Draft whole genome sequences of the periodontal pathobionts Porphyromonas gingivalis, Prevotella intermedia and Tannerella forsythia contain phase variable restriction modification systems

The periodontal strains used were collected during previous studies performed at the University of Cardiff (strains WW414, WW855 and WW2096), University of Bristol (WW2834, WW2842, WW2866, WW2881, WW28585, WW2903, WW2931, WW2952, WW3039, WW3040, and WW3102), King’s College London (WW5019, WW5127, and WW10960) and Queen Mary University of London (WW11663). Illumina sequencing was performed by the NUCLEUS Genomics Core Facility and data analysis used the Spectre2 and Alice2 High Performance Computing Facility at the University of Leicester. This work was in part funded by a grant from the BBSRC (BB/N002903/1) to MRO.Peer reviewedPublisher PD

Diurnal Differences in Intracellular Replication Within Splenic Macrophages Correlates With the Outcome of Pneumococcal Infection

Circadian rhythms affect the progression and severity of bacterial infections including those caused by Streptococcus pneumoniae, but the mechanisms responsible for this phenomenon remain largely elusive. Following advances in our understanding of the role of replication of S. pneumoniae within splenic macrophages, we sought to investigate whether events within the spleen correlate with differential outcomes of invasive pneumococcal infection. Utilising murine invasive pneumococcal disease (IPD) models, here we report that infection during the murine active phase (zeitgeber time 15; 15h after start of light cycle, 3h after start of dark cycle) resulted in significantly faster onset of septicaemia compared to rest phase (zeitgeber time 3; 3h after start of light cycle) infection. This correlated with significantly higher pneumococcal burden within the spleen of active phase-infected mice at early time points compared to rest phase-infected mice. Whole-section confocal microscopy analysis of these spleens revealed that the number of pneumococci is significantly higher exclusively within marginal zone metallophilic macrophages (MMMs) known to allow intracellular pneumococcal replication as a prerequisite step to the onset of septicaemia. Pneumococcal clusters within MMMs were more abundant and increased in size over time in active phase-infected mice compared to those in rest phase-infected mice which decreased in size and were present in a lower percentage of MMMs. This phenomenon preceded significantly higher levels of bacteraemia alongside serum IL-6 and TNF-alpha concentrations in active phase-infected mice following re-seeding of pneumococci into the blood. These data greatly advance our fundamental knowledge of pneumococcal infection by linking susceptibility to invasive pneumococcal infection to variation in the propensity of MMMs to allow persistence and replication of phagocytosed bacteria. These findings also outline a somewhat rare scenario whereby the active phase of an organism's circadian cycle plays a seemingly counterproductive role in the control of invasive infection

A Nuclear Export Signal in KHNYN Required for Its Antiviral Activity Evolved as ZAP Emerged in Tetrapods

The zinc finger antiviral protein (ZAP) inhibits viral replication by directly binding CpG dinucleotides in cytoplasmic viral RNA to inhibit protein synthesis and target the RNA for degradation. ZAP evolved in tetrapods and there are clear orthologs in reptiles, birds, and mammals. When ZAP emerged, other proteins may have evolved to become cofactors for its antiviral activity. KHNYN is a putative endoribonuclease that is required for ZAP to restrict retroviruses. To determine its evolutionary path after ZAP emerged, we compared KHNYN orthologs in mammals and reptiles to those in fish, which do not encode ZAP. This identified residues in KHNYN that are highly conserved in species that encode ZAP, including several in the CUBAN domain. The CUBAN domain interacts with NEDD8 and Cullin-RING E3 ubiquitin ligases. Deletion of the CUBAN domain decreased KHNYN antiviral activity, increased protein expression and increased nuclear localization. However, mutation of residues required for the CUBAN domain-NEDD8 interaction increased KHNYN abundance but did not affect its antiviral activity or cytoplasmic localization, indicating that Cullin-mediated degradation may control its homeostasis and regulation of protein turnover is separable from its antiviral activity. By contrast, the C-terminal residues in the CUBAN domain form a CRM1-dependent nuclear export signal (NES) that is required for its antiviral activity. Deletion or mutation of the NES increased KHNYN nuclear localization and decreased its interaction with ZAP. The final 2 positions of this NES are not present in fish KHNYN orthologs and we hypothesize their evolution allowed KHNYN to act as a ZAP cofactor. IMPORTANCE The interferon system is part of the innate immune response that inhibits viruses and other pathogens. This system emerged approximately 500 million years ago in early vertebrates. Since then, some genes have evolved to become antiviral interferon-stimulated genes (ISGs) while others evolved so their encoded protein could interact with proteins encoded by ISGs and contribute to their activity. However, this remains poorly characterized. ZAP is an ISG that arose during tetrapod evolution and inhibits viral replication. Because KHNYN interacts with ZAP and is required for its antiviral activity against retroviruses, we conducted an evolutionary analysis to determine how specific amino acids in KHNYN evolved after ZAP emerged. This identified a nuclear export signal that evolved in tetrapods and is required for KHNYN to traffic in the cell and interact with ZAP. Overall, specific residues in KHNYN evolved to allow it to act as a cofactor for ZAP antiviral activity

Rise in carriage of group W meningococci in university students in United Kingdom

