First come, first served : superinfection exclusion in Deformed wing virus is dependent upon sequence identity and not the order of virus acquisition

Funding: Biotechnology and Biological Sciences Research Council (BBSRC), BB/M00337X/2.Deformed wing virus (DWV) is the most important globally distributed pathogen of honey bees and, when vectored by the ectoparasite Varroa destructor, is associated with high levels of colony losses. Divergent DWV types may differ in their pathogenicity and are reported to exhibit superinfection exclusion upon sequential infections, an inevitability in a Varroa-infested colony. We used a reverse genetic approach to investigate competition and interactions between genetically distinct or related virus strains, analysing viral load over time, tissue distribution with reporter gene-expressing viruses and recombination between virus variants. Transient competition occurred irrespective of the order of virus acquisition, indicating no directionality or dominance. Over longer periods, the ability to compete with a pre-existing infection correlated with the genetic divergence of the inoculae. Genetic recombination was observed throughout the DWV genome with recombinants accounting for ~2% of the population as determined by deep sequencing. We propose that superinfection exclusion, if it occurs at all, is a consequence of a cross-reactive RNAi response to the viruses involved, explaining the lack of dominance of one virus type over another. A better understanding of the consequences of dual- and superinfection will inform development of cross-protective honey bee vaccines and landscape-scale DWV transmission and evolution.Publisher PDFPeer reviewe

Novel macrophage microbicidal responses against gram-positive bacteria

Antimicrobial resistance is a major global health threat, and there is growing interest in how modulation of the host immune response can enhance pathogen killing and reduce reliance on antimicrobials. One target cell is the macrophage; a key innate immune cell that possesses a range of microbicidal mechanisms and can combine responses for optimal pathogen killing. Streptococcus pneumoniae and Staphylococcus aureus are important gram-positive pathogens that represent differing intracellular burdens for the macrophage. A key macrophage microbicidal mechanism relevant to the killing of these pathogens is production of reactive oxygen species (ROS). While NADPH oxidase-derived ROS is an early response to infection, mitochondrial ROS (mROS) production is a later response and is enhanced during infection by alterations in mitochondrial dynamics. ROS and mROS can combine with other macrophage responses to facilitate pathogen killing, therefore the significance and potential for such interplay with other host defence mechanisms to enhance macrophage killing of pathogens such as S. pneumoniae and S. aureus is the focus of this thesis, with specific attention to mitochondrial-associated responses and the microbicidal and immunomodulatory host defence peptide cathelicidin. The data presented in this thesis show that expression of the CAMP gene, encoding cathelicidin, was upregulated by vitamin D in macrophages, was synergistically enhanced by bacterial infection or phenylbutyrate and was impaired by pro-inflammatory cytokines. Cathelicidin directly killed extracellular S. pneumoniae and contributed to early macrophage killing of intracellular S. aureus when bacterial burden was high. Mitochondrial adaptations to S. pneumoniae were more prevalent in macrophages during later stages of bacterial challenge and included increased mitochondrial fission and increased mROS production. Mitochondrial adaptations to S. aureus, which stresses macrophage microbicidal responses to a greater extent than S. pneumoniae, were observed during early stages of bacterial challenge. The regulators of canonical fission, dynamin-related protein 1 (Drp1) and mitochondrial fission factor (Mff), failed to influence overall levels of fission in the initial response to S. aureus. In contrast, Drp1 regulated localisation of mROS to intracellular S. aureus in a subset of macrophages, suggesting roles in mROS delivery to bacterial-containing phagolysosomes. In regard to mechanisms of mROS production, I have provided evidence that reverse electron transport (RET) occurs as an early response to S. pneumoniae challenge, but not late S. pneumoniae, or S. aureus challenge. S. aureus enhanced mROS production in macrophages, and while NADPH oxidase-derived ROS was the greater contributor to early killing of S. aureus, mROS also contributed to killing. Cathelicidin enhanced microbicidal responses against S. aureus particularly when NADPH oxidase-derived ROS generation was impaired, but also appeared to function as a brake on alterations in mitochondrial dynamics and mROS production in the presence of bacteria, therefore potentially regulating mitochondrial homeostasis. Results in this thesis demonstrate that macrophages use ROS, alterations in mitochondrial dynamics and mROS, and cathelicidin to combat S. pneumoniae and S. aureus infections with pathogen-dependent kinetics. Macrophages adapt responses to different pathogens to ensure a multi-layered immune response to clear pathogens. The work in this thesis provides greater insight into macrophage microbicidal responses to S. pneumoniae and S. aureus infection and could inform future therapeutic strategies to enhance macrophage microbicidal responses

