- Volume 1, Issue 1A, 2019

Arboviruses constitute a major public health problem, in particular mosquito-borne arboviruses that continue to emerge and re-emerge. Arbovirus infection of mammals is enhanced by the presence of a mosquito bite at the inoculation site, in comparison to virus experimentally administered by needle inoculation in the absence of a bite. Inflammatory responses to bites appear to be a key factor in this enhancement. However, the experimental inoculation of mosquito saliva with virus inoculum by needle, in the absence of bite trauma, also has the ability to enhance viral infections. In this study, we have studied the mechanistic basis for these observations. We have studied whether saliva from different mosquito species can enhance virus infection. Interestingly, while saliva from Aedes genus enhanced virus infection, An. gambiae saliva did not. Instead, An. gambiae saliva actively inhibited infection compared to inoculation with virus alone. This could partly explain why An. gambiae mosquitos are unsuitable vectors for transmitting most arboviruses. By comparing the effects that saliva of these different mosquitoes have at the bite site we have further specified which inflammatory responses at the inoculation site modulate arbovirus infection in the skin. Using an in vivo mouse model we have shown that An. gambiae causes significantly less oedema but a higher up-regulation of key inflammatory genes in the skin than A. aegypti. As such, we are providing important insights into how mosquito saliva modulates infection. A better understanding of this will aid the development of anti-viral treatments by targeting factors within the mosquito bite that are common to many distinct infections.

Human cytomegalovirus (HCMV) rapidly mutates during in vitro passage, and this strongly alters the way the virus spreads. In vivo HCMV spreads by direct cell-cell contact, as do recent clinical isolates. In contrast, passaged strains spread via cell-free virions. Because of this, cell-cell spread remains largely uncharacterised. We have developed a strain (Merlin) that retains a full length, wildtype genome. As a result, it mimics clinical HCMV and spreads by direct cell-cell contact, a method of spread that is more resistant to neutralising antibodies, and innate and intrinsic immunity. We now show that each cell-cell transfer is equivalent to an extremely high MOI infection, with up to 300 genomes delivered to each cell, potentially providing an explanation for the ‘immune-evasive’ properties of cell-cell transfer. Furthermore, infectious virions accumulate at cell-cell contacts between cells. This may represent a ‘virological synapse’ that protects virions from neutralising antibodies. Not only does Merlin enable us to characterise cell-cell spread, but it enables us to infect a wide range of clinically relevant cells with a virus expressing the complete complement of virus genes. In vivo, HCMV infects dendritic cells (DCs), but is never cleared, implying that it is able to subvert DC function. Therefore, we performed quantitative proteomic analysis of infected primary immature DCs, following cell-cell transfer. This quantified 7992 intracellular proteins, and 703 plasma membrane proteins. Over 99 proteins were downregulated following infection. Many of these are DC-specific, and have roles in regulating adaptive immunity. These viral-manipulations may therefore dramatically impact DC function.

Zika virus (ZIKV) is an arbovirus (family Flaviviridae) mainly transmitted by Aedes mosquitoes causing febrile illness and Zika congenital syndrome in infants if mothers were infected during pregnancy. ZIKV manipulates its host’s cellular machinery in order to facilitate infection and evade antiviral responses. The identification of host and vector proteins involved in these processes may lead to novel antiviral strategies. In this study, Ae. aegypti cell lines (AF5) stably expressing V5-tagged ZIKV capsid (C) or anchored capsid (AC) proteins were developed to investigate virus-vector protein interactions. To identify interaction partners, immunoprecipitation (IP) of V5-tagged C or AC was performed and subjected to proteomic analyses using nLC-MS/MS under label-free quantification conditions. A total of 148 and 53 mosquito protein interactors unique to C and AC were identified, respectively. Protein network and gene ontology analyses showed biological processes possibly important for ZIKV infection. To investigate further the role of these proteins during infection, 25 were chosen for dsRNA-based knockdown screen and infection with reporter virus (ZIKV-Nluc) in AF5 cells. Significant reduction in reporter virus signal was observed during knockdown of 6 interactors suggesting a pro-viral role for these proteins during infection. This was corroborated by conducting the same knockdown experiments but infecting with a clinical isolate of ZIKV (PE243), which showed reduced virus RNA levels and titre. Interestingly, three of the six proteins are part of the ubiquitin-proteasome pathway (UPP). Currently, functional experiments are underway to investigate the role of UPP during ZIKV infection.

