenic strains have been isolated from shower heads and hospital air conditioning units. Biofilms are notoriously difficult to eradicate and are associated with the development of increased antimicrobial resistance (AMR). Treatment for M. abscessus and chimaera infections often requires 2–3 antibiotics over 2 years. The question we want to address is whether the increased AMR and treatment time in NTM biofilm formation is due to lack of antibiotic penetration, resulting in non-therapeutic and AMR-generative levels, or the development of phenotypic and/or genetic resistance. In this project we will use Nano-SIMS (nano-scale secondary ion mass spectrometry) to measure penetration of the antibiotic bedaquiline (BDQ), used to treat NTM infections, into individual cells of NTM biofilms (M. abscessus and M. chimaera). Nano-SIMS maps the relative abundance of different ions down to the nano-scale and can be used to measure in profile through the biofilm. In addition, the minimum inhibitory concentration (MIC) and minimum duration for killing (MDK) of BDK will be measured to determine antibiotic susceptibility in biofilms and a planktonic growth control. Understanding the AMR generation and prolonged treatment in NTM biofilms could lead to improved mortality and morbidity. The development of novel treatment strategies could enhance treatment efficacy, reduce treatment duration and AMR development.

Bacterial antibiotic resistance is widely regarded to be one of the most pressing threats facing humanity. Finding new antibiotics is a vital research area and can now be supported by a vast reservoir of readily available ‘omic data on the back of the explosion of low cost sequencing technologies. Antimicrobial Peptides (AMPs): endogenous peptides that provide a fast and effective means of defence against pathogens as part of the innate immune response. The detection of AMPs in metagenomic data is a tantalising low-hanging fruit for computational biologists. Large reservoirs of existing sequences exist and are well annotated and understood. Post-computational wet-lab work is relatively cheap with spot synthesis of peptides cheaply available from a wide array of third party companies. A well organised screening program can screen 100 s of prospects a day against model bacterial organisms to test for activity and is one of the few areas of biological science that can scale to meet the data output from computational prediction toolkits. AMPLY, an in-house tool designed at Aberystwyth University, supported by Life Science Wales and working in collaboration with Tika Diagnostics at St. George’s Hospital (London) and Queen’s University (Belfast) is part of a next wave of computational drug discovery platforms and is already uncovering a treasure trove of novel AMPs in diverse microbial environments. However, AMPLY’s abilities extend beyond the analysis of ‘omic data alone and its predictive modelling has extracted a therapeutically viable novel AMP built from a string of ancient Welsh poetry.

Klebsiella pneumoniae (Kp) is an infamous cause of multi-drug resistant (MDR) healthcare-associated infections and several MDR clones are globally distributed. A small number of drug-susceptible clones have also become globally distributed, causing severe community-acquired infections. These ‘hypervirulent’ clones are distinguished by expression of highly serum-resistant K1/K2 capsules, plus high prevalence of acquired virulence determinants. While hypervirulence and drug resistance are usually mutually exclusive, there are now increasing reports of convergent strains that are both highly virulent and MDR – a potentially disastrous combination. To better understand the risks of MDR-virulence convergence, we leveraged a collection of >2200 Kp genomes to identify 28 common clones (n≥10 genomes each), and performed a genomic evolutionary comparison. Eight MDR and 6 hypervirulent clones were identified by acquired resistance and virulence gene prevalence. Chromosomal recombination, capsule locus diversity, pan-genome, plasmid and phage dynamics were compared. MDR clones were highly diverse, with frequent chromosomal recombination generating extensive capsule locus diversity. Additional pan-genome diversity was driven by frequent acquisition/loss of both plasmids and phage. In contrast, chromosomal recombination was rare in the hypervirulent clones, which were each associated with only a single capsule locus and showed significant reduction in pan-genome diversity, largely driven by a reduction in plasmid diversity. These data suggest that hypervirulent clones are subject to some sort of constraint for horizontal gene-transfer. Hence we predict MDR clones pose the greatest risk for MDR-virulence convergence because they are more likely to acquire virulence genes than hypervirulent clones are to acquire resistance genes.

