Moraxella catarrhalis evades neutrophil oxidative stress responses providing a safer niche for nontypeable Haemophilus influenzae

Moraxella catarrhalis and nontypeable Haemophilus influenzae (NTHi) are pathogenic bacteria frequently associated with exacerbation of chronic obstructive pulmonary disease (COPD), whose hallmark is inflammatory oxidative stress. Neutrophils produce reactive oxygen species (ROS) which can boost antimicrobial response by promoting neutrophil extracellular traps (NET) and autophagy. Here, we showed that M. catarrhalis induces less ROS and NET production in differentiated HL-60 cells compared to NTHi. It is also able to actively interfere with these responses in chemically activated cells in a phagocytosis and opsonin-independent and contact-dependent manner, possibly by engaging host immunosuppressive receptors. M. catarrhalis subverts the autophagic pathway of the phagocytic cells and survives intracellularly. It also promotes the survival of NTHi which is otherwise susceptible to the host antimicrobial arsenal. In-depth understanding of the immune evasion strategies exploited by these two human pathogens could suggest medical interventions to tackle COPD and potentially other diseases in which they co-exist

Structural basis for cooperativity of human monoclonal antibodies to meningococcal factor H-binding protein

Monoclonal antibody (mAb) cooperativity is a phenomenon triggered when mAbs couples promote increased bactericidal killing compared to individual partners. Cooperativity has been deeply investigated among mAbs elicited by factor H-binding protein (fHbp), a Neisseria meningitidis surface-exposed lipoprotein and one of the key antigens included in both serogroup B meningococcus vaccine Bexsero and Trumenba. Here we report the structural and functional characterization of two cooperative mAbs pairs isolated from Bexsero vaccines. The 3D electron microscopy structures of the human mAb-fHbp-mAb cooperative complexes indicate that the angle formed between the antigen binding fragments (fAbs) assume regular angle and that fHbp is able to bind simultaneously and stably the cooperative mAbs pairs and human factor H (fH) in vitro. These findings shed light on molecular basis of the antibody-based mechanism of protection driven by simultaneous recognition of the different epitopes of the fHbp and underline that cooperativity is crucial in vaccine efficacy

Structure analysis and site-directed mutagenesis of defined key residues and motives for pilus-related sortase C1 in group B Streptococcus

In group B Streptococcus (GBS), 3 structurally distinct types of pili have been discovered as potential virulence factors and vaccine candidates. The pilus-forming proteins are assembled into high-molecular-weight polymers via a transpeptidation mechanism mediated by specific class C sortases. Using a multidisciplinary approach including bioinformatics, structural and biochemical studies, and in vivo mutagenesis, we performed a broad characterization of GBS sortase C1 of pilus island 2a. The high-resolution X-ray structure of the enzyme revealed that the active site, into the \u3b2-barrel core of the enzyme, is made of the catalytic triad His157-Cys219-Arg228 and covered by a loop, known as the \u201clid.\u201d We show that the catalytic triad and the predicted N- and C-terminal transmembrane regions are required for the enzyme activity. Interestingly, by in vivo complementation mutagenesis studies, we found that the deletion of the entire lid loop or mutations in specific lid key residues had no effect on catalytic activity of the enzyme. In addition, kinetic characterizations of recombinant enzymes indicate that the lid mutants can still recognize and cleave the substrate-mimicking peptide at least as well as the wild-type protein.\u2014Cozzi, R., Malito, E., Nuccitelli, A., D\u2019Onofrio, M., Martinelli, M., Ferlenghi, I., Grandi, G., Telford, J. L., Maione, D., Rinaudo, C. D. Structure analysis and site-directed mutagenesis of defined key residues and motives for pilus-related sortase C1 in group B Streptococcus

RrgA is a pilus-associated adhesin in Streptococcus pneumoniae

Adherence to host cells is important in microbial colonization of a mucosal surface, and Streptococcus pneumoniae adherence was significantly enhanced by expression of an extracellular pilus composed of three subunits, RrgA, RrgB and RrgC. We sought to determine which subunit(s) confers adherence. Bacteria deficient in RrgA are significantly less adherent than wild-type organisms, while overexpression of RrgA enhances adherence. Recombinant monomeric RrgA binds to respiratory cells, as does RrgC with less affinity, and pre-incubation of epithelial cells with RrgA reduces adherence of wild-type piliated pneumococci. Non-adherent RrgA-negative, RrgB- and RrgC-positive organisms produce pili, suggesting that pilus-mediated adherence is due to expression of RrgA, rather than the pilus backbone itself. In contrast, RrgA-positive strains with disrupted rrgB and rrgC genes exhibit wild-type adherence despite failure to produce pili by Western blot or immunoelectron microscopy. The density of bacteria colonizing the upper respiratory tract of mice inoculated with piliated RrgA-negative pneumococci was significantly less compared with wild-type; in contrast, non-piliated pneumococci expressing non-polymeric RrgA had similar numbers of bacteria in the nasopharynx as piliated wild-type bacteria. These data suggest that RrgA is central in pilus-mediated adherence and disease, even in the absence of polymeric pilus production

