bluuebunny/crossref_metadata_2025 huggingface dataset:
Coriolis and centrifugal forces drive haltere deformations and influence spike timing
Authors: Mohren T. L. , Daniel T. L. , Eberle A. L. , Reinhall P. G. , Fox J. L.
Published: April 2019

The halteres of flies are mechanosensory organs that serve a crucial role in the control of agile flight, providing sensory input for rapid course corrections to perturbations. Derived from hind wings, halteres are actively flapped and are thus subject to a variety of inertial forces as the fly undergoes complex flight trajectories. Previous analyses of halteres modelled them as a point mass, showing that Coriolis forces lead to subtle deflections orthogonal to the plane of flapping. By design, these models could not consider the effects of force gradients associated with a mass distribution, nor could they reveal three-dimensional spatio-temporal patterns of strain that result from those forces. In addition, diversity in the geometry of halteres, such as shape and asymmetries, could not be simply modelled with a point mass on a massless rod. To study the effects of mass distributions and asymmetries, we examine the haltere subject to both flapping and body rotations using three-dimensional finite-element simulations. We focus on a set of simplified geometries, in which we vary the stalk and bulb shape. We find that haltere mass distribution gives rise to two unreported deformation modes: (i) halteres twist with a magnitude that strongly depends on stalk and bulb geometry and (ii) halteres with an asymmetric mass distribution experience out-of-plane bending due to centrifugal forces, independent of body rotation. Since local strains at the base of the haltere drive deformations of mechanosensory neurons, we combined measured neural encoding mechanisms with our structural analyses to predict the spatial and temporal patterns of neural activity. This activity depends on both the flapping and rotation dynamics, and we show how the timing of neural activity is a viable mechanism for rotation-rate encoding. Our results provide new insights in haltere dynamics and show the viability for timing-based encoding of fly body rotations by halteres.
Molecular identification of fungi isolated from coastal regions of Red Sea, Jeddah, Saudi Arabia
Authors: Alwakeel Suaad
Published: April 2019

To isolate fungal communities from coastal areas of the Red Sea in Saudi Arabia and identify and classify them by molecular techniques. Samples were collected from the seaside of the Red Sea in Jeddah, Saudi Arabia in March 2012 and stored in sterile screw cap bottles for further analysis. Phenotypic and genotypic characterization of fungal isolates was done using standard techniques. Eight fungal genera including Aspergillus, Penicillium, Thielavia, Fusarium, Emericella, Cladosporium, Scytalidium and Alternaria. Most isolated fungi showed significant growth on petroleum media and were thus considered capable of biodegradation of crude oil-based substances. The fungal genera isolated from the Red Sea had 97 –100 % similarity with the related fungi recorded in the GenBank in which they were deposited. The morphological and molecular structure of these marine fungal isolates closely resembles their terrestrial counterparts in the Genbank. The capabilities of these fungal species to utilize petroleum as a source of carbon speaks to future applications in which marine fungi may be utilized in the breakdown of petroleum-based waste in an ecologically efficient manner.
Chaga mushroom (inonotus obliquus) inhibits growth of both lung adenocarcinoma (A549) cells and Aspergillus fumigatus
Authors: Al-Tuwaijri Magda , Ibrahim Jehane
Published: April 2019

Lung tumors and infections remain the most common leading cause of mortality worldwide. Chaga mushroom (Inonotus obliquus) has long been considered the king of medicinal mushrooms which constitutes an inexhaustible source of active compounds which can affect the survival of tumor cells. It has been used widely for centuries as a dietary supplement and tea. In this sense, the aim of this study was to investigate in vitro cytotoxic capacity of water extracts of I. obliquus (Chaga mushroom) against the human lung cancer A549 cell line after 72 h incubation. In addition, the extracts were screened for antifungal activity on Aspergillus fumigatus species, a life-threatening cause of invasive pulmonary aspergillosis. The cytotoxic and the antimicrobial effects were performed using the MTT assay and the minimum inhibitory concentration (MIC) test, respectively. Owing to the noticeable effect on antiproliferation of hot-water extracts, especially those from I. obliquus, the extract could be of great potential to be used as an alternative cancer therapy. However, it was not proven to have antifungal effect against A. fumigatus fungi.
Structural elucidation of viral antagonism of innate immunity: the STAT1 interface
Authors: Moseley Gregory W. , Hossain Alamgir , Larrous Florence , Zhan Jingyu , Sethi Ashish , Ibrahim Youssef , Aloi Maria , Rawlinson Stephen M. , Lieu Kim G. , Mok Yee-Foong , Griffin Michael D. W. , Ito Naoto , Ose Toyoyuki , Bourhy Hervé , Gooley Paul R.
Published: April 2019