MenACWY conjugate vaccination was recently introduced in the UK for adolescents and 24 young adults to reduce disease due to Neisseria meningitidis group W (MenW). We 25 conducted a cross-sectional carriage study in first year university students. Despite 71% 26 MenACWY vaccine coverage, carriage of MenW, but not MenY, rose significantly in 27 students

Macrophages as a Replicative Niche During Systemic Bacterial Infection

Bloodborne bacterial pathogens are exposed to multiple macrophage-mediated clearance mechanisms in organs including the liver and spleen. Some pathogens – termed intracellular pathogens – are known to resist intracellular killing and persist within cells during the pathogenesis of infection. Extracellular pathogens are not widely considered to survive and replicate within macrophages. In this thesis, I report that two typically extracellular pathogens Streptococcus pneumoniae and Klebsiella pneumoniae have key phases of infection within tissue macrophages. For S. pneumoniae, I demonstrate that following infection of mice, bacteria can replicate within CD169-positive metallophilic macrophages and red pulp macrophages, but are efficiently cleared by SIGN-R1-positive marginal zone macrophages in the spleen. CD169+ macrophages were shown to be a critical safe haven for pneumococci prior to invasive disease, as blocking these cells with a monoclonal antibody prevented disease. Replicative foci within CD169-positive macrophages were shown to be hidden from neutrophil surveillance which may facilitate pneumococcal immune evasion in the early hours of infection. Instead, for K. pneumoniae, I demonstrate that hypervirulent strains (hvKp) – characterised by their hypermucoid capsules – replicated within splenic macrophages and Kupffer cells in the liver, while non-hv strains did not. Replication of hvKp within Kupffer cells formed a focal point for resistance to neutrophil-mediated killing, which led to the formation of tissue abscesses comparable to that which is observed in human disease. I developed a model of ex vivo human spleen perfusion, and porcine spleen-liver co-perfusion which allowed the translation of our murine findings for both pathogens to the human host. Together, this thesis identifies the within-macrophage niche as a safe haven for two bacterial species traditionally considered to be extracellular during the pathogenesis of infection. This work will open new research opportunities in the short term and facilitate the development of novel treatment strategies in the future.</div

Reprogramming of Cell Death Pathways by Bacterial Effectors as a Widespread Virulence Strategy

The modulation of programmed cell death (PCD) processes during bacterial infections is an evolving arms race between pathogens and their hosts. The initiation of apoptosis, necroptosis, and pyroptosis pathways are essential to immunity against many intracellular and extracellular bacteria. These cellular self-destructive mechanisms are used by the infected host to restrict and eliminate bacterial pathogens. Without a tight regulatory control, host cell death can become a double-edged sword. Inflammatory PCDs contribute to an effective immune response against pathogens, but unregulated inflammation aggravates the damage caused by bacterial infections. Thus, fine-tuning of these pathways is required to resolve infection while preserving the host immune homeostasis. In turn, bacterial pathogens have evolved secreted virulence factors or effector proteins that manipulate PCD pathways to promote infection. In this review, we discuss the importance of controlled cell death in immunity to bacterial infection. We also detail the mechanisms employed by type 3 secreted bacterial effectors to bypass these pathways and their importance in bacterial pathogenesis

Selective and non-selective bottlenecks as drivers of the evolution of hypermutable bacterial loci

Bottlenecks reduce the size of the gene pool within populations of all life forms with implications for their subsequent survival. Here, we examine the effects of bottlenecks on bacterial commensal-pathogens during transmission between, and dissemination within, hosts. By reducing genetic diversity, bottlenecks may alter individual or population-wide adaptive potential. A diverse range of hypermutable mechanisms have evolved in infectious agents that allow for rapid generation of genetic diversity in specific genomic loci as opposed to the variability arising from increased genomewide mutation rates. These localised hypermutable mechanisms include multi-gene phase variation (PV) of outer membrane components, multi-allele PV of restriction systems and recombination-driven antigenic variation. We review selected experimental and theoretical (mathematical) models pertaining to the hypothesis that localised hypermutation (LH) compensates for fitness losses caused by bottlenecks and discuss whether bottlenecks have driven the evolution of hypermutable loci

Flowchart and visual output of Phasome<i>It</i>.

<p><b>(A)</b> Outputs of Phasome<i>It</i> can be viewed visually on the index page. Green bars indicate there is an homopolymeric tract within the open reading frame; orange bars indicate there is an SSR close to the gene of interest (for example in a promoter region); grey bars indicate there is a non-PV gene homologous to a PV gene in that same homology grouping; the remaining coloured bars are indicative of SSRs other than homopolymers which can be further derived from the dataset below the visual output. <b>(B)</b> Gene groupings corresponding to the visual output are found in a table below. From here, functions, PV status in each strain and tract entries can be obtained for the grouping of interest. The full dataset from which this figure is derived, containing further phasome information not discussed in this manuscript are available (<a href="https://figshare.com/s/d31b7b0b6ca4aeeb48df" target="_blank">https://figshare.com/s/d31b7b0b6ca4aeeb48df</a>). A red outline shows highlights both the graphical and interactive outputs for the <i>opa</i> loci as an example.</p

Core phasome analysis of a subset of <i>Neisseria</i> species.

<p>Core phasome analysis of a subset of <i>Neisseria</i> species.</p