Enteropathogenic E. coli (EPEC) were the first E. coli strains linked to human disease (1945), and pose a serious health threat. The burden for public health changed over the past century as it did for other diarrheal diseases; whilst it is still endemic in large parts of South America, cases in Europe are commonly associated with recent travel. Despite its long-standing importance for global health and its significant impact on child mortality in Low- and Middle Income Countries (LMICs), we still have very limited understanding of what defines an EPEC beyond the diagnostic LEE island, or indeed if there are any shared characteristics, as most in-depth analyses of their pathogenic determinants are confined to few model strains. We present a study analysing ∼1300 whole-genome sequences combining published and newly sequenced datasets of EPEC and non-EPEC strains to map the evolutionary history and molecular determinants. Importantly, we have expanded the published data with whole-genome sequences of 300 historical and contemporary EPEC strains from a large collection of clinical isolates, mainly from Brazil and England, which enables us to compare the epidemiology in a high-income with a LMIC over an extended time frame. We furthermore present a molecular definition of EPEC, including mapping of phage islands and de-novo prediction of effectors in this large-scale dataset, as well as investigating the patterns of adhesins and other secretion systems, thus characterising different EPEC lineages which have emerged numerous times during the evolution of E. coli.

Throughout evolution, evolving objects (domains, genes, operons etc.) have continuously combined, forming new proteins, gene clusters, and genomes. Horizontal gene transfer, particularly among prokaryotes, has facilitated this combinatorial process. Thus, evolving objects that interact positively or synergistically with each other are expected to co-occur more often than by chance; conversely, evolving objects may avoid co-occurrence, indicating an antagonistic or redundant functionality between objects. In this work, we use methods adapted from graph theory to understand patterns of co-occurrence and exclusion in prokaryotes. We have implemented multi-level graph models in which each node (vertex) is a gene or species connected by an edge (relationship) to another node to display these coincidence relationships. Our method incorporates the phylogenetic distribution and synthenic distances of these genes, and we demonstrate how these concepts can be used to identify conserved clusters of vertical and horizontally inherited units of selection. We apply these multi-level graph models to a variety of datasets including prokaryotic pangenomes, and metagenomic sequencing datasets from human-associated microbial communities. We find evidence for genes that significantly co-occur with each other within each of these datasets; these genetic clusters include objects from characterized biological pathways but also include genes with unknown functions. Further, we identify genes that exclude each other, indicating evolving objects with antagonistic or redundant biological functions. This work represents a different approach to understanding the evolution of prokaryotes and allows us to draw novel hypotheses as to the potential role of these genetic clusters in prokaryote biology.

Pangenome analysis can help reveal functional and physiological differences between organisms by linking genes present in their genomes with phenotypic characteristics. While this works well for closely-related organisms and large numbers of isolates, analysis of species with a large pangenome may be problematic as optimal sample size increases with intraspecies diversity. Organisms such as E. coli exhibit high intraspecies diversity, resulting in an exceptionally large pangenome, pushing the limits of what constitutes a species. However, the phylogeny of the mobile elements can differ strongly from the phylogeny of the rest of the genome, reflecting how rapidly these elements can be gained or lost. We suggest when studying adaptations on a short genetic time-scale (ie, within species or strain), the mobile regions of the genome may be more informative than the more stable parts of the genome. To this end, we developed a pipeline, Horizontally Acquired Partial Pangenome of Inserted Elements (or ‘happie’), which allows researchers to study the mobile pangenome. Thus, happie could be used as a proxy to detect genes associated with a particular trait amidst the noise of a diverse genetic background. We aim to use happie to compare a panel of known soil-persistent E. coli isolates and a curated panel of non-soil-persistent isolates. This will allow us to determine whether specific mobile elements (or genes carried on those elements) are associated with soil persistence, which would indicate that the cost of harbouring the mobile element was less than the advantage conferred.