To evade innate immunity, many viruses express interferon-antagonists that target STATs, critical mediators of immune signalling. Virus-STAT interfaces may provide new therapeutic targets but progress is hindered by a lack of direct structural data, owing to poor tractability of antagonists/full-length STATs for structural/biophysical approaches. By applying cross-saturation transfer NMR, we report the first direct structural analysis of binding of full-length STAT1 to an interferon-antagonist of a human pathogenic virus, the first such study of the virus-host interface. Analysis using mutation of the interface, biophysical characterization, immune signalling/protein-protein interaction assays including PCA, reverse genetics and animal infection demonstrated the significance of this interface in immune signaling suppression, and in disease caused by a pathogenic field-strain lyssavirus. Importantly, NMR/mutagenesis also revealed that the interface comprises multiple surfaces/domains in both the viral and cellular partners, indicating that antagonism involves extensive interactions consistent with a multifaceted inhibitory mechanism, distinct from ‘simple’ mechanisms such as tethering. Furthermore, by elucidating the spatial relationship of interactions critical to immune evasion and replication, the data provide insight into how ostensibly simple viruses can regulate these central functions via a single multifunctional protein. These data provide novel insights into fundamental viral biology, and potential exploitation of these mechanisms as new targets for antivirals and vaccine development. The study also demonstrates the power of biophysical/NMR approaches to elucidate the atomic interface of full-length STATs with regulatory proteins, providing a framework for studies to reveal immune evasion mechanisms of other pathogens in their full complexity.
Detoxifying potentials of two indigenous adsorbents: imarsil and activated charcoal in the reduction of aflatoxin in vegetable oils consumed in Nigeria
Authors: Eniola Oni , Flora Oluwafemi , Sarafadeen Kareem , Adebukunolami Omemu
Published: April 2019

Food contamination with aflatoxin is more prevalent in tropical regions where environmental conditions such as high temperature and humidity prevail, which favour the growth of toxigenic fungiand accumulation of these toxins in food and feeds represent a major threat to human and animal health. In lieu of the previously known adsorbents, adsorption studies of Aflatoxin (AF) were performed using inexpensive, readily available and local adsorbents Imarsil and activated charcoal (AC). Fifteen edible oils were purchases from open markets in Nigeria and screened for aflatoxin using High Performance Liquid Chromatography (HPLC). Thirteen out of the fifteen vegetable oil samples were positive to aflatoxin at the following concentration(172, 123, 195, 142, 46, 107, 96, 116, 22, 33, 228, 17 and 4) ng/kg while two had no detectable AF. Atsix different concentrations (0.5, 1, 1.5, 2, 2.5 and 3%) of Imarsil and activated charcoal (AC) with contact time of 1,2 and 3 h at room temperature (37 °C), the aflatoxin-adsorbing capabilities depend on the adsorbent concentrations and contact time. Imarsil demostrated 100 % adsorption efficiency within one hour. At AF contamination rates of 96–228 ng/kg, activated charcoal was not effective while Imarsil had 100 % removal efficiency within 3 h witha significant reduction (P<0.05) observed at the highest contamination rate and adsorbent concentration. AC demonstrated very mild adsorption activity. Results from this study indicated that Industrial incorporation of Imarsilinto the oil refining process would reduce greatly the menace of aflatoxicosis. Hence, the use of Imarsilshould be encouraged.
Investigating the efficacy and improved stability against Staphylococcus aureus of Lynronne-1D, a modified rumen microbiome derived antimicrobial peptide
Authors: Lawther Katie , Oyama Linda , Huws Sharon
Published: April 2019

Antimicrobial resistant infections are at a crisis point, posing a massive threat to human health with an anticipated annual mortality of 10 million people by 2050. Antimicrobial peptides (AMPs) are a promising solution to treat drug resistant infections, with the highly competitive ecosystem of the rumen microbiome being a fruitful resource for mining AMPs. In this study, we aimed to improve the stability of a known rumen AMP with great therapeutic potential, Lynronne-1. This AMP is efficacious against topical skin infections of Methicillin resistant Staphylococcus aureus but has no activity in systemic infections when administered intravenously due to degradation by peptidases. To overcome this hurdle and improve its use intravenously, we substituted the L isoforms N and C terminal amino acid residues to d-isoforms, thereby increasing the stability of the peptide in the presence of trypsin by three-fold. The activity of the modified peptide, named Lynronne-1D against S. aureus was subsequently investigated. Lynronne-1D retained its antimicrobial activity with an MIC of 8 µg ml−1 against S. aureus and improved MICs (>4 fold) in Gram-negative bacteria strains. The peptide had rapid and potent bactericidal activity causing a ≥6 log c.f.u./ml reduction in viable S. aureus cells within 30 min of treatment. It induced membrane permeabilization within 5 min and successfully prevented biofilm formation by S. aureus cells. Lynronne-1D was also non-cytotoxic to mammalian blood cells. The improved properties of Lynronne-1D over the original peptide makes it a promising therapeutic agent for the treatment of systemic infections of S. aureus.
Regulation of exploratory growth and antibiotic production in Streptomyces venezuelae by the two-component system CutRS
Authors: McLean Thomas C. , Wilkinson Barrie , Hutchings Matthew I.
Published: April 2019

