A family of conserved bacterial virulence factors dampens interferon responses by blocking calcium signaling.

Interferons (IFNs) induce an antimicrobial state, protecting tissues from infection. Many viruses inhibit IFN signaling, but whether bacterial pathogens evade IFN responses remains unclear. Here, we demonstrate that the Shigella OspC family of type-III-secreted effectors blocks IFN signaling independently of its cell death inhibitory activity. Rather, IFN inhibition was mediated by the binding of OspC1 and OspC3 to the Ca2+ sensor calmodulin (CaM), blocking CaM kinase II and downstream JAK/STAT signaling. The growth of Shigella lacking OspC1 and OspC3 was attenuated in epithelial cells and in a murine model of infection. This phenotype was rescued in both models by the depletion of IFN receptors. OspC homologs conserved in additional pathogens not only bound CaM but also inhibited IFN, suggesting a widespread virulence strategy. These findings reveal a conserved but previously undescribed molecular mechanism of IFN inhibition and demonstrate the critical role of Ca2+ and IFN targeting in bacterial pathogenesis.

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.

Variable disruption of epithelial monolayers by Neisseria meningitidis carriage isolates of the hypervirulent MenW cc11 and MenY cc23 lineages.

Colonization of mucosal tissues by Neisseria meningitidis requires adhesion mediated by the type IV pilus and multiple outer-membrane proteins. Penetration of the mucosa and invasion of epithelial cells are thought to contribute to host persistence and invasive disease. Using Calu-3 cell monolayers grown at an air-liquid interface, we examined adhesion, invasion and monolayer disruption by carriage isolates of two clonal complexes of N. meningitidis. Carriage isolates of both the serogroup Y cc23 and the hypervirulent serogroup W cc11 lineages exhibited high levels of cellular adhesion, and a variable disruption phenotype across independent isolates. Inactivation of the gene encoding the main pilus sub-unit in multiple cc11 isolates abrogated both adhesive capacity and ability to disrupt epithelial monolayers. Contrastingly, inactivation of the phase-variable opa or nadA genes reduced adhesion and invasion, but not disruption of monolayer integrity. Adherence of tissue-disruptive meningococci correlated with loss of staining for the tight junction protein, occludin. Intriguingly, in a pilus-negative strain background, we observed compensatory ON switching of opa genes, which facilitated continued adhesion. We conclude that disruption of epithelial monolayers occurs in multiple meningococcal lineages but can vary during carriage and is intimately linked to pilus-mediated adhesion.

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 genome-wide 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.

Random sorting of Campylobacter jejuni phase variants due to a narrow bottleneck during colonization of broiler chickens.

Phase variation (PV), involving stochastic switches in gene expression, is exploited by the human pathogen Campylobacter jejuni to adapt to different environmental and host niches. Phase-variable genes of C. jejuni modulate expression of multiple surface determinants, and hence may influence host colonization. Population bottlenecks can rapidly remove the diversity generated by PV, and strict single-cell bottlenecks can lead to propagation of PV states with highly divergent phenotypes. Using a combination of high-throughput fragment size analysis and comparison with in vivo and in silico bottleneck models, we have characterized a narrow population bottleneck during the experimental colonization of broiler chickens with C. jejuni strain 81-176. We identified high levels of variation in five PV genes in the inoculum, and subsequently, massively decreased population diversity following colonization. Each bird contained a dominant five-gene phasotype that was present in the inoculum indicative of random sorting through a narrow, non-selective bottleneck during colonization. These results are evidence of the potential for confounding effects of PV on in vivo studies of Campylobacter colonization factors and poultry vaccine studies. Our results are also an argument for population bottlenecks as mediators of stochastic variability in the propensity to survive through the food chain and cause clinical human disease.

Ca2+-calmodulin signalling at the host-pathogen interface.