Systemic salmonellosis encompasses typhoid and paratyphoid fever, and invasive non-typhoidal salmonellosis, with high mortality and morbidity amongst children and the immunocompromised in low-resource settings. Immunisation efforts remain hampered by the unavailability of safe vaccines with cross-protectivity against causative Salmonella strains. Characterisation of the within-host Salmonella dynamics in the naïve and immunised host can elucidate the mechanisms by which different vaccine types exert their protective effect, and help in vaccine selection and design. Experimental data tracking the changes in bacterial population composition in the different tissues of the host at different timepoints can be coupled with mechanistic mathematical models to estimate the parameters governing the processes of bacterial replication, killing and inter-organ migration. Using a recently described minimisation-divergence estimation approach, we extend a three-compartmental mechanistic model and re-analyse existing datasets to better characterise the bacterial migratory processes between the blood, liver and spleen in the early stages of infection, and the overall Salmonella dynamics in the later phases in the naïve host. We apply the same model to published data from mice immunised with either a live-attenuated or killed whole-cell vaccine to identify their in vivo differential impacts on Salmonella migration, replication and death. Finally, we identify alternative experimental designs to improve the statistical qualities of the mathematical model and allow better inference of parameters governing the unobserved processes of bacterial dynamics.

Infections account for >20 % of all deaths worldwide, and are particularly problematic in low-middle income countries (LMICs), with bacterial infections killing approximately 5 million people annually. The crisis of antimicrobial resistance means our options for controlling infections are narrowing. Vaccines are a cost-effective approach to prevent infectious disease. However, there are many bacterial infections against which we lack any licensed vaccine. The BactiVac Network (birmingham.ac.uk/bactivac), was launched in August 2017 and is led by Profs Cal MacLennan and Adam Cunningham. BactiVac is a global bacterial vaccinology network established to accelerate the development of vaccines against bacterial infections, particularly those relevant to LMICs. BactiVac brings together academic, industrial and other partners involved in vaccine research against human and animal bacterial infections from the UK and LMICs. The Network fosters partnership and provides catalyst pump-priming project and training funding to encourage cross-collaboration across our membership. Full details are available on our website. BactiVac has >600 members across >60 countries with 39 % based in LMICs and 10 % in industry. Membership is free – join us at bit.ly/apply BactiVac Benefits of membership include: Access to catalyst funding – third round for pump-priming projects closes 5 May 2019, open call for training awards Invitation to subsidised Annual Network Meetings–next meeting will be held in March 2020 Access to Members’ Directory – to develop collaborations and access distinct expertise. We encourage you to become a member, be involved in this initiative, and to be part of its growing success now and in the future.

Reverse vaccinology 2.0 (RV 2.0), in which the cloning and recombinant expression of antigen-specific antibodies is followed by determination of their functional activity, is a valuable approach that can unravel novel vaccine antigens, or reinforce the vaccine candidacy of already known antigens. Invasive meningococcal disease (IMD) remains a serious source of concern even with the availability of vaccines. Incomplete strain coverage is a limitation of current vaccines, hence efforts to identify candidate antigens that will compose supplementary or replacement vaccines are necessitated. In this proof-of-principle study, we sought to assess the applicability of RV 2.0 to anti-meningococcal vaccine antigen discovery. Antibody-secreting cells (ASCs) obtained from a patient convalescing from serogroup B (MenB) IMD, were isolated and sorted singly using FACS. The specificity and functionality of each antibody produced by individual ASCs were assessed in ELISA and bactericidal assays, respectively. Eight cross-reactive anti-meningococcal antibodies were successfully cloned; three of these mediated complement-dependent killing of antigenically-heterologous MenB strains. Western blot data shows binding of these three bactericidal antibodies to a ∼35 kDa antigen. None of the three bactericidal antibodies were reactive with a target in current vaccine formulations strongly suggesting that the ∼35 kDa antigen does not compose available vaccines. Unequivocal determination of the identity of the ∼35 kDa antigen is ongoing. Given the need for antigens that would compose improved or novel anti-meningococcal vaccines, this study shows that the RV 2.0 approach has the potential to be a powerful tool in the identification of functionally-immunogenic anti-meningococcal antigens.

