- Volume 1, Issue 1A, 2019

Many honey bee colonies suffer large losses due to colony collapse disorder. This phenomenon, which has dramatically increased in frequency since 2006, has led to widespread efforts in sequencing honey bee pathogens, including RNA viruses such as deformed wing virus. However, honey bees coexist with a number of other arthropods, whose viruses are less thoroughly characterised. Many viruses currently classified as honey bee pathogens may therefore have a much wider host range. In particular, ants, which like bees are members of the Hymenoptera order, often coexist with bees and the two groups have previously been shown to exchange viruses. Parasitism by Varroamites, known to act as effective vectors for a number of RNA viruses, is also almost ubiquitous amongst honey bees, but little is known about viruses endemic to mites. We have previously demonstrated that it is possible to detect and characterise viral RNA in publicly available RNA-seq datasets. There are over 3000 such datasets for diverse Hymenoptera and mite species. We have developed a computational pipeline to identify viral transcripts in these datasets. This pipeline performs quality control, removes low complexity reads and reads generated from host RNA and various known contaminants, assembles the remaining reads into transcripts and detects the presence of regions with homology to known RNA viruses. Viral fragments identified with this pipeline will be examined phylogenetically to identify novel pathogens, clarify host range and specificity, and characterise transmission patterns.

Mitogen-activated protein kinase (MAPK) pathways are conserved signalling cascades in eukaryotes which regulate a myriad of processes in fungi from sexual reproduction to stress responses. These pathways rely on recruitment of three kinases on a scaffold protein to facilitate efficient kinase phosphorylation and subsequent downstream signalling to the nucleus. The model filamentous fungus Aspergillus nidulans utilises a MAPK pathway termed the pheromone module to regulate both development and secondary metabolism. This complex consists of the MAP3K (SteC), MAP2K (MkkB), MAPK (MpkB) and adaptor protein SteD. To date, there has been no scaffold protein identified for this MAPK pathway. In this study, we characterised a protein termed HamE, which we propose as a scaffold that regulates kinase phosphorylation and signalling in the pheromone module. Mass spectrometry analysis and BIFC experiments revealed that HamE physically interacts with both MkkB and MpkB and transiently interacts with SteC. Deletion of hamE or any of the pheromone module kinases results in reduced sporulation and complete abolishment of cleistothecia production. Mutants also exhibited reductions in expression of secondary metabolite gene clusters, including the velvet complex and sterigmatocystin genes. HamE acts as a positive regulator of MpkB phosphorylation, allowing for HamE to subsequently regulate development and secondary metabolism.

The twin-arginine translocation (Tat) machinery mediates the transport of folded proteins across the cytoplasmic membranes of prokaryotes and thylakoid membranes of chloroplasts. In Gram-negative bacteria three integral membrane components, TatA, TatB and TatC, are essential for generating an active complex. Most Gram-positive bacteria however, have a minimalist Tat complex formed solely from TatA and TatC subunits. Bacillus subtilis encodes two TatAC systems; TatAdCd and TatAyCy. These complexes operate in parallel but have differing substrate specificities. To date, little is known about how the B. subtilis TatAC-complexes assemble, however, the Escherichia coli TatABC complex is well studied and a substrate-triggered positional exchange of TatA and TatB has been identified as the first step in the assembly of an active complex. In this study, we aim to identify site specific interactions taking place between the B. subtilis Tat components and how these interactions differ whilst the complex is engaged in transport of a substrate. Molecular modelling was used to identify likely interaction interfaces between TatA and TatC, and site-directed mutagenesis was used to generate single cysteine variants of both proteins. Subsequently in vivo disulfide crosslinking was undertaken in both E. coli and B.subtilis, in the presence and absence of overproduced Tat substrates, to determine changes that occur between resting state and activated complexes. Overall, this work will enable us to outline a possible mechanism of assembly in TatAC-complexes of Gram-positive bacteria and identify whether the two distinct translocases assemble in a similar fashion.