Viruses in the picornavirus family comprise a single molecule of positive sense RNA contained within a simple non-enveloped capsid. The mechanism for RNA packaging is not well understood. We have developed a novel and simple approach to identify predicted RNA secondary structures involved in genome packaging in the picornavirus foot-and-mouth disease virus (FMDV). By interrogating deep sequencing data generated from both packaged and unpackaged populations of RNA, we have determined multiple regions of the genome with constrained variation in the packaged population. Predicted secondary structures of these regions revealed stem-loops with conservation of structure and a common motif at the loop. Disruption of these features resulted in attenuation of virus growth in cell culture due to a reduction in assembly of mature virions. To further test the function of these putative packaging signals (PPS), we have developed a trans-encapsidation assay using subgenomic replicons expressing GFP, helper virus and flow cytometry. The results of these studies provide evidence for the involvement of predicted RNA structures in picornavirus packaging and offer readily transferable methodologies for identifying packaging requirements in many other viruses.

Human Cytomegalovirus (HCMV) is a master immune regulator, encoding multiple proteins that modulate a variety of immune signalling pathways. We previously performed a systematic proteomic analysis of temporal changes in host and viral proteins throughout the course of infection and determined that HCMV downregulates >900 host proteins. HCMV is the largest human herpesvirus, potentially encoding hundreds of ORFs. Identification of which individual gene targets a given cellular factor can therefore be challenging. To facilitate the mapping of viral gene functions, we employed a panel of HCMV mutants, each deleted in contiguous gene blocks dispensable for virus replication in vitro. Three proteomic screens of these mutants were performed, with each mutant represented in at least duplicate. From these data we have defined the genetic loci responsible for targeting >250 host proteins. Bioinformatic enrichment analysis on the targets of each mutant virus enabled attribution of novel functions to blocks of uncharacterised genes. Our approach was validated from analysis of the US1-11 genetic locus, which confirmed that the major function of US1-11 genes is the regulation of MHC class I molecules and other cell surface receptors. The data also suggests that the major functions of the poorly characterised blocks RL1-6 and US29-34A are the regulation of secreted proteins and the regulation of a family of cell surface adhesion molecules respectively. Overall this approach can be used to gain global insights into HCMV gene function, the study of which has previously been only been possible on a single gene basis.

Herpesviruses are large and complex DNA viruses that are composed of an icosahedral capsid, a proteinaceous layer termed the tegument, and a glycoprotein rich lipid envelope. One important area of host-pathogen interaction that is still poorly understood is the extensive change to intracellular organelles and cellular morphology that occur within the infected cell during active virus replication. In order to characterise the spatiotemporal dynamics of host cell remodelling caused by herpesvirus infection, we use novel multiparametric fluorescence microscopy methods compatible with live-cell imaging. In addition, we apply expansion microscopy to map 3D rearrangement in great detail. The remodelling of the host cell is correlated to the stage of virus replication which can highly vary between individual cells. Therefore, we have constructed a recombinant reporter virus that expresses eYFP-tagged ICP0, a multifunctional immediate early tegument protein, as well as mCherry-tagged glycoprotein C (gC), a late protein that is a major component of the viral envelope. The sequential expression of these two viral proteins provides us with an intrinsic time stamp for the stage of virus infection in each cell. With this fluorescent reporter virus, we are able to describe the remodeling of- the three-dimensional architecture of microtubules and the actin network,- compartments of the secretory and endocytic pathways which are intimately linked to viral envelope protein synthesis, maturation and transport,- and key antiviral and inflammatory signalling platforms (mitochondria and peroxisomes).