Extra-intestinal pathogenic E. coli (ExPEC) is one of the leading causes of bacteraemia and urinary tract infections (UTI) worldwide. At present, there is no licensed vaccine available and with the increasing incidence of multidrug resistance (MDR) the demand for developing alternatives to antibiotics is paramount. We have developed in vitro functional antibody assays to fast track screening of patient convalescent sera from ExPEC infections to identify potential vaccine antigens. A functional immunological assay will also facilitate the assessment of vaccine candidates against the many disease-causing serotypes circulating in the population. Outer membrane vesicles (OMVs) purified from a mutant (ΔlpxM) MDR O25 (ST131) strain were used to immunise mice to raise antiserum against the bacterial strain. Immunised mice were protected in a mouse model of E. coli bacteraemia. The antiserum from protected animals was used to develop an antibody-mediated bacterial killing assay and a complement deposition assay (CDA). The antiserum mediated killing with human IgG and IgM-depleted plasma as the complement source, and also showed deposition of C3b and C5b-9 onto the bacterial surface determined by flow cytometry. This may be a useful screening tool for mining for new antigens and in assessing the antisera raised against the candidates for cross serotype protection.

Campylobacter is the leading cause of food-borne gastroenteritis worldwide and the most common cause of infection in humans have been linked to the consumption of contaminated poultry meat. Previous poultry vaccines have provided short term reduction in Campylobacter intestinal load in chickens but have limited commercial efficacy because of high diversity and rapid evolution of strains. Genome-wide association study (GWAS) of isolates from humans and chicken have identified genes associated with survival of Campylobacter through the poultry processing chain. These genes represent targets for vaccine design that would be selectively neutral within the chicken gut and therefore could be sustained in the population. We designed an autogenous vaccine based on isolates with survival-associated genes and monitored the efficacy in the chicken and through processing. First, we sampled for Campylobacter across five broiler farms feeding into one abattoir in Norfolk and characterized genomic diversity using next generation sequencing (NGS), including the identification of survival genes. These data were then used in a predictive model to identify the minimum number of strains to be used in vaccine design based on known immunogenicity of the strains. Second, the vaccine was developed and given at two time-points to a whole farm of breeder chickens. Third, vaccine efficacy was monitored at three time points post-vaccination in vaccinated breeders and their progeny. The cfu/g reduction in the ceacum and neck skin were determined. This study demonstrates the potential for NGS in autogenous vaccine design to reduce harmful strains of bacteria that are carried commensally in livestock.

This study determined the in-ovo antiviral effect of crude Methanolic leaf extract of Cymbopogon citratus on Newcastle disease virus. Cold extraction was carried out using analytical grade methanol. Phytochemical screening of the crude extract was carried out using standard procedures. Antiviral assay was carried out in nine-day old specific pathogen free embryonated hens’ eggs in three designs made of five eggs per group (Virus with extract, virus only and un-inoculated groups) with concentrations ranging from 12.5 to 100 mg ml−1. Egg toxicity of the extract was determined for concentrations of 12.5, 25, 50, 100, 200, 300 and 400 mg ml−1. Inoculated eggs were incubated at 37 °C and observed daily for 96 h for embryo survival and mortality. Spot haemagglutination was carried out on bacteria-free allantoic fluid from the embryonated eggs to detect the presence of the virus. Phytochemical assay revealed the presence of saponins, flavonoids, steroids, terpenes, phlebotannins and terpenoids. Mild toxicity was observed at concentrations of 100 mg ml−1 and above. There was no haemagglutination of fluid from the eggs inoculated with a combination of Virus and extract at concentrations of 50 and 100 mg ml−1. The current findings demonstrated that leaf extract of Cymbopogon citratus has potential medicinal value as well as antiviral activity against Newcastle disease virus in-vivo. The specific mechanism of action remains to be studied to further elucidate on its potential as a therapeutic product for the treatment of Newcastle Disease.