The architecture of amyloid-like peptide fibrils revealed by X-ray scattering, diffraction and electron microscopy

Structural analysis of protein fibrillation is inherently challenging. Given the crucial role of fibrils in amyloid diseases, method advancement is urgently needed. A hybrid modelling approach is presented enabling detailed analysis of a highly ordered and hierarchically organized fibril of the GNNQQNY peptide fragment of a yeast prion protein. Data from small-angle X-ray solution scattering, fibre diffraction and electron microscopy are combined with existing high-resolution X-ray crystallographic structures to investigate the fibrillation process and the hierarchical fibril structure of the peptide fragment. The elongation of these fibrils proceeds without the accumulation of any detectable amount of intermediate oligomeric species, as is otherwise reported for, for example, glucagon, insulin and [alpha]-synuclein. Ribbons constituted of linearly arranged protofilaments are formed. An additional hierarchical layer is generated via the pairing of ribbons during fibril maturation. Based on the complementary data, a quasi-atomic resolution model of the protofilament peptide arrangement is suggested. The peptide structure appears in a [beta]-sheet arrangement reminiscent of the [beta]-zipper structures evident from high-resolution crystal structures, with specific differences in the relative peptide orientation. The complexity of protein fibrillation and structure emphasizes the need to use multiple complementary methods

Defining composition and function of the rhizosphere microbiota of barley genotypes exposed to growth-limiting nitrogen supplies

The microbiota populating the rhizosphere, the interface between roots and soil, can modulate plant growth, development, and health. These microbial communities are not stochastically assembled from the surrounding soil, but their composition and putative function are controlled, at least partially, by the host plant. Here, we use the staple cereal barley as a model to gain novel insights into the impact of differential applications of nitrogen, a rate-limiting step for global crop production, on the host genetic control of the rhizosphere microbiota. Using a high-throughput amplicon sequencing survey, we determined that nitrogen availability for plant uptake is a factor promoting the selective enrichment of individual taxa in the rhizosphere of wild and domesticated barley genotypes. Shotgun sequencing and metagenome-assembled genomes revealed that this taxonomic diversification is mirrored by a functional specialization, manifested by the differential enrichment of multiple Gene Ontology terms, of the microbiota of plants exposed to nitrogen conditions limiting barley growth. Finally, a plant soil feedback experiment revealed that host control of the barley microbiota underpins the assembly of a phylogenetically diverse group of bacteria putatively required to sustain plant performance under nitrogen-limiting supplies. Taken together, our observations indicate that under nitrogen conditions limiting plant growth, host-microbe and microbe-microbe interactions fine-tune the host genetic selection of the barley microbiota at both taxonomic and functional levels. The disruption of these recruitment cues negatively impacts plant growth

Quasi-continuous Interpolation Scheme for Pathways between Distant Configurations

A quasi-continuous interpolation (QCI) scheme is introduced for characterizing physically realistic initial pathways from which to initiate transition state searches and construct kinetic transition networks. Applications are presented for peptides, proteins, and a morphological transformation in an atomic cluster. The first step in each case involves end point alignment, and we describe the use of a shortest augmenting path algorithm for optimizing permutational isomers. The QCI procedure then employs an interpolating potential, which preserves the covalent bonding framework for the biomolecules and includes repulsive terms between unconstrained atoms. This potential is used to identify an interpolating path by minimizing contributions from a connected set of images, including terms corresponding to minima in the interatomic distances between them. This procedure detects unphysical geometries in the line segments between images. The most difficult cases, where linear interpolation would involve chain crossings, are treated by growing the structure an atom at a time using the interpolating potential. To test the QCI procedure, we carry through a series of benchmark calculations where the initial interpolation is coupled to explicit transition state searches to produce complete pathways between specified local minima.This work was supported by the Engineering and Physical Sciences Research Council [grant number EP/H042660/1]This document is the unedited Author’s version of a Submitted Work that was subsequently accepted for publication in the Journal of Chemical Theory and Computation, copyright © American Chemical Society after peer review. To access the final edited and published work see http://dx.doi.org/10.1021/ct300483

Mapping the Conformational Dynamics and Pathways of Spontaneous Steric Zipper Peptide Oligomerization

The process of protein misfolding and self-assembly into various, polymorphic aggregates is associated with a number of important neurodegenerative diseases. Only recently, crystal structures of several short peptides have provided detailed structural insights into -sheet rich aggregates, known as amyloid fibrils. Knowledge about early events of the formation and interconversion of small oligomeric states, an inevitable step in the cascade of peptide self-assembly, however, remains still limited