The predominance of specific bacteria within the Crohn’s disease intestine remains poorly understood with little evidence uncovered to support a selective pressure underlying their presence. Intestinal ethanolamine is readily accessible during periods of intestinal inflammation, and enables pathogens to outcompete the host microbiota under such circumstances. Here we show that the intestinal short chain fatty acid propionic acid stimulates increased ethanolamine degradation by one such Crohn’s disease associated pathogen, adherent-invasive Escherichia coli (AIEC). This degradation occurs within bacterial microcompartments that are subsequently excreted in outer membrane vesicles. Additionally ethanolamine, added extracellularly at concentrations comparable to those in the human intestine, is accessible to intracellular AIEC and stimulates significant increases in growth within macrophages. Finally, expression of the operon for ethanolamine degradation (eut) is increased in children with active Crohn’s disease compared to healthy controls. After clinical remission was seen with exclusive enteral nutrition treatment, Crohn’s disease patient’s exhibit significantly reduced eutexpression. Our data indicates a role for ethanolamine metabolism in facilitating AIEC colonization of the Crohn’s disease intestine and warrants further study of its potential use as an indicator of inflammatory status in Crohn’s disease.

Biofilms are communities of microorganisms that attach to various surfaces and are widely associated with infection for animals and plants. Our investigation is focussed on a current and growing concern: the distribution and formation of biofilms in washing machines. Many countries wash clothes at reduced temperatures around 30 to 40 °C degrees 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 into the environment. P. aeruginosa is one of the predominant bacteria found in washing machines and is highly resistant to many antibiotics. Little is known about environmental microniches present in biofilms. In this work, 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 biofilm formation. Here, we use novel pH-sensitive optical nanosensors that penetrate P. aeruginosa biofilms and emit fluorescence in response to variation in pH. Confocal laser scanning microscopy revealed that the nanosensors can penetrate biofilms within minutes and interact with the biofilm structure. Different washing detergents were tested resulting in altered biofilm formation and killing abilities. Using time lapse imaging, pH changes were tracked in real time at a microcolony and single cell level which will ultimately facilitate monitoring of environmental changes induced as biocides penetrate biofilms, underpinning the development of more effective antimicrobials to limit the emergence of AMR.

Lyme disease (LD) is a multisystem infection caused by tick-borne spirochaetes of the Borrelia burgdorferii sensu lato group. UK and US laboratory diagnosis of LD involves the two-tier serological approach. The negative predictive value of the test has been challenged, particularly in early stage LD. There is considerable interest, therefore, in the development of improved diagnostic tests. The main aim of the project is to identify new markers that could form the basis for improved tests. A mass spectrometry biomarker discovery study was undertaken on LD positive and negative residual diagnostic samples from UK LD testing by Public Health England and a cohort of patient samples from collaboration with a research group in the Czech Republic. A ‘related-disease control group’ including serum samples from syphilis, leptospirosis and chronic fatigue syndrome was also included. Several proteins were found at a significantly higher or lower in abundance in the ld-positive patients compared with ld-negative. Of particular interest was Lipocalin-2 (LCN2), a protein involved in immunity. LCN2 has previously been found in increased abundance in mice exposed to B. burgdorferi. Further analysis of LD samples using Illumina RNA sequencing revealed further markers of interest. Transcriptomic analysis including Ingenuity Pathway Analysis (IPA) gave insights into the host response to LD infection. Proteins of interest from proteomic and transcriptomic analysis were taken forward for further analysis by WB or ELISA in a larger sample set.

