An unbiased genetic screen reveals the polygenic nature of the influenza virus anti-interferon response

UNLABELLED: Influenza A viruses counteract the cellular innate immune response at several steps, including blocking RIG I-dependent activation of interferon (IFN) transcription, interferon (IFN)-dependent upregulation of IFN-stimulated genes (ISGs), and the activity of various ISG products; the multifunctional NS1 protein is responsible for most of these activities. To determine the importance of other viral genes in the interplay between the virus and the host IFN response, we characterized populations and selected mutants of wild-type viruses selected by passage through non-IFN-responsive cells. We reasoned that, by allowing replication to occur in the absence of the selection pressure exerted by IFN, the virus could mutate at positions that would normally be restricted and could thus find new optimal sequence solutions. Deep sequencing of selected virus populations and individual virus mutants indicated that nonsynonymous mutations occurred at many phylogenetically conserved positions in nearly all virus genes. Most individual mutants selected for further characterization induced IFN and ISGs and were unable to counteract the effects of exogenous IFN, yet only one contained a mutation in NS1. The relevance of these mutations for the virus phenotype was verified by reverse genetics. Of note, several virus mutants expressing intact NS1 proteins exhibited alterations in the M1/M2 proteins and accumulated large amounts of deleted genomic RNAs but nonetheless replicated to high titers. This suggests that the overproduction of IFN inducers by these viruses can override NS1-mediated IFN modulation. Altogether, the results suggest that influenza viruses replicating in IFN-competent cells have tuned their complete genomes to evade the cellular innate immune system and that serial replication in non-IFN-responsive cells allows the virus to relax from these constraints and find a new genome consensus within its sequence space.

IMPORTANCE: In natural virus infections, the production of interferons leads to an antiviral state in cells that effectively limits virus replication. The interferon response places considerable selection pressure on viruses, and they have evolved a variety of ways to evade it. Although the influenza virus NS1 protein is a powerful interferon antagonist, the contributions of other viral genes to interferon evasion have not been well characterized. Here, we examined the effects of alleviating the selection pressure exerted by interferon by serially passaging influenza viruses in cells unable to respond to interferon. Viruses that grew to high titers had mutations at many normally conserved positions in nearly all genes and were not restricted to the NS1 gene. Our results demonstrate that influenza viruses have fine-tuned their entire genomes to evade the interferon response, and by removing interferon-mediated constraints, viruses can mutate at genome positions normally restricted by the interferon response.

Relative contributions of lipooligosaccharide inner and outer core modifications to nontypeable Haemophilus influenzae pathogenesis

Nontypeable Haemophilus influenzae (NTHi) is a frequent commensal of the human nasopharynx that causes opportunistic infection in immunocompromised individuals. Existing evidence associates lipooligosaccharide (LOS) with disease, but the specific and relative contributions of NTHi LOS modifications to virulence properties of the bacterium have not been comprehensively addressed. Using NTHi strain 375, an isolate for which the detailed LOS structure has been determined, we compared systematically a set of isogenic mutant strains expressing sequentially truncated LOS. The relative contributions of 2-keto-3-deoxyoctulosonic acid, the triheptose inner core, oligosaccharide extensions on heptoses I and III, phosphorylcholine, digalactose, and sialic acid to NTHi resistance to antimicrobial peptides (AMP), self-aggregation, biofilm formation, cultured human respiratory epithelial infection, and murine pulmonary infection were assessed. We show that opsX, lgtF, lpsA, lic1, and lic2A contribute to bacterial resistance to AMP; lic1 is related to NTHi self-aggregation; lgtF, lic1, and siaB are involved in biofilm growth; opsX and lgtF participate in epithelial infection; and opsX, lgtF, and lpsA contribute to lung infection. Depending on the phenotype, the involvement of these LOS modifications occurs at different extents, independently or having an additive effect in combination. We discuss the relative contribution of LOS epitopes to NTHi virulence and frame a range of pathogenic traits in the context of infection.