Multiple eukaryotic cell processes are modulated by calcium ions (Ca2+). As such, Ca2+ is emerging as a crucial regulator of innate immunity in multicellular organisms. In particular, recent studies have identified roles of Ca2+ signalling at the host-bacteria interface. Following microbial exposure, Ca2+ signals mobilised from the extracellular milieu or intracellular stores are transduced into cell physiological responses. However, during infection with host-adapted pathogens, Ca2+ signals are often atypical, due to the activities of virulence factors, with varied consequences for both the pathogen and the host cell. In this review, we describe the Ca2+-dependent host factors regulating antibacterial immunity, in addition to bacterial effectors that promote, inhibit, or co-opt Ca2+-calmodulin signalling to promote infection.

Analyzing Macrophage Infection at the Organ Level.

Classical in vivo infection models are oftentimes associated with speculation due to the many physiological factors that are unseen or not accounted for when analyzing experimental outputs, especially when solely utilizing the classic approach of tissue-derived colony-forming unit (CFU) enumeration. To better understand the steps and natural progression of bacterial infection, the pathophysiology of individual organs with which the bacteria interact in their natural course of infection must be considered. In this case, it is not only important to isolate organs as much as possible from additional physiological processes, but to also consider the dynamics of the bacteria at the cellular level within these organs of interest. Here, we describe in detail two models, ex vivo porcine liver and spleen coperfusion and a murine infection model, and the numerous associated experimental outputs produced by these models that can be taken and used together to investigate the pathogen-host interactions within tissues in depth.

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-α 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.

Interaction of Klebsiella pneumoniae with tissue macrophages in a mouse infection model and ex-vivo pig organ perfusions: an exploratory investigation.

BACKGROUND: Hypervirulent Klebsiella pneumoniae (hvKp) strains of capsule type K1 and K2 cause invasive infections associated with hepatic abscesses, which can be difficult to treat and are frequently associated with relapsing infections. Other K pneumoniae strains (non-hvKp), including lineages that have acquired carbapenem resistance, do not manifest this pathology. In this work we aimed to test the hypothesis that within-macrophage replication is a key mechanism underpinning abscess formation in hvKp infections. METHODS: In this exploratory investigation, to study the pathophysiology of abscess formation, mice were intravenously infected with 106 colony forming units (CFU) of either hvKp isolates (six strains) or non-hvKp isolates (seven strains). Intracellular bacterial replication and neutrophil influx in liver and spleen was quantified by fluorescence microscopy of sliced cryopreserved organs of mice collected 30 min, 6 h, and 24 h after infection with the aim to provide data of bacterial association to Kupffer cells in the liver and to the different tissue macrophages in the spleen. Microbiological and microscopy analysis of an ex-vivo model of pig liver and spleen infection were used to confirm within-macrophage replication. Pig organs were perfused with heparinised, autologous pig's blood and injected with 6·5 × 107 CFU of hvKp K2 sequence type 25 strain GMR151. Blood and tissue biopsies collected before infection and 30 min, 1 h, 2 h, 3 h, 4 h, and 5 h after infection were used to measure bacterial counts and to identify the subcellular localisation of bacteria by immunohistochemistry analysis. FINDINGS: We show that hvKp resisted phagocyte-mediated clearance and replicated in mouse liver macrophages to form clusters 6 h after infection, with a mean of 7·0 bacteria per Kupffer cell (SD 6·2); however, non-hvKp were efficiently cleared (mean 1·5 bacteria per cell [SD 1·1]). HvKp infection promoted neutrophil recruitment to sites of infection, which in the liver resulted in histopathological signs of abscess formation as early as 24 h post-infection. Experiments in pig organs which share a high functional and anatomical resemblance to human organs, provided strong evidence for the propensity of hvKp to replicate within the hepatic macrophages. INTERPRETATION: These findings show subversion of innate immune processes in the liver by K pneumoniae and resistance to Kupffer cell mediated clearance as an explanation for the propensity of hvKp strains to cause hepatic abscesses. FUNDING: University of Oxford and a Royal Society Wolfson grant funded biosafety facility.

Splenic macrophages as the source of bacteraemia during pneumococcal pneumonia.