Antibiotic production and cellular development in bacteria are intimately linked to the extracellular environment. One key mechanism by which bacteria recognise and respond to these external cues is through two-component regulatory systems (2CS). Consisting of a membrane-bound sensor kinase and a cognate DNA-binding response regulator, these 2CSs are essential in the response to a myriad of signals including antibiotic attack, microbial interaction and nutrient availability. The soil-associated filamentous actinobacteria Streptomyces spp. are prolific antibiotic producers with a large number of 2CS. In addition to their complex life cycle a new developmental stage has recently been described termed exploration. When exploring, the streptomycetes rapidly expand via non-branching vegetative hyphae when contacted by fungi, a juxtaposition to the canonical lifecycle which ends in sporulation. Fifteen of the fifty-six 2CS in the model organism Streptomyces venezuelae are highly conserved throughout Streptomycetaceae. Having developed and screened a 2CS operon deletion library within S. venezuelae we determined that one of these, cutRS, is involved in co-ordinating exploration and antibiotic production. The cutRS deletion mutant displays unrepressed exploration and overproduction of chloramphenicol. With a greater understanding of how Streptomyces spp. identify and respond to external signals we can imitate and subvert these systems. Using this we aim to activate cryptic biosynthetic gene clusters enabling the discovery of novel antimicrobial products which may prove beneficial in the clinical setting.
Utilising novel antimicrobial peptides identified from the rumen microbiome as a potential treatment for multiple A. baumannii strains
Authors: Alexander Peter , Oyama Linda , Huws Sharon
Published: April 2019

Antibiotic and antimicrobial resistance is a global issue, not only for medical and veterinary treatment, but also for economic development. One of the critical bacterial pathogens known to develop resistance to antimicrobial agents is Acinetobacter baumannii, one of the ESKAPE pathogens. A. baumannii is an aerobic Gram-negative coccobacillus bacterium associated with bacteraemia, urinary tract infections and ventilator-associated pneumonia, and is typically viewed as an opportunistic pathogen. Previous research has shown that clinical strains of A. baumannii have susceptibility to novel antimicrobial peptides, specifically those identified from a rumen metagenomic dataset. Using standardised 96 well MIC testing, 3 antimicrobial peptides (Lynronne-1, Lynronne-2, Lynronne-3, identified as part of a previous research project by the Huws Lab) were compared against multiple strains of A. baumannii, in order to show efficacy of treatment against a wider variety of strains. Of the strains tested, a number were clinical isolates with demonstrated resistance (imipenem resistant, OXO-23/OXO-50). The results show that the antimicrobial peptides had noticeable inhibitory effects on the bacterial growth. There was also variation between the 3 peptides utilised, with Lynronne-1 appearing to have the lowest MIC over the majority of the strains tested. Further research as part of this project will utilise other identified antimicrobial peptides, including rationally designed peptides, as well as potentially using another gastrointestinal microbiome metagenomic dataset to identify a greater number of novel antimicrobial peptides.
iPSCs derived from endothelial progenitors model latency and reactivation during human cytomegalovirus infection
Authors: Poole Emma , Huang Chris , Forbester Jessica , Shnayder Miri , Nachson Aharon , Kweider Baraa , Basaj Anna , Smith Daniel , Jackson Sarah , Kiskin Fedir , Roche Kate , Murphy Eain , Wills Mark , Dougan Gordon , Stern-Ginossar Noam , Rana Amer , Sinclair John
Published: April 2019

To date, models of human cytomegalovirus (HCMV) latency and reactivation have depended on the use of primary myeloid cells, which have limited availability, are difficult to culture and are challenging to genetically modify. We now show that induced pluripotent stem cells (iPSCs) derived from circulating late outgrowth endothelial progenitors (EPC) can be differentiated down the myeloid lineage, where HCMV latent carriage and reactivation is known to occur in vivo, and act as a model to allow the interrogation of viral and cellular factors involved in latency and reactivation of this persistent human pathogen. In contrast, monocytes generated from iPSCs derived from de-differentiated fibroblasts failed to support HCMV latent carriage. These iPSCs derived from EPCs may also be suitable for in depth genetic interrogation of other viruses which also infect cells of the myeloid lineage, such as HIV and Zika.
Antimicrobial activity of Zamzam water against Salmonella typhii
in vitro
Authors: Utami Amalia Tri
Published: April 2019

Objective

To determine the antimicrobial effect of zam zam water against Salmonella typhi in vitro. The antimicrobial effect was measured from the minimum inhibitory concentration (MIC) and the minimal bactericidal concentration (MBC) of zamzam water against Salmonella typhi.

Design

This experimental study used post-test only control group design with four time repetition. Step one was cultivating bacteria in liquid medium with various concentration of extract, that was 1 %, 1.25 %, 1.5 %, 1.75 %, 2 % with two control, extract control and bacterial control.