Modeling Klebsiella pneumoniae pathogenesis by infection of the wax moth Galleria mellonella

The implementation of infection models that approximate human disease is essential for understanding pathogenesis at the molecular level and for testing new therapies before they are entered into clinical stages. Insects are increasingly being used as surrogate hosts because they share, with mammals, essential aspects of the innate immune response to infections. We examined whether the larva of the wax moth Galleria mellonella could be used as a host model to conceptually approximate Klebsiella pneumoniae-triggered pneumonia. We report that the G. mellonella model is capable of distinguishing between pathogenic and nonpathogenic Klebsiella strains. Moreover, K. pneumoniae infection of G. mellonella models some of the known features of Klebsiella-induced pneumonia, i.e., cell death associated with bacterial replication, avoidance of phagocytosis by phagocytes, and the attenuation of host defense responses, chiefly the production of antimicrobial factors. Similar to the case for the mouse pneumonia model, activation of innate responses improved G. mellonella survival against subsequent Klebsiella challenge. Virulence factors necessary in the mouse pneumonia model were also implicated in the Galleria model. We found that mutants lacking capsule polysaccharide, lipid A decorations, or the outer membrane proteins OmpA and OmpK36 were attenuated in Galleria. All mutants activated G. mellonella defensive responses. The Galleria model also allowed us to monitor Klebsiella gene expression. The expression levels of cps and the loci implicated in lipid A remodeling peaked during the first hours postinfection, in a PhoPQ- and PmrAB-governed process. Taken together, these results support the utility of G. mellonella as a surrogate host for assessing infections with K. pneumoniae.

Burkholderia cenocepacia Lipopolysaccharide Modification and Flagellin Glycosylation Affect Virulence but Not Innate Immune Recognition in Plants

Burkholderia cenocepacia causes opportunistic infections in plants, insects, animals, and humans, suggesting that “virulence” depends on the host and its innate susceptibility to infection. We hypothesized that modifications in key bacterial molecules recognized by the innate immune system modulate host responses to B. cenocepacia. Indeed, modification of lipo- polysaccharide (LPS) with 4-amino-4-deoxy-L-arabinose and flagellin glycosylation attenuates B. cenocepacia infection in Arabi- dopsis thaliana and Galleria mellonella insect larvae. However, B. cenocepacia LPS and flagellin triggered rapid bursts of nitric oxide and reactive oxygen species in A. thaliana leading to activation of the PR-1 defense gene. These responses were drastically reduced in plants with fls2 (flagellin FLS2 host receptor kinase), Atnoa1 (nitric oxide-associated protein 1), and dnd1-1 (reduced production of nitric oxide) null mutations. Together, our results indicate that LPS modification and flagellin glycosylation do not affect recognition by plant receptors but are required for bacteria to establish overt infection.

A further investigation of the kinetic demixing/decomposition of La0.6Sr0.4Co0.2Fe0.8O3- δ oxygen separation membranes

Kinetic demixing and decomposition were studied on three La_0.6Sr_0.4Co_0.2Fe_0.8O_{3- δ} oxygen-separation hollow fibre membrane modules, which were operated under a 0.21/0.009bar oxygen partial pressure difference at 950°C for 1128, 3672 and 5512h, respectively. The post-operation membranes were characterized by Secondary Ion Mass Spectrometry, Scanning Electronic Microscope, Energy Dispersive Spectrum and X-ray Diffraction. The occurrence of kinetic demixing and decomposition was confirmed through the microstructural evolution of the membranes. Secondary-phase grains were found on the air-side surface of the membranes after the long-term operation and Co and Fe enrichment as well as La depletion was found on the surface and in the bulk at the air side. Cation diffusivities were found to be in the order Co>Fe>Sr>La. Kinetic demixing and decomposition rates of the membranes at the air side were found to be self-accelerating with time; the role of A-site deficiency in the perovskite lattice in the bulk near the air side surface is implicated in the mechanism. The oxygen permeability was not affected by the kinetic demixing and decomposition of the material during long-term operation (up to 5512h), however, we may expect permeability to be affected by secondary phase formation on the air-side surface at even longer operational times. © 2010 Elsevier B.V.