This was a cross sectional and observational study, conducted in the Department of Microbiology, Sylhet MAG Osmani Medical College, Sylhet. The study period was from July 2010 to June 2011 in objective to explore the Varicella-Zoster virus immune status (VZV-IgG) in pregnant mothers and neonates up to six months post birth. For this purpose, 60 pregnant women, 60 new born babies and 60 infants aged six months were selected. The mean age of the pregnant woman was 28.8 (SD±4.7) years. The sex of new born babies and six months aged infants was identical [29 (48.3 %) male vs 31 (51.7 %) male; P=0.715]. Seroprevalence rate of IgG antibody of Varicella-Zoster Virus in Pregnant, mothers 81.7%, in new born babies 78.3 % and in infants aged six months were 10.0 %. The seropositivity of VZV IgG level in both pregnant mothers and newborn babies were almost similar but the infant aged six months were significantly lower than that of new born babies (p This showed that a significant proportion of Bangladeshi pregnant mother is susceptible to varicella and infant aged six months is highly susceptible to varicella. Any vaccination strategy must have to take into account these epidemiological variability of the country.

The lyssavirus genus is a diverse group of viruses all capable of causing an invariably fatal disease known as rabies, most commonly caused by the prototype species rabies virus (RABV). Alongside RABV the lyssavirus genus currently contains 15 other viruses capable of causing rabies. These viruses are broadly categorised into phylogroups according to the predicted level of vaccine protection, with protection from current vaccines and therapeutics afforded against phylogroup I but not II or III. Current evidence suggests that for a protective neutralising antibody response against RABV a neutralising antibody titre of 0.5 IU ml−1 is sufficient. This arbitrary value has been developed and promoted as a serological cut-off based on the reactivity of defined sera with a standardised dose of RABV. Studies using cross protection assays, have suggested that for protection against more divergent members of the genus, even those in phylogroup I, 10-fold or greater than the 0.5 IU ml−1 antibody titres are required. The continued discovery of novel lyssaviruses globally warrants an in-depth assessment of the protective titre required to protect against all the lyssaviruses to inform occupationally high-risk groups (e.g., scientists, bat workers and speleologists). Based on live virus neutralization assays, a minimum of 7 distinct lyssavirus glycoprotein antigens would have to be included in any pan-lyssavirus vaccine. Certainly, representative immunogens from all lyssavirus species characterized in phylogroups II and III are required to stimulate a pan-lyssavirus response.

Avian influenza is highly contagious poultry disease and the mortality can reach 100 %, leading to huge economic burden and threat to food security. Vaccination is the most effective strategy for prevention and control of influenza. Recombinant vector vaccines are effective promising vaccines capable of immunizing against multiple pathogens. Traditional methods for constructing recombinant vaccines involve homologous recombination or bacterial artificial chromosomes but these methods can be time-consuming and labour-intensive. The clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system is a recently developed gene editing technology which has proven beneficial to gene modification and offers an alternative for constructing recombinant vaccines. There are mainly two methods used for gene insertion; error-prone non-homologous end joining (NHEJ) and the high-fidelity homology-directed repair (HDR) pathway. Owing to its high fidelity, most studies focus on using HDR for vaccine development but NHEJ offers some attractive advantages through its high efficiency. In our study, both HDR and NHEJ dependent CRISPR/Cas9 systems were explored for the rapid generation of recombinant influenza vaccines using duck enteritis virus and herpesvirus of turkeys as vectors.