Results

The MIC (Minimal Inhibition Concentration) is 1.5 % concentration of extract. Step two was plating in NAP (Nutrient Agar Plate) medium. The MBC (Minimal Bactericidal Concentration) is 1.75 % concentration of zamzam water. The result of experiment was knew there are different average of Salmonella typhi colony from every group. The result experiment was analyzed by One Way Anova Test. The hypothesis test of MBC show significant differentiation, and then was continued with regression test. The conclusion of this study was zamzam water have antimicrobial activity against Salmonella typhii
in vitro.
Discovering novel antimicrobials from Streptomyces formicae, a symbiont of fungus farming plant ants, using CRISPR/Cas9 genome editing
Authors: Devine Rebecca , Qin Zhiwei , Wilkinson Barrie , Hutchings Matt
Published: April 2019

Finding new antimicrobial compounds is vital to combat the growing threat of resistance. Most currently used antibiotics originate from actinomycetes discovered more than half a century ago. We recently reported the new species Streptomyces formicae, isolated from the African fungus-farming plant-ant, Tetraponera penzigi. S. formicae produces a novel family of polyketide antibiotics, the formicamycins, that have potent activity against resistant pathogens including MRSA and vancomycin-resistant Enterococci (VRE). Using CRISPR/Cas9, we have identified and characterised the genes responsible for formicamycin biosynthesis in the native producer. In addition, we used cappable RNA- and ChIP-sequencing to determine the transcriptional organisation of the pathway. We exploited this information to generate multiple mutants of S. formicae that overproduce formicamycins and their biosynthetic intermediates, some of which also have bioactivity. Furthermore, the potential for novel chemistry from S. formicae is not limited to the formicamycin pathway; antiSMASH analysis shows this talented strain contains at least 45 secondary metabolite biosynthetic gene clusters (BGCs). Under standard laboratory conditions, wild-type S. formicae also exhibits antifungal activity against the drug resistant Lamentospora prolificans, and when the formicamycin BGC is deleted, the strain produces even more potent antibacterial activity against MRSA. To identify the biosynthetic pathways for these metabolites, entire BGCs up to 208 kbp were deleted using CRISPR. Overall, this work demonstrates that searching under-explored environments for new species combined with genome editing is a promising route towards finding novel anti-invectives.
Investigating alternative AUG usage in avian Influenza A virus segment 2
Authors: Billington Elizabeth , Pinto Rute Maria , Shelton Holly , Digard Paul
Published: April 2019

Influenza A viruses (IAV) have a segmented, negative sense RNA genome. PB1-F2 is an IAV accessory protein encoded by segment 2, in the +1 reading frame. IAVs from avian hosts generally encode full length PB1-F2s, which contrasts with human IAVs which frequently have C-terminal truncations. Many reported activities of PB1-F2, including innate immune antagonism, require motifs in its C-terminal domain. Full length PB1-F2 is translated from AUG 4 of segment two, but one or more of AUGs 7, 8 and 9 may also serve as independent initiation codons for the C-terminal domain. Products from the AUGs 7-9 are expressed during infection by a vaccine strain IAV, but their presence or absence had no effect on virus growth in vitro. We generated a panel of isogenic viruses, containing segment 2 from an avian H5N1 IAV, which differed in the presence or absence of the various AUG start codons in segment 2. No difference in growth kinetics in vitro or viral polymerase activity, measured using a mini-replicon assay, was observed for any of these mutants. However a significant difference in mean plaque size on MDCK cells was seen when individual changes were made to any of AUGs 7-9, suggesting a subtle effect on virus fitness possibly caused by loss of expression of PB1-F2 C-terminal fragments. In addition structural predictions suggest that the AUG mutations will affect secondary structure of full length PB1-F2. Our works suggests segment 2 protein expression from multiple AUGs could impact of the virus replication cycle.
The effect of antibiotic and nutrient limitation to antibiotic resistant bacteria in single-cell level
Authors: Liu Li , Kreft Jan , Vigolo Daniele
Published: April 2019

Heterogeneity in bacterial populations can manifest in various ways, such as resistant cells, which can be observed in harsh environments after the use of antibiotics. Many studies have looked at the evolution of resistance and the effect of inhibitory and sub-inhibitory concentrations of antibiotics by batch culture measurements without considering the heterogeneity of bacterial populations. But antibiotic susceptibility and fitness costs of resistance mutations or plasmids are affected by the growth rate and physiology of individual cells. Single-cell analysis in microfluidic systems has opened up new possibilities enabling us to investigate the various putative mechanisms behind the persistence phenomenon required direct observation under the microscope. In this study, we use a gradient mixer and a novel micro-chemostat, to create concentration gradients of growth substrates and/or antibiotics to study the effect of nutrient and antibiotic concentration on individual cells growing under constant and defined conditions in cell-sized channels. The single-cell elongation, morphology and growth rate of ribosome-targeting antibiotics resistant E. coli was tracked by combining the microfluidics, microscope phase contrast imaging and fluorescent tag in high throughput mode. A mechanistic cellular model was used to describe the reaction between antibiotics and ribosome and the resulting effects on bacterial growth; then we linked the intracellular chemical-reaction kinetics processes to the population level and predicted the behaviour of population responses. Our approach has enabled the investigation of single-cell individuality and predictions of population dynamics under different environment.
Characterisation of extended spectrum beta-lactamases (ESBL) resistance in multi-drug resistant Salmonella concord
Authors: Lee Winnie , Greig David , Day Martin , Chattaway Marie , Nair Satheesh
Published: April 2019

