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

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

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

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

Distribution of phase variable genes between phase variable modules.

<p>Distribution of phase variable genes between phase variable modules.</p

Tract length distribution in different <i>Neisseria</i> species.

<p>Data are represented by heat maps. Colour intensity represents the percentage that a given tract length comprises of the total number of identified tracts of that type for each species. ‘-’ is indicative of no identified repeats of the given length. Information on the numbers of strains for each species, and numbers of tract lengths analysed can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196675#pone.0196675.t001" target="_blank">Table 1</a>.</p

Phasome analysis of pathogenic and commensal Neisseria species expands the known repertoire of phase variable genes, and highlights common adaptive strategies

Pathogenic Neisseria are responsible for significantly higher levels of morbidity and mortality than their commensal relatives despite having similar genetic contents. Neisseria possess a disparate arsenal of surface determinants that facilitate host colonisation and evasion of the immune response during persistent carriage. Adaptation to rapid changes in these hostile host environments is enabled by phase variation (PV) involving high frequency, stochastic switches in expression of surface determinants. In this study, we analysed 89 complete and 79 partial genomes, from the NCBI and Neisseria PubMLST databases, representative of multiple pathogenic and commensal species of Neisseria using PhasomeIt, a new program that identifies putatively phase-variable genes and homology groups by the presence of simple sequence repeats (SSR). We detected a repertoire of 884 putative PV loci with maxima of 54 and 47 per genome in gonococcal and meningococcal isolates, respectively. Most commensal species encoded a lower number of PV genes (between 5 and 30) except N. lactamica wherein the potential for PV (36–82 loci) was higher, implying that PV is an adaptive mechanism for persistence in this species. We also characterised the repeat types and numbers in both pathogenic and commensal species. Conservation of SSR-mediated PV was frequently observed in outer membrane proteins or modifiers of outer membrane determinants. Intermittent and weak selection for evolution of SSR-mediated PV was suggested by poor conservation of tracts with novel PV genes often occurring in only one isolate. Finally, we describe core phasomes—the conserved repertoires of phase-variable genes—for each species that identify overlapping but distinctive adaptive strategies for the pathogenic and commensal members of the Neisseria genus

Range of phase variable genes identified in each species.

<p>Data shown the median, range, upper and lower quartile number of PV genes, as indicated by presence of a repeat tract. These data exclude gene groupings which contain dinucleotide repeat tracts, due to the insufficient evidence of phase variation associated with dinucleotide repeats in the literature, and the loci discussed herein. Statistical analysis were performed with a Kruskal-Wallis test with Dunn’s multiple comparisons. NS; not significant, ***; p-value of <0.0005.</p