Extracellular DNA impedes the transport of vancomycin in Staphylococcus epidermidis biofilms pre-exposed to sub-inhibitory concentrations of vancomycin

Staphylococcus epidermidis biofilm formation is responsible for the persistence of orthopedic implant infections. Previous studies have shown that exposure of S. epidermidis biofilms to sub-MICs of antibiotics induced an increased level of biofilm persistence. BODIPY FL-vancomycin (a fluorescent vancomycin conjugate) and confocal microscopy were used to show that the penetration of vancomycin through sub-MIC-vancomycin-treated S. epidermidis biofilms was impeded compared to that of control, untreated biofilms. Further experiments showed an increase in the extracellular DNA (eDNA) concentration in biofilms preexposed to sub-MIC vancomycin, suggesting a potential role for eDNA in the hindrance of vancomycin activity. Exogenously added, S. epidermidis DNA increased the planktonic vancomycin MIC and protected biofilm cells from lethal vancomycin concentrations. Finally, isothermal titration calorimetry (ITC) revealed that the binding constant of DNA and vancomycin was 100-fold higher than the previously reported binding constant of vancomycin and its intended cellular D-Ala-D-Ala peptide target. This study provides an explanation of the eDNA-based mechanism of antibiotic tolerance in sub-MIC-vancomycin-treated S. epidermidis biofilms, which might be an important factor for the persistence of biofilm infections.

基于局部损伤混凝土模型的FRP-混凝土界面有限元分析研究

A meso-scale finite element model is presented for investigating the FRP-concrete bond behaviour under static and dynamic loadings. It adopts a local concrete damage model. A large number of single shear tests under static pull-off loading were modeled. It is demonstrated that the developed model can satisfactorily simulate the static debonding behaviour, in terms of the load-carrying capacity and the local bond-slip behaviour. A preliminary study of the effect of the dynamic loading rate on the debonding behaviour was also conducted by considering a dynamic increase factor for the concrete strength as a function of strain rate. It is shown that a higher loading rate leads to a higher load-carrying capacity, a longer effective bond length, and a larger damaged area of concrete in the single shear loading scenario.

Chp8, a diguanylate cyclase from Pseudomonas syringae pv tomato DC3000 suppresses the PAMP flagellin, increases EPS and promotes plant immune evasion.

The bacterial plant pathogen Pseudomonas syringae causes disease in a wide range of plants. The associated decrease in crop yields results in economic losses and threatens global food security. Competition exists between the plant immune system and the pathogen, the basic principles of which can be applied to animal infection pathways. P. syringae uses a type III secretion system (T3SS) to deliver virulence factors into the plant that promote survival of the bacterium. The P. syringae T3SS is a product of the hypersensitive response and pathogenicity (hrp) and hypersensitive response and conserved (hrc) gene cluster, which is strictly controlled by the codependent enhancer-binding proteins HrpR and HrpS. Through a combination of bacterial gene regulation and phenotypic studies, plant infection assays, and plant hormone quantifications, we now report that Chp8 (i) is embedded in the Hrp regulon and expressed in response to plant signals and HrpRS, (ii) is a functional diguanylate cyclase, (iii) decreases the expression of the major pathogen-associated molecular pattern (PAMP) flagellin and increases extracellular polysaccharides (EPS), and (iv) impacts the salicylic acid/jasmonic acid hormonal immune response and disease progression. We propose that Chp8 expression dampens PAMP-triggered immunity during early plant infection.