Salmonellosis in children from Ethiopia is caused mainly by Salmonella Concord, which are highly invasive, multi-drug resistant and extended-spectrum β-lactamase (ESBL) producers. S. Concord infections have been observed in children adopted from Ethiopia, now living in Europe and United States. S. Concord infections are present in parents of these adopted children, posing a significant dilemma for treatment. Data from Salmonella isolates are stored in Public Health England’s in-house Gastro Data Warehouse (GDW) database. Resistance profiles for 37 pure S. Concord isolates were determined using agar incorporation methods using EUCAST guidelines. The minimum inhibitory concentration (MIC) was determined for 18 antimicrobial agents and ESBL resistance was confirmed using Double Disk Synergy and ESBL E-test methods. ESBL resistance was present in 8 isolates with resistance most commonly seen against penicillins and cephalosporins. Isolates 408 and 537, and isolate 527 displayed resistance to 78 % and 56 % of the antibiotics respectively. Drug resistant regions in isolates 408, 527 and 537 that have been previously sequenced by an Illumina HiSeq 2500 were characterised. ESBL genes blaCTXM-15 was present in isolate 527 and 408 and blaSHV-12 was found in isolate 537. Replicons from plasmids, InCI2, InCFIB, InCF2 and IncH2 were located in the assembled sequences of the three previously sequenced isolates. In conclusion, the possible mechanisms causing spread of ESBL resistance in S. Concord is most likely due to acquisition of plasmids through horizontal gene transfer from various Enterobacteriaceae, however further research must be conducted to confirm this to advance antimicrobial research in this area.
HIV-1 Vpr accessory protein interacts with REAF and mitigates its associated anti-viral activity
Authors: Gibbons Joseph , Marno Kelly , Pike Rebecca , Lee Jason , Jones Christopher , Ogunkolade William , Pardieu Claire , Warnes Gary , Rowley Paul , Sloan Richard , McKnight Aine
Published: April 2019

The accessory protein Vpr of Human Immunodeficiency Virus type 1 (HIV-1) enhances replication of the virus in macrophages. Virus particle packaged Vpr is released in target cells shortly after entry, suggesting it is required early in infection. Why it is required for infection of macrophages and not cycling T-cells and why it induces G2/M arrest in cycling cells are unknown. Here we observe, by co-immunoprecipitation assay, an interaction between Vpr and endogenous REAF (RNA-associated Early-stage Antiviral Factor, RPRD2), a protein shown previously to potently restrict HIV infection. After HIV-1 infects macrophages, within 30 min of viral entry, Vpr induces the degradation of REAF. Subsequently, as replication continues, REAF expression is upregulated – a response which is curtailed by Vpr. REAF is more highly expressed in differentiated macrophages than in cycling T-cells. Expression in cycling cells is cell-cycle dependent and knockdown induces cell-cycle perturbation. Therefore, our results support the long held hypothesis that Vpr induces the degradation of a factor involved in the cell cycle that impedes HIV infection in macrophages.
Correlating prophage presence in Helicobacter pylori with restriction-modification systems
Authors: Crawford Liam , Zea Roxana Zamudio , Millard Andrew , Beleza Sandra , Oggioni Marco
Published: April 2019

The human pathogen Helicobacter pylori colonises approximately half of the global population and infectioncan lead to a range of gastric diseases. The temperate bacteriophages in H. pylorihave been poorly characterised, most likely due to a very high number and strain-to-strain variability of restriction modification (RM) systems, which can be easily more than 20 in any strain. This work aims to study the prevalence of bacteriophages and RM systems in over 460 strains of H. pyloriisolated from 184 gastric samples from asymptomatic subjects. H. pyloriprophages were identified using the PHASTER tool. The gold standard RM systems were downloaded from Rebase and the RM genes were identified in the 460 genomes using Blast+. The analysis for phage genomes showed 57 intact bacteriophages, ranging from 12 to 30 Kb in length, in 25 subjects (14 %). Approximately half of the bacteriophages were shown to have integrated in the Lipid A biosynthesis gene lpxD. Using 107 RM system genes, we built a presence/absence matrix of the genes in all our H. pylori genomes, which was ordered according to a maximum likelihood gene presence/absence tree generated by FastTree. This clustering revealed the presence of multiple pairs of strains, one strain carrying an intact prophage whereas the other is lacking a prophage but containing the same RM systems, potentially allowing for a prophage donor and recipient pair. The availability of this panel of donor and recipient strain pairs is an ideal starting point to study the molecular biology of bacteriophages in H. pylori.
Signal-integration through PhoPQ enables Salmonella to adapt to heterogeneous tissue microenvironments
Authors: Cianfanelli Francesca Romana , Bumann Dirk
Published: April 2019