BACKGROUND: Severe community-acquired pneumococcal pneumonia is commonly associated with bacteraemia. Although it is assumed that the bacteraemia solely derives from pneumococci entering the blood from the lungs it is unknown if other organs are important in the pathogenesis of bacteraemia. Using three models, we tested the relevance of the spleen in pneumonia-associated bacteraemia. METHODS: We used human spleens perfused ex vivo to explore permissiveness to bacterial replication, a non-human primate model to check for splenic involvement during pneumonia and a mouse pneumonia-bacteraemia model to demonstrate that splenic involvement correlates with invasive disease. FINDINGS: Here we present evidence that the spleen is the reservoir of bacteraemia during pneumonia. We found that in the human spleen infected with pneumococci, clusters with increasing number of bacteria were detectable within macrophages. These clusters also were detected in non-human primates. When intranasally infected mice were treated with a non-therapeutic dose of azithromycin, which had no effect on pneumonia but concentrated inside splenic macrophages, bacteria were absent from the spleen and blood and importantly mice had no signs of disease. INTERPRETATION: We conclude that the bacterial load in the spleen, and not lung, correlates with the occurrence of bacteraemia. This supports the hypothesis that the spleen, and not the lungs, is the major source of bacteria during systemic infection associated with pneumococcal pneumonia; a finding that provides a mechanistic basis for using combination therapies including macrolides in the treatment of severe community-acquired pneumococcal pneumonia. FUNDING: Oxford University, Wolfson Foundation, MRC, NIH, NIHR, and MRC and BBSRC studentships supported the work.

Meningococcal core and accessory phasomes vary by clonal complex.

Neisseria meningitidis is a Gram-negative human commensal pathogen, with extensive phenotypic plasticity afforded by phase-variable (PV) gene expression. Phase variation is a stochastic switch in gene expression from an ON to an OFF state, mediated by localized hypermutation of simple sequence repeats (SSRs). Circulating N. meningitidis clones vary in propensity to cause disease, with some clonal complexes (ccs) classified as hypervirulent and others as carriage-associated. We examined the PV gene repertoires, or phasome, of these lineages in order to determine whether phase variation contributes to disease propensity. We analysed 3328 genomes representative of nine circulating meningococcal ccs with PhasomeIt, a tool that identifies PV genes by the presence of SSRs and homologous gene clusters. The presence, absence and functions of all identified PV gene clusters were confirmed by annotation or blast searches within the Neisseria PubMLST database. While no significant differences were detected in the number of PV genes or the core, conserved phasome content between hypervirulent and carriage lineages, individual ccs exhibited major variations in PV gene numbers. Phylogenetic clusters produced by phasome or core genome analyses were similar, indicating co-evolution of PV genes with the core genome. While conservation of PV clusters is high, with 76 % present in all meningococcal isolates, maintenance of an SSR is variable, ranging from conserved in all isolates to present only in a single cc, indicating differing evolutionary trajectories for each lineage. Diverse functional groups of PV genes were present across the meningococcal lineages; however, the majority directly or indirectly influence bacterial surface antigens and could impact on future vaccine development. Finally, we observe that meningococci have open pan phasomes, indicating ongoing evolution of PV gene content and a significant potential for adaptive changes in this clinically relevant genus.

An ex vivo porcine spleen perfusion as a model of bacterial sepsis.

An ex vivo, porcine spleen perfusion model was established to study the early events occurring in the spleen prior to the onset of bacterial sepsis, using organs retrieved from animals slaughtered for food production. Porcine spleens were harvested from adult pigs and connected to a normothermic extracorporeal perfusion circuit. A constant perfusion of heparinized blood was performed for 6 hours. After injection of Streptococcus pneumoniae to the circuit serial samples of both blood and spleen biopsies were collected and analysed. Functionality of the perfused organs was assessed by monitoring the blood-gas parameters, flow rate and filtering capability of the organ. Interestingly, we observed full clearance of bacteria from the blood and an increase in bacterial counts in the spleen. Classical histology and immunohistochemistry on biopsies also confirmed no major damages in the organ architecture and changes in the immune cell distribution, other than the presence of clusters of pneumococci. A time-course study confirmed that each focus of infection derived from the replication of single pneumococcal cells within splenic macrophages. The model proposed - in line with the 3Rs principles - has utility in the replacement of experimental animals in infection research. Murine models are prevalently used to study pneumococcal infections, but are often not predictive for humans due to substantial differences in the immune systems of the two species. This model is designed to overcome these limitations, since porcine immunology and splenic architecture in particular, closely resemble those of humans.