Bacterial flagella are assembled from thousands of protein subunits that are unfolded and exported via a specialized type III secretion system. Subunit export is fuelled by the proton motive force (PMF) facilitated by a cytoplasmic ATPase complex comprising FliH, FliI and FliJ, which are evolutionarily related to components of the F1 ATPase. The FliJ stalk component of the ATPase binds the export gate protein FlhA, allowing it to utilise ΔΨ to drive highly efficient subunit export. What is unclear is how FliJ activation of FlhA is regulated to prevent constitutive proton influx when there are no subunits available. FliJ-mediated export gate activation could be regulated by other proteins that bind FliJ. We have shown that FliJ recruits unladen export chaperones, transferring them to their cognate subunits to create a local cycle of chaperone-subunit binding. To investigate whether chaperones also regulate FliJ activation of FlhA, we sought to isolate chaperone variants that were defective in FliJ binding but retained their ability to bind subunits and other export components. Disruption of chaperone-FliJ binding attenuated motility and cognate subunit export. To test whether chaperones blocked the FlhA-FliJ interaction, we developed in vitro and in vivo competition assays. Our data showed that chaperones and FlhA compete for a common binding site on FliJ, and that unladen chaperones, which would be present in the cell when subunit levels are low, disrupt the FliJ-FlhA interaction, preventing activation of the export gate. This provides a mechanism whereby the export gate is only activated when subunits are available.

Microbial production strains need to operate under sub- optimal growth conditions such as low pH, high osmolarity and thermal stress. The capacity to carry out industrial fermentations at higher temperatures reduces the risk of bacterial contamination and lowers cooling costs. We want to understand the basis of thermotolerance in the industrial yeast Kluyveromyces marxianus. As part of the EU-funded project, CHASSY, K. marxianus was grown in chemostat cultures under different stress conditions and a multi-omics analysis performed to study a range of stress responses, including elevated temperature (40 °C). Transcriptomes were generated from steady state cultures growing at identical growth rates under different stress conditions and gene set enrichment analysis (GSEA) performed. A range of functions were identified as being specifically expressed at higher temperatures and these are now being further investigated. One example is the temperature-specific expression of two putative hexose transport genes. Subsequent mutational inactivation using CRISPR and heterologous complementation established that at least one of these two genes is required for growth at (40 °C). We are now trying to determine the substrates for, and the precise function of, these genes. We also developed a ribosome profiling pipeline for K. marxianus and are using this to investigate the translational response to temperature stress. The combined study of both transcription and translation at steady state and as a culture responds to a temperature shift will give a comprehensive view of the basis of thermotolerance in K. marxianus and should identify strategies to exploit this in biotechnological processes.

Due to their intrinsic thermodynamic properties, RNA can misfold easily in cells. One way to mitigate RNA misfolding is through the actions of RNA chaperones, which bind and unwind structured RNA molecules and thereby offer opportunities for these misfolded species to refold properly. Such rescue activity has implications for the fitness effects of individual mutations-- at least mutations that compromise RNA folding or structure might be buffered by RNA chaperones. However, little is known about the rules governing such mutation buffering. Here, we describe how a model RNA chaperone, the DEAD-box RNA helicase CYT-19, affects the fitness effects of mutations in a model structured RNA, the Tetrahymenagroup I intron, whose self-splicing activity is dependent on its structure. We performed deep mutational scanning on the P1ex region of the intron which is critical for its self-splicing activity, and assayed differential splicing activity of all possible P1ex mutants in the presence and absence of CYT-19 to identify mutations that are buffered by RNA chaperone activity. I will discuss the properties of the chaperone-dependent and chaperone-independent mutation pools. Our results highlight that, to understand RNA robustness in vivo, we need to consider how mutational fitness effects are modulated by RNA chaperones and other trans-acting factors.