Adherent Invasive Escherichia coli (AIEC) is a non-diarrhoeagenic intestinal E. coli patho type with a putativea etiological role in Crohn’s Disease (CD). AIEC pathogenes is ischaracterised by adhesion to, invasion of, and replication within intestinal epithelial cells and macrophages, and biofilm formation. Using a heterologous expression screen, we identified a gene in the AIEC LF82 genome encoding a protein which self-assembles into filaments in HeLa cells, which we hypothesised was a novel pilin or biofilm matrix component, designated bcmA (biofilm coupled to motility in AIEC). Using a crystal violet-based assay, we found LF82ΔbcmA have defective biofilm formation, which can be fully complemented by episomal bcmA expression. Microscopic analysis of LF82 biofilms demonstrated LF82ΔbcmA form patchy, sparse biofilms, and revealed an intracellular localisation for GFP-tagged bcmA, suggesting the protein is not a surface-exposed adhesin or biofilm matrix component. We therefore assessed the role of bcmA in flagellar-mediated motility and found that – despite displaying wild-type flagellar morphologies – LF82ΔbcmA have profound swimming and swarming defects. Work in a Caenorhabdit is elegans infection model suggests bcmA is not required for full virulence; however, preliminary evidence suggests LF82ΔbcmA have defective C. elegans gut colonisation. Taken together, our data demonstrates roles for bcmA in AIEC host colonisation via an undefined role in motility. bcmA is highly conserved among pathogenic gammaproteobacteria, including Salmonella Typhi, Shigella boydii and Klebsiella pneumoniae. This suggests bcmA may not only be significant for AIEC pathogenesis, but may also represent an important virulence determinant in several major gammaproteobacterial pathogens.

Cryptococcus neoformans is an opportunistic fungal infection that causes cryptococcal meningitis in immunocompromised individuals. Macrophages play a critical role in determining the outcome of infection, and can either phagocytose and kill the cryptococcal cells, or disseminate infection. While it is known that macrophages impact the progression of cryptococcal disease, it is not known how the macrophage intracellular niche contributes to complex infection outcomes. Clinical and experimental studies have identified potential genetic differences, in both host and pathogen, but statistical robustness has been difficult to achieve due the large variability in the outcome of infections. Therefore, in attempt to quantitatively explain this variability, we have used a zebrafish model of cryptococcal infection where we can directly relate the initial level of fungal infection with final infection outcome. We find that at low levels of initial fungal burden the outcome of infection is stochastic, while over high ranges of initial infection the outcome is linearly related to the initial burden, but with a further stochastic component that contributes to increased variability. Using these experimental data and data from clinical trials we have generated a computational simulation of in vivo cryptococcal infections which allows us to consider different infection variables and how they alter the progression of cryptococcosis. By combining such computational simulations with our experimental models we demonstrate that the macrophage intracellular niche determines the unknown stochastic component, independent of fungal burden. Using this knowledge, we can better identify the molecular, population genetic, and clinical parameters associated with the outcome of cryptococcosis.

Apicomplexans are human and animal protozoan pathogens responsible for diseases including malaria, cryptosporidiosis and toxoplasmosis. As obligate intracellular parasites they are highly organised cells with numerous novel and specialised sub-compartments that form the basis of their invasion biology, host defence evasion, and novel metabolic traits. However, our understanding of these cells is highly constrained by our limited knowledge of the locations and functions of most of the cell’s proteome. Even in the best-studied apicomplexans (Plasmodium spp. and Toxoplasma gondii) only a small fraction of proteins’ locations have been experimentally determined, with most assignments based on predictions from orthologues in distant relatives. Moreover, many parasite proteins are annotated as ‘hypotheticals’, for example 4113 of 8121 Toxoplasma proteins, and many are unique to parasites stymying even predictions of location or function by comparative biology. To address this deficit in our basic understanding of the compositional organisation of the apicomplexan cell, we have used a spatial proteomics method called hyper LOPIT to simultaneously capture the steady-state subcellular association of thousands of proteins in the apicomplexan Toxoplasma. These protein atlases reveal: extensive protein association networks throughout the cell providing testable hypotheses of their function; conservation and novelty of compartment proteomes between apicomplexans; differential selective pressures across the different cell compartments; and clear instances of protein relocation from one organelle to a different one during apicomplexan speciation. This new, global view of the organisation of the apicomplexan cell proteome provides a much more complete framework for understanding the mechanisms of function and biology of these cells.