Dengue Virus Inhibition of Autophagic Flux and Dependency of Viral Replication on Proteasomal Degradation of the Autophagy Receptor p62

Autophagic flux involves formation of autophagosomes and their degradation by lysosomes. Autophagy can either promote or restrict viral replication. In the case of Dengue virus (DENV) several studies report that autophagy supports the viral replication cycle, and describe an increase of autophagic vesicles (AVs) following infection. However, it is unknown how autophagic flux is altered to result in increased AVs. To address this question, and gain insight into the role of autophagy during DENV infection, we established an unbiased, image-based flow cytometry approach to quantify autophagic flux under normal growth conditions and in response to activation by nutrient deprivation or the mTOR inhibitor Torin1. We found that DENV induced an initial activation of autophagic flux, followed by inhibition of general and specific autophagy. Early after infection, basal and activated autophagic flux was enhanced. However, during established replication, basal and Torin1-activated autophagic flux was blocked, while autophagic flux activated by nutrient deprivation was reduced, indicating a block to AV formation and reduced AV degradation capacity. During late infection AV levels increased as a result of inefficient fusion of autophagosomes with lysosomes. Additionally, endo-lysosomal trafficking was suppressed, while lysosomal activities were increased. We further determined that DENV infection progressively reduced levels of the autophagy receptor SQSTM1/p62 via proteasomal degradation. Importantly, stable over-expression of p62 significantly suppressed DENV replication suggesting a novel role for p62 as viral restriction factor. Overall our findings indicate that in the course of DENV infection, autophagy shifts from a supporting to an anti-viral role, which is countered by DENV.

IMPORTANCE: Autophagic flux is a dynamic process starting with the formation of autophagosomes and ending with their degradation after fusion with lysosomes. Autophagy impacts the replication cycle of many viruses. However, thus far the dynamics of autophagy in case of Dengue virus (DENV) infections has not been systematically quantified. Therefore, we employed high-content, imaging-based flow cytometry to quantify autophagic flux and endo-lysosomal trafficking in response to DENV infection. We report that DENV induced an initial activation of autophagic flux, followed by inhibition of general and specific autophagy. Further, lysosomal activity was increased, but endo-lysosomal trafficking was suppressed confirming the block of autophagic flux. Importantly, we provide evidence that p62, an autophagy receptor, restrict DENV replication and was specifically depleted in DENV-infected cells via increased proteasomal degradation. These results suggest that during DENV infection autophagy shifts from a pro- to an antiviral cellular process, which is counteracted by the virus.

Induction and antagonism of antiviral responses in respiratory syncytial virus-infected pediatric airway epithelium.

Airway epithelium is the primary target of many respiratory viruses. However, virus induction and antagonism of host responses by human airway epithelium remains poorly understood. To address this, we developed a model of respiratory syncytial virus (RSV) infection based on well- differentiated pediatric primary bronchial epithelial cell cultures (WD-PBECs) that mimics hallmarks of RSV disease in infants. RSV is the most important respiratory viral pathogen in young infants worldwide. We found that RSV induces a potent antiviral state in WD-PBECs that was mediated in part by secreted factors, including interferon lambda-1 (IFNλ1)/IL-29. In contrast, type I interferons were not detected following RSV infection of WD-PBECs., Interferon (IFN) responses in RSV-infected WD-PBECs reflected those in lower airway samples from RSV-hospitalized infants. In view of the prominence of IL-29, we determined whether recombinant IL-29 treatment of WD-PBECs before or after infection abrogated RSV replication. Interestingly, IL-29 demonstrated prophylactic, but not therapeutic, potential against RSV. The absence of therapeutic potential reflected effective RSV antagonism of IFN-mediated antiviral responses in infected cells. Our data are consistent with RSV non-structural proteins 1 and/or 2 perturbing the Jak-STAT signaling pathway, with concomitant reduced expression of antiviral effector molecules, such as MxA/B. Antagonism of Jak-STAT signaling was restricted to RSV-infected cells in WD-PBEC cultures. Importantly, our study provides the rationale to further explore IL-29 as a novel RSV prophylactic.