The development of optical microscopy has been traditionally driven by the needs of eukaryotic cell biology. Therefore, there are a number of unmet requirements for the application of advanced microscopy techniques to the field of microbiology. Conventional techniques, such as widefield epi-fluorescence and confocal laser scanning microscopy, are common-place in most laboratories, however these methods have trade-offs in terms of their attainable resolution and limited imaging volume. Here we present the application of several optical microscopy methods with the aim addressing the unmet needs of the field; increasing spatial resolution and sampling volume. We demonstrate the use of Interference Reflection Microscopy (IRM) for investigating the morphology and gliding motility of Myxococcus xanthus. This label-free technique provides super-resolution in the axial plane where changes in cell shape on the order of 100 nm can be detected in live cells. Using IRM we show novel insights into the gliding behaviour of these bacteria. We also present the application of the Mesolens to microbiology. The Mesolens is a large optical microscope with the unique combination of a low magnification and a high numerical aperture which results in an imaging volume >100 mm 3 with isotropic sub-cellular resolution. We demonstrate the use of the Mesolens to image live bacterial communities at multiple spatial scales simultaneously and offer new insights for bacterial community dynamics and biofilm architecture. Our work details novel applications of advanced microscopy to the field, and in doing so fills the technology gap which has previously restricted the study of complex microbial behaviours.

Chitin is an essential structural polysaccharide component of the cell walls and septa of fungi. Recent reports have suggested that Candida cells can resist killing by echinocandins by up-regulation of chitin synthesis thereby sustaining cell wall integrity both in vitro and in vivo (Lee et al. 2012). This increase in chitin content seen in C. albicans cells that are less susceptible to caspofungin is coordinated simultaneously by the PKC, Ca2+/calcineurin and HOG pathways (Munro et al. 2007, Walker et al. 2008). However, when echinocandins are removed, the chitin content quickly returns to basal levels, suggesting that elevated chitin cell wall content represents a fitness cost. We show here that those cells that die in the presence of caspofungin often have supra-normal chitin levels rather than low chitin levels, and therefore that having too much chitin in the cell wall may be detrimental for viability. Chitin content may therefore need to be clamped at levels that enable cells to survive cell wall stresses but are not so high that they negatively affect cell viability.

CRISPR-associated Rossmann fold (CARF) domain signalling underpins the modulation of CRISPR-Cas systems. Unlike the majority of known CARF domains associated with nuclease activity in CRISPR-Cas systems, the CARF domain of the transcriptional regulator RtcR modulates the opposite function by activating an RNA end sealing system. The Rtc RNA repair system in Escherichia coli consists ofthe universally conserved RNA cyclase RtcA and RNA ligase RtcB, and is known to be induced by antibiotics and oxidative stress. We aim to investigate the CARF domain mediated transcriptional regulation of the Rtc system in vivo and in vitro. A reporter based assay confirmed that the RtcR CARF domain has a negative regulatory effect on RtcR activity and subsequent induction of the rtcBA operon. Both predictive modelling and site-directed mutagenesis suggest the Rtc induction is not due to oxidation of cysteines present in the RtcR regulatory CARF domain. Interestingly, the enzymes RtcA and RtcB, products of the actions of RtcR as a transcription regulator, are required for RtcR to stimulate expression of the rtcBA operon and directly bind to the RtcR CARF domain as shown in vivo by two-hybrid and in vitro by gel filtration. Given that CARF domains are known to be activated by RNA binding, a range of RNA molecules linked to the Rtc system or CARF domains in general are currently being investigated as potential inducers in an in vitro transcription system. Together, our data indicate an expanded range for CARF domain signalling, including via protein-protein interactions.