Mechanosensitive channels are required for bacteria cells to survive hypoosmotic shock (transition from high to low osmolarity environment). YnaI is one of the mechanosensitive channels found amongst many bacterial species including Salmonella Typhimurium. Previous studies have suggested that S. Typhimurium YnaI may be implicated in host colonization during infection of farmed animals. Disruption of ynaI impaired intestinal colonization in pigs, cattle and chicken. To investigate S. Typhimurium YnaI structure and function, the S.Typhimurium ynaI was cloned into a plasmid, followed by physiological characterization of the S. Typhimurium YnaI constructs expressed in an E. coli channel-less mutant strain. To further understand the role of YnaI in S. Typhimurium pathogenesis, S. Typhimurium ynaI was deleted and the ability of mutant to survive and replicate in host cells was investigated. The S. Typhimurium YnaI channel has unique characteristics when expressed in E. coli: S. Typhimurium YnaI channel conferred almost complete protection against 0.3 M NaCl hypoosmotic shock when overexpressed, but interestingly, high level expression of S. Typhimurium YnaI inhibited growth in two different complex media and in minimal media. Deletion of ynaI from S. Typhimurium led to increased internalization in macrophages and epithelial cells. Data derived from this study reveals novel characteristics of S. Typhimurium YnaI which may provide insights into other functions of the S. Typhimurium YnaI channel.

The biocide triclosan is used extensively in household and hospital settings, resulting in chronic exposure to the biocide in individuals that use triclosan-containing products. Triclosan is thought to induce antibiotic tolerance and alter biofilm formation, although the underlying mechanisms causing these changes are yet to be elucidated. If true, the widely used biocide may contribute to antibiotic treatment failures, and therefore requires investigation. To determine how triclosan induces antibiotic tolerance, Staphylococcus aureus was pre-treated with triclosan prior to treatment with the clinically relevant antibiotics ciprofloxacin, rifampicin, and vancomycin. Planktonic S. aureus cultures pre-treated with triclosan had 1000 fold higher viable counts compared to non triclosan pre-treated cultures. Inspection of biofilms by live/dead staining found that triclosan pre-treatment protected S. aureus biofilms from treatment with otherwise lethal doses of ciprofloxacin, rifampicin, or vancomycin. Biofilms of mutant strains with a defective stringent response were not protected from antibiotic treatment, even in the presence of triclosan. Interestingly, stringent response mutants still exhibited triclosan-induced antibiotic tolerance in planktonic culture, but mutants with a defective agr quorum sensing system did not. Confocal laser scanning microscopy revealed that incubation of S. aureus with triclosan altered biofilm structure, resulting in increased proportions of polysaccharide in the biofilm matrix that could potentially mediate protection against antibiotics. Neither the stringent response mutants nor agr mutants influenced triclosan-induced biofilm changes, suggesting another, currently uncharacterised response. We suggest that triclosan triggers multiple global regulatory systems in S. aureus, subsequently inducing tolerance to multiple antibiotic classes and altering biofilm structure.