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.

PhasomeIt: an 'omics' approach to cataloguing the potential breadth of phase variation in the genus Campylobacter.

Hypermutable simple sequence repeats (SSRs) are drivers of phase variation (PV) whose stochastic, high-frequency, reversible switches in gene expression are a common feature of several pathogenic bacterial species, including the human pathogen Campylobacter jejuni. Here we examine the distribution and conservation of known and putative SSR-driven phase variable genes - the phasome - in the genus Campylobacter. PhasomeIt, a new program, was specifically designed for rapid identification of SSR-mediated PV. This program detects the location, type and repeat number of every SSR. Each SSR is linked to a specific gene and its putative expression state. Other outputs include conservation of SSR-driven phase-variable genes and the 'core phasome' - the minimal set of PV genes in a phylogenetic grouping. Analysis of 77 complete Campylobacter genome sequences detected a 'core phasome' of conserved PV genes in each species and a large number of rare PV genes with few, or no, homologues in other genome sequences. Analysis of a set of partial genome sequences, with food-chain-associated metadata, detected evidence of a weak link between phasome and source host for disease-causing isolates of sequence type (ST)-828 but not the ST-21 or ST-45 complexes. Investigation of the phasomes in the genus Campylobacter provided evidence of overlapping but distinctive mechanisms of PV-mediated adaptation to specific niches. This suggests that the phasome could be involved in host adaptation and spread of campylobacters. Finally, this tool is malleable and will have utility for studying the distribution and genic effects of other repetitive elements in diverse bacterial species.

Intracellular replication of Streptococcus pneumoniae inside splenic macrophages serves as a reservoir for septicaemia.

Bacterial septicaemia is a major cause of mortality, but its pathogenesis remains poorly understood. In experimental pneumococcal murine intravenous infection, an initial reduction of bacteria in the blood is followed hours later by a fatal septicaemia. These events represent a population bottleneck driven by efficient clearance of pneumococci by splenic macrophages and neutrophils, but as we show in this study, accompanied by occasional intracellular replication of bacteria that are taken up by a subset of CD169+ splenic macrophages. In this model, proliferation of these sequestered bacteria provides a reservoir for dissemination of pneumococci into the bloodstream, as demonstrated by its prevention using an anti-CD169 monoclonal antibody treatment. Intracellular replication of pneumococci within CD169+ splenic macrophages was also observed in an ex vivo porcine spleen, where the microanatomy is comparable with humans. We also showed that macrolides, which effectively penetrate macrophages, prevented septicaemia, whereas beta-lactams, with inefficient intracellular penetration, failed to prevent dissemination to the blood. Our findings define a shift in our understanding of the pneumococcus from an exclusively extracellular pathogen to one with an intracellular phase. These findings open the door to the development of treatments that target this early, previously unrecognized intracellular phase of bacterial sepsis.

Draft Whole-Genome Sequences of Periodontal Pathobionts Porphyromonas gingivalis, Prevotella intermedia, and Tannerella forsythia Contain Phase-Variable Restriction-Modification Systems.

Periodontal disease comprises mild to severe inflammatory host responses to oral bacteria that can cause destruction of the tooth-supporting tissue. We report genome sequences for 18 clinical isolates of Porphyromonas gingivalis, Prevotella intermedia, and Tannerella forsythia, Gram-negative obligate anaerobes that play a role in the periodontal disease process.