Burkholderia cenocepacia (Bc) is an environmental opportunistic pathogen that causes persistent, often severe, lung infections in individuals with cystic fibrosis and other underlying diseases. Rho GTPases are central molecular switches that regulate cytoskeletal dynamics, trafficking, immune responses and cell proliferation in eukaryotic cells. Many microbes produce proteins that target Rho GTPase signalling. Bc employs a type VI secretion system (T6SS) to survive in macrophages by disarming Rho GTPases and causing actin cytoskeletal defects. Bc protein TecA is a non-VgrG T6SS effector that is responsible for actin disruption. TecA and other bacterial homologs bear a cysteine protease-like catalytic triad, which inactivates Rho GTPases by deamidating a conserved asparagine in the GTPase switch-I region. RhoA deamidation induces Pyrin inflammasome activation1. Our goal is to determine the detailed TecA secretion mechanism and the interacting partners inside the bacterial cytoplasm and inside the macrophages. In this study, we found by Co-IP/MS analysis that TecA interacts with the T6SS tube protein HcP, the membrane anchored TssM and with the elongation factor Tu. We also found that TecA is secreted in the absence of HcP and the secretion mechanism is discussed. 1. Aubert, D.F., X. Hao, J. Yang, X. Shi, W. Gao, L. Li, F. Bisaro, S. Chen, M.A. Valvano, and F. Shao. 2016. A Burkholderia Type VI Effector Deamidates Rho GTPases to Activate the Pyrin Inflammasome. Cell Host and Microbe 19 : 664–674.

Microbial eukaryotes (parasites/protists) are widely distributed and are common inhabitants of the gastro-intestinal (GI) tract of humans and animals. Some species, including Giardia, Entamoeba and Cryptosporidium are associated with symptomatic gastro-intestinal illness. However, others, for example Blastocystis, have questionable pathogenicity as they can be found in symptomatic and asymptomatic individuals. The aim of this study is to investigate selected protists which present health concerns to humans or animals. To date, approximately 180 faecal samples from 33 mammalian species, four bird species and one reptile across two wildlife parks in the Southeast England have been collected. A combination of cell culturing techniques, microscopy and molecular biology have been carried out to positively identify different protists including Blastocystis, Cryptosporidium, Eimeria, Entamoeba, Giardia and Isospora. Preliminary data show over fifty percent of the animals are sequence positive for at least one species, with approximately thirty percent exhibiting co-habitation with two or more different species. This study provides one of the first thorough investigations into distribution and prevalence of GI tract protists in wildlife parks in the UK. As a result, it has enhanced our awareness regarding what may constitute a normal eukaryotic component of the gut microbiome, in addition to aiding conservation efforts by examining the impact captivity has on an animal’s microbiome and potential implications this may have on their release.

In recent years, multiple studies have examined the bacterial communities present in the gastric microbiota during the progression to gastric cancer (GC). Although Helicobacter pylori is the biggest risk factor for GC, the microbiota of GC comprises a decreased load of H. pylori and an enrichment of bacteria, such as Prevotella spp., Veillonella spp., Actinomyces spp. However, interactions between H. pylori and these cancer-associated bacteria is hugely understudied. Here, we have used hypoxic growth conditions (5 % O₂) to investigate polymicrobial interactions between cancer-associated bacteria and H. pylori in vitro. We found that whilst the co-culture of H. pylori with Prevotella spp. and Veillonella spp. had no effect on growth of either bacteria, Actinomyces oris completely inhibited the growth of H. pylori. Moreover, A. oris did not inhibit the growth of other Gram-negative pathogens such as Salmonella Typhimurium and E. coli, whilst there was a slight growth inhibition of Campylobacter jejuni. Furthermore, ultrafiltration of A. oris culture supernatants revealed that inhibition is mediated by a secreted factor larger than 5 kDa, which can be heat inactivated. Interestingly, Actinomyces viscosus can also specifically kill H. pylori suggesting that this inhibition could be conserved across the Actinomyces genus. We are currently identifying the inhibitory factor responsible for inhibiting H. pylori growth. Furthermore, we are investigating whether A. oris can clear gastric H. pylori infection in a mouse model of infection and the implications of this on gastric carcinogenesis. In conclusion, whilst data-rich microbiota studies continue to thrive, it is imperative that we understand the mechanisms underpinning changes to the gastric microbiota and whether these bacteria are drivers or ‘passengers’ of gastric carcinogenesis.