Enteric fever is major health issue in developing countries and it is becoming progressively untreatable due to increase of antimicrobial resistance. The causative agent, Salmonella enterica, replicates in host phagocytes in various organs and regulates expression of hundreds of genes in response to host signals. PhoPQ is one of the key regulators and essential for virulence in humans and in a mouse typhoid fever model. The sensor kinase PhoQ responds to diverse stimuli (Mg2+ limitation, low pH, cationic antimicrobial peptides, high osmolarity, and, indirectly, to reducing conditions). However, which signals are predominant in vivo remains unclear. To address this issue, we determined the activity of the PhoPQ regulon using a chromosomally encoded fusion of the PhoP-dependent phoNp promoter to gfp-ova and we quantified single-cell fluorescence levels of Salmonella in spleen of infected mice using flow cytometry. The results show extensive heterogeneity in PhoP-activities in the Salmonella population. Comparison of mutants with sensing defects suggests that, in vivo, PhoQ responds to a combination of antimicrobial peptides, acidic pH and low Mg2+, but not to reducing conditions. Negative feedbacks have also only a minor impact. Single-cell analysis of phoNp and asrp promoters suggests differential environmental pH as a major driver of heterogeneous PhoP activities. A combination of immunohistochemistry and proteomics of Salmonella from infected samples was used to validate this hypothesis. Together, our data show how Salmonella uses the PhoPQ system to integrate various host signals in order to tune expression of virulence factors to the diverse tissue microenvironments that this pathogen inhabits.
The role of acetyl-phosphate in the pathogenesis of Neisseria gonorrhoeae
Authors: Felix Diaz Parga Ernesto , Shaw Jonathan G.
Published: April 2019

Acetyl-phosphate (AcP), an intermediate from the phosphotransacetylase-acetate kinase (PTA-AK) pathway, has shown to be critical in pathogenic bacteria for the general metabolism and synthesis of virulence factors. Lysine acetylation is a post-translational modification (PTM) that occurs enzymatically and non-enzymatically by the addition of an acetyl residue from acetyl coenzyme A and AcP, respectively. Neisseria gonorrhoeae, the etiologic agent of gonorrhoea, has been shown to use AcP for lysine acetylation, however, the role that AcP has in the pathogenesis and how acetylation is regulated has not been discerned. The concentration of AcP was altered in N. gonorrhoeae MS11 by interrupting the genes involved in the PTA-AK pathway, pta and ackA, and the gene that encodes for a lysine deacetylase family protein, kdac. AcP concentrations were increased in ΔackA and decreased in Δpta resulting in modulation of lysine acetylation. Growth on glucose, lactate or pyruvate were investigated. In aerobic conditions, ΔackA mutant solely grew in glucose, while the Δpta mutant grew in glucose and lactate. In microaerophilic conditions, ΔackA and Δpta mutants solely grew in presence of glucose. The virulence of ΔackA and Δpta was tested by infecting larvae of Galleria mellonella. WT killed 50 % population (n=15) after 6 days and ΔackA after 24 h, however, Δpta after 6 days it only killed 10 %. Taken together, our results show AcP as an important metabolite for the metabolism and virulence of N. gonorrhoeae.
Fitting linear mixed models to a highly-structured dataset effectively controls for population structure in bacterial genome-wide association studies
Authors: Kidman Samuel , Ukor Emem-Fong , Floto Andres , Parkhill Julian
Published: April 2019

Cystic fibrosis is an inherited, autosomal recessive disease that causes an accumulation of viscous mucus within the lung, leading to chronic bacterial infections. Pseudomonas aeruginosa is a Gram-negative, opportunistic pathogen that can invade the cystic fibrosis lung, and is the most common bacteria isolated from cystic fibrosis patients. In the largest study of its kind, ∼4200 P. aeruginosa isolates were collected from nine cystic fibrosis patients. The isolates were whole-genome sequenced and screened for 14 virulence-related phenotypes. We aim to associate phenotype with genotype by fitting both linear models and linear mixed models association tests. During this study, linear mixed models were found to effectively control for strong population structure signals, allowing relevant associations to be uncovered. When sub-sampling the dataset into unstructured, single-patient groups, fitting linear mixed models were found to associate genotype with phenotype as effectively as fitting linear models. We also found that aggregating non-synonymous SNPs in the same gene to a single knockout mutation increases the power of the association tests to identify relevant associations. The future direction of this study will involve filtering the results to identify any phenotypically-relevant associations. These putative associations will then be experimentally verified.
The application of CRISPR/Cas9 system in the generation of viral vectored avian influenza vaccines
Authors: Chang Pengxiang , Sadeyen Jean-Remy , Sealy Joshua , Bhat Sushant , Iqbal Munir
Published: April 2019

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.
Analysis of the novel role of NS5A domain I in the assembly of infectious hepatitis C virus particles
Authors: Chen Shucheng , Harris Mark
Published: April 2019