Generation and Characterization of ALX-0171, a Potent Novel Therapeutic Nanobody for the Treatment of Respiratory Syncytial Virus Infection

Respiratory Syncytial Virus (RSV) is an important causative agent of lower respiratory tract infections in infants and elderly. Its fusion (F) protein is critical for virus infection. It is targeted by several investigational antivirals and by palivizumab, a humanised monoclonal antibody used prophylactically in infants considered at high risk of severe RSV disease. ALX-0171 is a trimeric Nanobody that binds the antigenic site II of RSV F-protein with subnanomolar affinity. ALX-0171 demonstrated superior in vitro neutralisation compared to palivizumab against prototypic RSV A and B strains. Moreover, ALX-0171 completely blocked replication below limit of detection in 87% of the viruses tested versus 18% for palivizumab at a fixed concentration. Importantly, ALX-0171 was highly effective in reducing both nasal and lung RSV titers when delivered prophylactically or therapeutically directly to the lungs of cotton rats. ALX-0171 represents a potent novel antiviral compound with significant potential to treat RSV-mediated disease.

Fasciola hepatica from naturally infected sheep and cattle in Great Britain are diploid

Diploid (2n = 2x = 20) and triploid (2n = 3x = 30) Fasciola hepatica have been reported in the UK, and in Asia diploid, triploid and mixoploid (2x/3x) Fasciola spp. exist but there is little information to indicate how common triploidy is, particularly in UK fluke. Here the ploidy of 565 adult F. hepatica from 66 naturally infected British sheep and 150 adult F. hepatica from 35 naturally infected British cattle was determined. All 715 of these parasites were diploid, based on observation of 10 bivalent chromosomes and sperm (n = 335) or, since triploids are aspermic, sperm alone (n = 380). This constitutes the first extensive analysis of the ploidy of F. hepatica field isolates from Great Britain and shows that most F. hepatica isolated from cattle and sheep are diploid and have the capacity to sexually reproduce. These data suggest that triploidy, and by extension parthenogenesis, is rare or non-existent in wild British F. hepatica populations. Given that F. hepatica is the only species of Fasciola present in Britain our results indicate that the parasite is predominantly diploid in areas where F. hepatica exists in isolation and suggests that triploidy may only originate in natural populations where co-infection of F. hepatica and its sister species Fasciola gigantica commonly occurs.

Genome Expression Profiling-Based Identification and Administration Efficacy of Host-Directed Antimicrobial Drugs against Respiratory Infection by Nontypeable Haemophilus influenzae

Therapies that are safe, effective, and not vulnerable to developing resistance are highly desirable to counteract bacterial infections. Host-directed therapeutics is an antimicrobial approach alternative to conventional antibiotics based on perturbing host pathways subverted by pathogens during their life cycle by using host-directed drugs. In this study, we identified and evaluated the efficacy of a panel of host-directed drugs against respiratory infection by nontypeable Haemophilus influenzae (NTHi). NTHi is an opportunistic pathogen that is an important cause of exacerbation of chronic obstructive pulmonary disease (COPD). We screened for host genes differentially expressed upon infection by the clinical isolate NTHi375 by analyzing cell whole-genome expression profiling and identified a repertoire of host target candidates that were pharmacologically modulated. Based on the proposed relationship between NTHi intracellular location and persistence, we hypothesized that drugs perturbing host pathways used by NTHi to enter epithelial cells could have antimicrobial potential against NTHi infection. Interfering drugs were tested for their effects on bacterial and cellular viability, on NTHi-epithelial cell interplay, and on mouse pulmonary infection. Glucocorticoids and statins lacked in vitro and/or in vivo efficacy. Conversely, the sirtuin-1 activator resveratrol showed a bactericidal effect against NTHi, and the PDE4 inhibitor rolipram showed therapeutic efficacy by lowering NTHi375 counts intracellularly and in the lungs of infected mice. PDE4 inhibition is currently prescribed in COPD, and resveratrol is an attractive geroprotector for COPD treatment. Together, these results expand our knowledge of NTHi-triggered host subversion and frame the antimicrobial potential of rolipram and resveratrol against NTHi respiratory infection.