Tick borne diseases (TBDs) have significant impact on the health and welfare of domestic animals and humans. There is extremely little data on the prevalence of tick species, TBDs or their impact in Nigeria. Nigeria’s scenario is further worsened by lack of basic diagnostic facilities and treatment, compared with the average person’s income. The multiple climate zones and animal husbandry practices in Nigeria also make it difficult to extrapolate studies from one zone to the other five geopolitical zones. TBDs reported in Nigerian dogs include Anaplasma species. (A. platys A. omatnenne), Babesia species (B. rossi, B. canis and B. gibsoni), Theileria species. (T. equi, T. sable), Ehrlichia sp. (E. canis, E. ruminantum), Hepatozoon species (H. canis) and Candidatus Neoehrlichia mikurensis. The prevalence of zoonotic pathogens of dogs such as Borrelia sp., in humans in West Africa also indicates that these are likely to present in dogs in Nigeria. This main study aim is to identify ticks taken from dogs in Nigeria using morphological and molecular methods, determine which host species the ticks have fed on, and identify pathogens that they are carrying. It also aims to compare molecular and point of care diagnostics for TBDs in blood from Nigerian dogs. The overall objective is to provide robust data on which tick species and TBDs are present in Nigerian dogs and the potential zoonotic or epizootic risk.

Staphylococcus aureus subsp. anaerobius is a microaerophilic, catalase-negative bacterium responsible for abscess pathology (Morel’s disease) in small ruminants. We performed whole-genome sequencing to a collection of isolates taken in Europe and Africa over the last 30 years, and carried out an evolutionary genomic analysis to understand the molecular bases of its host adaptation and restricted metabolism. Phylodynamic analyses showed that S. anaerobius emerged from a Staphylococcus aureus progenitor about 1000 years ago (716–1184), with an evolutionary rate of ∼1.2 SNPs/year (approximately 10-fold slower than S. aureus clones), before differentiating into two distinct lineages separating African and European isolates. The S. anaerobius genome displays signatures of extreme adaptation to a highly specific niche, with 205 pseudogenes that together represent over 10 % of the genome and affect many metabolic and pathogenic pathways. In addition, S. anaerobius contains 87 highly similar insertion sequences (IS) located in intergenic regions. Our functional analysis suggests that the IS transcription could affect the expression of their flanking genes, using at least two complementary strategies: antisense RNA or modification of promoter regions. We propose that the IS-mediated control of gene expression underpins an orchestrated mechanism of host adaptation. Remarkably, we also identified 6 large genomic transversions (size range: 70–346 kb) flanked by IS, presumably the result of homologous recombination. In summary, S. anaerobius evolved from S. aureus undergoing restrictive host specialisation, which shaped its genome through widespread pseudogenisation, accumulation of IS that modulate gene expression, and large chromosomal rearrangements.

Mastitis is a disease of the mammary gland which affects most mammals and is one of the costliest ongoing challenges in modern dairy farming. The most significant challenges in mastitis management are the speed and accuracy of diagnosis and the use of antibiotics. Current mastitis diagnosis in veterinary practices in the United Kingdom utilises culture-based techniques. However, this approach has some limitations including; processing time, species selection biases and culture failure. This ongoing PhD project seeks to assess the utility of high-throughput sequencing technology in addressing the current challenges in mastitis diagnosis. Samples were obtained from cattle from farms in North Yorkshire, United Kingdom. Cattle were selected from monthly somatic cell count records as mastitis positive cases with a cell count between 250 000 and 550 000 cells ml−1 or control animals with a count below 200 000 cells. Mastitis positive cases were further split into bacteriology positive and negative groups. During sampling a cow-side somatic cell test (California Milk Test) was performed to confirm the monthly database readings. All farms sampled were conventional dairy farms with comparable management systems. Samples were collected by trained veterinarians following a pre-defined protocol designed to limit sample contamination. Microbial community diversity, richness and composition will be compared between sample groups to survey for variations which may explain why culture fails in culture negative subclinical cases. 131 sample animals have been assigned to study groups with samples collected, DNA extracted and prepared for sequencing on the Illumina MiSeq platform with results expected in January 2019.

