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

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

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

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

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.<br/

Evidence for and against deformed wing virus spillover from honey bees to bumble bees : a reverse genetic analysis

Funding: This work was supported by grant funding from BBSRC BB/M00337X/2 and BB/I000828/1. This research was also supported by the United States Department of Agriculture (USDA) National Institute of Food and Agriculture (NIFA) grant 2017-06481 (EVR).Deformed wing virus (DWV) is a persistent pathogen of European honey bees and the major contributor to overwintering colony losses. The prevalence of DWV in honey bees has led to significant concerns about spillover of the virus to other pollinating species. Bumble bees are both a major group of wild and commercially-reared pollinators. Several studies have reported pathogen spillover of DWV from honey bees to bumble bees, but evidence of a sustained viral infection characterized by virus replication and accumulation has yet to be demonstrated. Here we investigate the infectivity and transmission of DWV in bumble bees using the buff-tailed bumble bee Bombus terrestris as a model. We apply a reverse genetics approach combined with controlled laboratory conditions to detect and monitor DWV infection. A novel reverse genetics system for three representative DWV variants, including the two master variants of DWV - type A and B - was used. Our results directly confirm DWV replication in bumble bees but also demonstrate striking resistance to infection by certain transmission routes. Bumble bees may support DWV replication but it is not clear how infection could occur under natural environmental conditions.Publisher PDFPeer reviewe

Green bees:reverse genetic analysis of deformed wing virus transmission, replication, and tropism

Environmental and agricultural pollination services by honey bees, Apis mellifera, and honey production are compromised by high levels of annual colony losses globally. The majority are associated with disease caused by deformed wing virus (DWV), a positive-strand RNA virus, exacerbated by the ectoparasitic mite Varroa destructor. To improve honey bee health, a better understanding of virus transmission and pathogenesis is needed which requires the development of tools to study virus replication, transmission, and localisation. We report the use of reverse genetic (RG) systems for the predominant genetically distinct variants of DWV to address these questions. All RG-recovered viruses replicate within 24 h post-inoculation of pupae and could recapitulate the characteristic symptoms of DWV disease upon eclosion. Larvae were significantly less susceptible but could be infected orally and subsequently developed disease. Using genetically tagged RG DWV and an in vitro Varroa feeding system, we demonstrate virus replication in the mite by accumulation of tagged negative-strand viral replication intermediates. We additionally apply a modified DWV genome expressing a fluorescent reporter protein for direct in vivo observation of virus distribution in injected pupae or fed larvae. Using this, we demonstrate extensive sites of virus replication in a range of pupal tissues and organs and in the nascent wing buds in larvae fed high levels of virus, indicative of a direct association between virus replication and pathogenesis. These studies provide insights into virus replication kinetics, tropism, transmission, and pathogenesis, and produce new tools to help develop the understanding needed to control DWV-mediated colony losses