Hepatitis C virus (HCV) is an enveloped virus with a positive-sense, single-stranded RNA of approximately 9.6 kb, a member of the genus Hepaci virus within the family Flaviviridae. The genome contains a single large open reading frame encoding a 3000 residue polyprotein. The non-structural 5A protein (NS5A) is a highly phosphorylated protein, whichis comprised of three domains (I, II and III). Previously, we demonstrated that two residues within NS5A domain I (V67 and P145) play critical roles in HCV assembly challenging the dogma that NS5A domain I exclusively participated in genome replication. In this study, we identified 8 surface exposed residues of domain Iwhich were located in close proximity to V67 and P145. The mutants were cloned into a JFH-1 derived subgenomic replicons (mSGR-luc-JFH1) to confirm whether they are required in genome replication. The results of luciferase assay suggested that I52A exhibited the same phenotype as V67A and P145A and is a further candidate for regulating assembly of HCV. In parallel we sought to investigate whether domain I was involved in the interaction of NS5A with cyclophilin A (CypA), a cellular peptidyl-prolyl isomerase required for HCV replication. CypA can be inhibited by cyclosporin A (CsA), which also inhibits HCV genome replication. Surprisingly, all three mutant replicons (I52A, V67A and P145A) were more sensitive to CsA treatment than wildtype, suggesting that domain I does indeed interact with CypA. Ongoing studies will therefore investigate the roles of both domain I and CypA in genome replication and assembly of infectious HCV particles.
Encapsidation of viral RNA in Picornavirales: studies on cowpea mosaic virus demonstrate dependence on viral replication
Authors: Peyret Hadrien , Kruse Inga , Saxena Pooja , Lomonossoff George
Published: April 2019

To elucidate linkage between replication and encapsidation in Picornavirales, we have taken advantage of the bipartite nature of the plant-infecting member of the order, cowpea mosaic virus (CPMV), to decouple the two processes. RNA-free virus-like particles (eVLPs) can be generated by transiently co-expressing the RNA-2-encoded coat protein precursor (VP60) with the RNA-1-encoded 24K protease, in the absence of the replication machinery (Saunders et al., 2009). We have made use of the ability to produce assembled capsids of CPMV in the absence of replication to examine the putative linkage between RNA replication and packaging in the Picornavirales. We show here that the remarkable specificity of packaging observed in CPMV is due to a functional linking between the two processes of viral replication and encapsidation. We have created a series of mutant RNA-1 and RNA-2 molecules and have assessed the effect of the mutations on both the replication and packaging of the viral RNAs. We demonstrate that mutations that affect replication have a concomitant impact on encapsidation, and that RNA-1-mediated replication is required for encapsidation of both RNA-1 and RNA-2. This close coupling between replication and encapsidation provides a means for the specific packaging of viral RNAs. Moreover, we demonstrate that this feature of CPMV can be used to specifically encapsidate custom RNA by placing a sequence of choice between the RNA-2 sequences required for replication, which opens the door to novel research and therapeutic applications in the field of custom RNA packaging and delivery technologies.
The use of microbiological methods to reduce aflatoxin M1 in cheese
Authors: Gonçalves Bruna , Henck Jennifer , Uliana Rômulo , Kamimura Eliana , Oliveira Carlos , Corassin Carlos
Published: April 2019

Studies have shown evidence of human exposure to aflatoxin M1 due to the consumption of contaminated milk and dairy products (mainly cheeses). This poses a great risk to public health, since milk and milk products are frequently consumed by a portion of the population considered immunosuppressed, children and the elderly. Knowledge of the negative impacts of aflatoxins on health and economics has led to investigations of strategies to prevent their formation in food, as well as to eliminate, inactivate or reduce the bioavailability of these toxins in contaminated products This study evaluated the effect of microbiological methods using lactic acid bacteria on aflatoxin M1 (AFM1) reduction in Minas Frescal cheese (typical Brazilian product, being among the most consumed cheeses in Brazil) spiked with 1 µg l−1 AFM1. Inactivated lactic acid bacteria (0, 5%, v/v de L. rhamnosus e L. lactis) were added during the cheese production process. Nine cheeses were produced, divided into three treatments: negative controls (without AFM1 or lactic acid bacteria), positive controls (AFM1 only), and lactic acid bacteria+AFM1. Samples of cheese were collected on days 2, 10, 20 and 30 after the date of production and submitted to composition analyses and determination of AFM1 by high performance liquid chromatography. The reductions of AFM1 in cheese by lactic acid bacteria at the end of the trial indicate a potential application of inactivated lactic acid bacteria in reducing the bioavailability of AFM1 in Minas frescal cheese without physical-chemical and microbiological modifications during the 30 day experimental period.
Mutasynthesis of novel prodiginines derived from the antibiotic prodigiosin by exploiting substrate specificity of H2MAP oxidases PigB and HapB
Authors: Couturier Maxime , Bhalara Hiral , Salmond George P.C. , Leeper Finian J.
Published: April 2019

The natural product, prodigiosin, caught researchers’ interest more than a century ago because of its bright red color. Today, interest in this tripyrrolic secondary metabolite remains strong due to its biological effects, including potent antibiotic activity against various Gram-positive bacteria. Further exploration of the potential of this class of molecules requires libraries of analogues. Yet, the total synthesis of prodigiosin-like compounds (prodiginines) proves challenging. This can be overcome by highjacking the bacterial biosynthetic machinery via mutasynthesis. Although a number of different bacteria produce prodigiosin, its biosynthetic pathway is well conserved. The final precursors, MAP and MBC, are always produced independently, before a terminal condensation reaction. Our work focuses on the last step of the formation of MAP: the oxidation of H2MAP. We were able to isolate and characterize HapB, the enzyme catalyzing this reaction in Hahella chejuensis. In addition, we showed that some modifications of alkyl substituents on the C2 and C3 positions of H2MAP did not alter HapB activity significantly. We then fed these analogues of H2MAP into Serratia ΔpigD, a mutant of Serratia ATCC sp.39006 which does not produce any endogenous H2MAP. As expected from the in vitro testing, chain elongation past two carbons on the C2 position could not be accepted whereas all substrates with a modification on the C3 position restored pigmentation, leading to the formation of eight novel prodiginines.
Food or a free ride? The ability of a marine microbial community to degrade plastics
Authors: Wright Robyn , Gibson Matthew , Christie-Oleza Joseph
Published: April 2019