Dendritic cells (DCs) are the sentinels of the immune system, responsible for recognising invading pathogens and priming the adaptive immune system for appropriate responses. Hence they are considered potential targets for vaccines against pathogens such as foot-and-mouth disease virus (FMDV). Little is known of the events of FMDV replication in bovine moDCs. Present work therefore sought to characterize FMDV and its immune complex (IC) replication in bovine moDCs in vitro. A chimeric heparin sulphate FMDV (O1M) was used in this study. Immuno-fluorescence microscopy (IFM) and quantitative RT-PCR was used to analyse viral replication at 0–6, 8, 16 and 24 hpi. Plaque assays were used to investigate the yields of live virus produced in moDCs at 0, 4, 8 and 24 hpi. FMDV and IC FMDV could infect moDC. In moDC infected with FMDV alone, or with immune-complexed (IC) FMDV, replication was observed by IFM between 2–4 and 1–16 hpi. In contrast, for both FMDV and FMDV IC infections RT-PCR analyses showed viral replication peaked at 4 hpi and then decreased between 8 to 24 hpi. Plaque assays using supernatants of the infected moDC showed no evidence of an increase in viral titre at 24 hpi. The detection of viral nsp (3AB and derivatives) suggests replication of FMDV persists for longer in moDCs when entry is mediated by IC. However, the lack of increase in virus yield suggests replication is abortive. One possible explanation for this difference could be that bovine moDCs are able to recognise non-immune complexed FMDV more rapidly.

Bovine ephemeral fever virus (BEFV) [Family Rhabdoviridae: genus Ephemerovirus] causes a transient febrile illness in cattle that results in economic losses and morbidity, with occasional mortality. Epidemics of the disease have been particularly costly in countries of the Middle East, including Israel. The virus is considered a vector-borne pathogen although the exact relationship with a number of blood-feeding arthropods has not been established. In order to improve developments in diagnostic detection, phylogeographic investigations and virus-vector relationships of BEFV we have derived the first genome of this virus from an isolate from Israel and used this to assess recent outbreaks of disease. We have also investigated the vector competence of BEFV with a number of target arthropod species. The complete genome sequence of BEFV (Israel strain 1) is 14 850 base pairs in length and shows over 95 % identity with the only other Middle Eastern genome for BEFV from Turkey. A wider phylogeny shows that these viruses form a clade, previously described as cluster II, which is clearly distinct from BEFV strains derived from China, Japan and Australia. However, there is a distinct separation of those viruses from Israel to others in the Middle East. Preliminary vector competence studies suggest that at least three species of mosquito that are potential transmission vectors of the virus are incapable of infection with, or transmission of, BEFV. Current studies are ongoing to assess other blood-feeding insect species as potential vectors.

Mycobacterium bovisis the causative agent of bovine tuberculosis (bTB), one of the most costly and persistent agricultural infectious diseases still widespread in England today. However, not all parts of England are created equally with respect to the incidence of bTB. In 2012 the Department for the Environment, Food and Rural Affairs (DEFRA) divided the country into three risk areas; the high, low and edge risk areas. The perpetuation of the bTB epidemic is often blamed on the known wildlife host of M. bovis, the European badger (Meles meles). Despite this, no large scale studies examining both the prevalence and transmission patterns of M. bovis in this species has been undertaken. Here we describe the results of a major survey of 600 road-kill badgers from 6 counties within the edge risk area of England. Mycobacterium like colonies were isolated from over 80 carcasses of which, 65 were confirmed to be tuberculosis complex positive by PCR. These isolates were spoligotyped, VNTR typed and then subsequently whole-genome sequenced. We describe a SNP based transmission analysis of the sequenced isolates that provides a higher degree of resolution between the badgers compared to the described molecular typing methods.