Neisseria meningitidis (meningococcus) is a major meningitis-causing bacteria and is known for its ability to breach blood-brain barrier (BBB). Meningococcus binds to Laminin receptor (LAMR) on the surface of endothelium, which is part of the BBB. The meningococcal surface proteins PorA and PilQ were previously identified as bacterial ligands responsible for binding and, subsequently, the LAMR-binding moiety of PorA was localised to its fourth extracellular loop (PorA-Loop4). Using a circularised peptide corresponding to PorA-Loop 4 from N. meningitidis MC58, the PorA-LAMR interaction induced specific cellular responses in human brain microvascular endothelial cells (HBMECs) including G1 cell cycle arrest. Flow cytometric analysis indicated that the treatment of HBMECs with PorA-Loop4 for 24 h caused a significant reduction of cells (20 %) at S-phase and a corresponding increase (23 %) in G1 population. Immunoblotting and quantitative real time PCR (qRT-PCR) analysis suggested that a blockade in Akt signalling (key proteins including Akt, GSK-3β, CyclinD1, and CDK4) contributes to the G1 arrest. Immunoblotting showed that the expression of phosphorylated GSK-3β and CDK4 were significantly increased in treated HBMECs. In contrast, the expression of phosphorylated Akt and Cyclin D1 were decreased following treatment. Transcriptome analysis using qRT-PCR confirmed that treatment of HBMECs with PorA-Loop4 peptide for 2, 4, 8, or 24 h increased gene expression of CDK4, and decreased expression of Cyclin D1. Immunofluorescent imaging of Akt, GSK-3β, CyclinD1, and CDK4 in Loop4-treated HBMECs are consistent with qRT PCR and immunoblot results. The data confirm that PorA-Loop4 induce G1 arrest through the Akt signalling pathway via Akt/GSK-3β/CyclinD1/CDK4.

We have designed a new Gram-negative antimicrobial- it is an oligonucleotide Transcription Factor Decoy (TFD) that binds to and inhibits bacterial transcription factors controlling genes essential for growth and pathogenicity. The TFD is highly effective in vitro and in vivo, tested in Galleria mellonella survival models and in a mouse model of intra-abdominal infection. TFDs are formulated as nanoparticles, composed of a proprietary lipidic molecule (CM2), that binds to essential prokaryotic phospholipids, such as Cardiolipin, to deliver the oligonucleotide across the bacterial membrane. Studies with models of bacterial membranes showed that translocation was dependent on the presence of Cardiolipin but occurred in the absence of ATP or a pH gradient. Flow Cytometry studies found that the efficiency of TFD delivery to bacterial cells was high, and Live/Dead staining confirmed that cells were not being lysed by the nanoparticles. Further investigation of the mechanism of delivery used a proteomics and metabolomics study of the response of E. coli to nanoparticulate delivery. A number of highly induced transcription factors were identified consistent with stress induced by disruption of respiratory centres bound by Cardiolipin within the membrane. A TFD was designed to inhibit one of the induced transcription factors. This TFD was shown to bind tightly to its cognate transcription factor, have a potent MIC in vitro and be efficacious in murine models of infection. Hence, the work demonstrates that TFDs can be rationally designed to create new antimicrobials to efficiently target bacterial transcription factors.

Burkholderia cepacia complex (Bcc) is a group of 22 closely related species of Gram-negative bacteria that cause chronic infections in people with cystic fibrosis (CF) that are rarely eradicated. The high-level antibiotic resistance and poorly understood mechanisms by which Bcc survive and persist during chronic infection mean that combatting these chronic infections is particularly challenging. We have previously found that a group of 20 low-oxygen-activated (lxa) encoded proteins were consistently upregulated in sequential Bcc isolates in two chronically infected CF patients over time of infection. Many of these proteins have not previously been studied in Bcc but the consistent upregulation over time of infection suggests that they play an important role during chronic infection. Two particular proteins of interest within the lxalocus that were consistently upregulated were a universal stress protein (USP) and a phospholipid binding protein (PBP). Single gene deletion mutants (Δusp and Δpbp) in the wildtype Bcc strain K56-2 (WT) both showed a 90 % reduction in attachment to CFBE41o- cells compared to WT. There was also a 5-fold reduction in the virulence of Δpbp in the acute infection model Galleria mellonella (P<0.005) and an increased sensitivity of Δusp to peroxide-induced oxidative stress (P<0.0001) and low pH (P<0.05) relative to WT. A reduction in both uptake and survival of Δusp in U937 macrophage-like cell line compared to WT, suggests the USP plays a role in the intra-macrophage survival of Bcc. Overall, these proteins, previously associated with low-oxygen conditions may play a considerable role in Bcc pathogenesis and its adaption during chronic infection.