Recalcitrant polymers are widely distributed in the environment. This includes natural polymers, such as chitin, but also synthetic polymers, which are becoming increasingly abundant, and for which biodegradation is uncertain. Distribution of labour in microbial communities commonly evolves in nature, particularly for arduous processes, suggesting that a community may be better at degrading recalcitrant compounds than individual microorganisms. Previous work, carried out by us, showed that a microbial community could be selected for the efficient degradation of chitin, but if the community was left for too long then the function decayed as the active members were replaced by cheaters and grazers. Here we aimed to determine whether (i) a marine microbial community may be capable of degrading the common packaging plastic poly(ethylene terephthalate) (PET), (ii) whether we see the same pattern of community succession as we do with chitin and (iii) if this community may be better able to degrade PET than an individual organism. We incubated several different types of PET with the natural microbial community found colonising coastal marine debris, and characterised the microbial community succession across the incubation period. We show an enrichment of taxa that have previously been shown to be capable of the degradation of recalcitrant compounds, and we show that this community is capable of growing faster than an individual organism. We are currently determining the correlation between microbial community structure and the concentration of breakdown products from PET, as well as the mechanisms that are used for this degradation.
Studying the mammalian adaptation potential of influenza A viruses in a single infection cycle
Authors: Khalid Zakaria Mohammad , Frise Rebecca , Peacock Thomas , Barclay Wendy S , Shelton Holly
Published: April 2019

Various influenza A viruses (AIVs) have infected humans in the last decade. These infections result from spill over into humans at times of AIV poultry outbreaks with severe clinical consequences for those infected. These AIVs do not have the ability to be efficiently spread among humans via respiratory droplets or aerosols and subsequently cause a pandemic. However, adaptation of these avian influenza viruses to humans by mutation or reassortment may change this. We know that AIVs require adaptation of several critical characteristics in order to effectively spread between humans. Therefore, in order for human pandemic emergence, AIV that crosses into a human host must already be sufficiently able, or must rapidly adapt to overcome these constraints to transmit to a subsequent human host before infection is cleared by an immune response. In this study, we used mice and analysed the mutations acquired by avian origin H7N9 and H9N2 isolates in a temporal fashion during a single infection cycle in individual hosts. Viruses recovered from infected mice lungs were subjected to Next Generation sequencing (NGS) to identify mammalian adaptation enhancing mutations. Moreover, the timing of these mutations was correlated to the host response, with particular emphasis on cytokine profile and innate immune responses during the time prior to and following viral sequence changes to help understand the virus-host dynamics and the host environment favouring adaptation. Furthermore, understanding of the determinants and mechanisms of adaptation and transmission may aid in assessing the risks posed by avian influenza A viruses to human health.
The VP4 protein from a very virulent IBDV strain antagonises type I IFN responses to a greater extent than the VP4 from a classical strain
Authors: Dulwich Katherine , Giotis Efstathios , Skinner Michael , Broadbent Andrew
Published: April 2019

Infectious bursal disease virus (IBDV) infects B cells in the bursa of Fabricius (BF) causing morbidity, mortality and immunosuppression in infected chickens. Classical strains (e.g. F52/70) cause 1–2 % mortality whereas very virulent strains (e.g. UK661) cause over 60 % mortality for reasons that remain poorly understood. We inoculated birds with either F52/70 or UK661, and found that the expression of pro-inflammatory and type I IFN-related genes was significantly down-regulated in UK661 compared to F52/70 infected birds (P<0.05), despite no statistically significant difference in peak virus titres between the two strains. This was also observed in vitro in an immortalised B cell line where UK661 caused significantly reduced IFNβ and Mx1 expression compared to F52/70 (P<0.05). The IBDV protease (VP4) has previously been reported to act as a type I IFN antagonist, although it remains unknown whether this is characteristic of all IBDVs or a strain-specific phenomenon. Using a luciferase reporter assay, we compared the IFNβ production in DF-1 cells in response to poly I:C stimulation in the presence of eGFP-VP4 expression plasmids, finding UK661 VP4 was able to down-regulate IFNβ production to a greater extent than F52/70 VP4 (P<0.01). There are 9 amino acid differences between the two VP4 proteins and we are identifying those contributing to the observed phenotype. Taken together, our data suggest that the VP4 protein in very virulent IBDV strains evolved a greater ability to antagonise type I IFN responses than classical strains which may, in part, explain their enhanced virulence.