Arbidol (arb, umifenovir) is used clinically in several countries as an anti-influenza virus drug. We have previously shown that arb inhibits many viruses including hepatitis C virus, Ebola and Zika, and that the primary mode of action appears to be via inhibition of virus entry and/or fusion of viral membranes with intracellular endosomal membranes. We have also shown that arb is a good inhibitor of (non-enveloped) poliovirus types 1 and 3. Here, we evaluate the antiviral potential of arb against another picornavirus, foot-and-mouth disease virus (FMDV), an important veterinary pathogen. Sub-cytotoxic concentrations of arb inhibited the replication of FMDV replicon RNA. arb inhibition of FMDV RNA replication was not a result of generalised inhibition of uptake of cargo (e.g. plasmid DNA or RNA), nor did arb inhibit FMDV replication when added at 4 h post-transfection of FMDV replicon RNA. FMDV replication was blocked by the replication inhibitor guanidium hydrochloride (GuHCl). Upon GuHCl removal, FMDV replication was restored, and arb suppressed this recovery of virus replication. For other picornaviruses, recovery of virus replication upon GuHCl removal has been shown to require translation. However, arb did not suppress cap- or internal ribosome entry site (IRES)-dependent translation. arb also inhibited infectious equine rhinitis associated virus (ERAV), a close relative of FMDV. Testing of arb against infectious FMDV is in progress. Collectively, the data demonstrate that arb inhibits certain picornaviruses by a mechanism that appears to be independent of effects on virus entry but involves inhibition of genome replication.

Nipah virus (NiV) is a zoonotic paramyxovirus that causes severe and often fatal respiratory and neurological disease in humans. Since 1998, NiV outbreaks have occurred in Malaysia, Bangladesh and India. NiV poses a significant pandemic threat due to its broad host range and the widespread distribution of its natural host, the Pteropus fruit bat. Despite this, there are currently no licenced therapeutics or vaccines for use in either humans or livestock. Aiming to develop a safe and inexpensive vaccine to protect livestock in future outbreaks and to prevent onward transmission to humans, we are evaluating the immunogenicity and efficacy of three candidate vaccines in pigs. These vaccines are based on the NiV G or F surface glycoproteins, proteins which are essential for mediating virus-cell or cell-cell entry. The candidate vaccines include: (i) a recombinant soluble NiV G protein subunit, (ii) a recombinant molecular clamp stabilised NiV F protein subunit and (iii) a replication deficient, adenoviral vectored NiV G protein. We have developed low biocontainment assays to characterise the antibody responses to NiV and to aid identification of immune correlates of protection through quantification of both antigen-specific and neutralising antibody responses to our vaccine candidates. This includes anti-NiV F/G indirect ELISAs, a microneutralisation test using pseudotyped particles, and a microfusion inhibition test using a quantifiable cell-cell fusion assay. Using these assays we have demonstrated that all three of our novel vaccines are immunogenic in pigs and capable of generating a robust antibody response, with evidence for neutralising antibodies.

Constituent proteins that reside within promyelocytic leukaemia nuclear bodies (PML-NBs) are known to play an important role in cellular restriction of herpesvirus gene expression as a component of the intrinsic antiviral immune response to DNA virus infection. However, the precise mechanism(s) of PML-NB genome detection, entrapment, and silencing remain unknown. We have recently reported a sensitive method of viral genome detection that enables the visualisation of infecting EdC (5-Ethynyl-2’-deoxycytidine) labelled HSV-1 genomes (HSV-1EdC). We report that ultraviolet light (UV) irradiation of HSV-1EdC virions inhibits the nuclear recognition of viral DNA (vDNA) by core PML-NB constituent proteins (PML, Sp100, and Daxx). This impairment of recruitment was independent of UV source, but dependent on dose and time course of UV irradiation that correlated with a loss in viral genome decompaction upon nuclear entry. Moreover, UV treatment promoted premature uncoating of viral genomes within the cytoplasm of infected cells in a dose-dependent manner. Our data highlights the importance of genome decompaction in PML-NB sensing of infecting viral genomes. Additionally, it uncovers a previously undescribed mechanism of action for the induction of innate immune defences during herpesvirus infection that have historically utilised UV inactivation to promote the activation of cellular pattern recognition receptors (PPRs) and induction of interferon stimulated gene (ISG) expression.