Capturing a Flavivirus Pre-Fusion Intermediate

During cell entry of flaviviruses, low endosomal pH triggers the rearrangement of the viral surface glycoproteins to a fusion-active state that allows the release of the infectious RNA into the cytoplasm. In this work, West Nile virus was complexed with Fab fragments of the neutralizing mAb E16 and was subsequently exposed to low pH, trapping the virions in a pre-fusion intermediate state. The structure of the complex was studied by cryo-electron microscopy and provides the first structural glimpse of a flavivirus fusion intermediate near physiological conditions. A radial expansion of the outer protein layer of the virion was observed compared to the structure at pH 8. The resulting ∼60 Å-wide shell of low density between lipid bilayer and outer protein layer is likely traversed by the stem region of the E glycoprotein. By using antibody fragments, we have captured a structural intermediate of a virus that likely occurs during cell entry. The trapping of structural transition states by antibody fragments will be applicable for other processes in the flavivirus life cycle and delineating other cellular events that involve conformational rearrangements

Small-Molecule Inhibitors of Dengue-Virus Entry

Flavivirus envelope protein (E) mediates membrane fusion and viral entry from endosomes. A low-pH induced, dimer-to-trimer rearrangement and reconfiguration of the membrane-proximal “stem" of the E ectodomain draw together the viral and cellular membranes. We found stem-derived peptides from dengue virus (DV) bind stem-less E trimer and mimic the stem-reconfiguration step in the fusion pathway. We adapted this experiment as a high-throughput screen for small molecules that block peptide binding and thus may inhibit viral entry. A compound identified in this screen, 1662G07, and a number of its analogs reversibly inhibit DV infectivity. They do so by binding the prefusion, dimeric E on the virion surface, before adsorption to a cell. They also block viral fusion with liposomes. Structure-activity relationship studies have led to analogs with submicromolar IC90s against DV2, and certain analogs are active against DV serotypes 1,2, and 4. The compounds do not inhibit the closely related Kunjin virus. We propose that they bind in a previously identified, E-protein pocket, exposed on the virion surface and although this pocket is closed in the postfusion trimer, its mouth is fully accessible. Examination of the E-trimer coordinates (PDB 1OK8) shows that conformational fluctuations around the hinge could open the pocket without dissociating the trimer or otherwise generating molecular collisions. We propose that compounds such as 1662G07 trap the sE trimer in a “pocket-open" state, which has lost affinity for the stem peptide and cannot support the final “zipping up" of the stem

Analysis of Epitopes on Dengue Virus Envelope Protein Recognized by Monoclonal Antibodies and Polyclonal Human Sera by a High Throughput Assay

Dengue virus is the leading cause of arboviral diseases worldwide. The envelope protein is the major target of neutralizing antibodies and vaccine development. While previous studies have reported several epitopes on envelope protein, the possibility of interdomain epitopes and the relationship of epitopes to neutralizing potency remain unexplored. We developed a high throughput dot blot assay by using 67 alanine mutants of surface-exposed envelope residues as a systematic approach to identify epitopes recognized by mouse monoclonal antibodies and polyclonal human sera. Our results suggested the presence of interdomain epitopes more frequent than previously appreciated. Compared with monoclonal antibodies generated by traditional protocol, the potent neutralizing monoclonal antibodies generated by a new protocol showed several unique features of their epitopes. Moreover, the predominant epitopes of antibodies against envelope protein in polyclonal sera can be identified by this assay. These findings have implications for future development of epitope-specific diagnostics and epitope-based dengue vaccine, and add to our understanding of humoral immune responses to dengue virus at the epitope level

Supramolecular Organization of the Repetitive Backbone Unit of the Streptococcus pneumoniae Pilus

Streptococcus pneumoniae, like many other Gram-positive bacteria, assembles long filamentous pili on their surface through which they adhere to host cells. Pneumococcal pili are formed by a backbone, consisting of the repetition of the major component RrgB, and two accessory proteins (RrgA and RrgC). Here we reconstruct by transmission electron microscopy and single particle image reconstruction method the three dimensional arrangement of two neighbouring RrgB molecules, which represent the minimal repetitive structural domain of the native pilus. The crystal structure of the D2-D4 domains of RrgB was solved at 1.6 Å resolution. Rigid-body fitting of the X-ray coordinates into the electron density map enabled us to define the arrangement of the backbone subunits into the S. pneumoniae native pilus. The quantitative fitting provide evidence that the pneumococcal pilus consists uniquely of RrgB monomers assembled in a head-to-tail organization. The presence of short intra-subunit linker regions connecting neighbouring domains provides the molecular basis for the intrinsic pilus flexibility

Illustrating and homology modeling the proteins of the Zika virus

The Zika virus (ZIKV) is a flavivirus of the family Flaviviridae, which is similar to dengue virus, yellow fever and West Nile virus. Recent outbreaks in South America, Latin America, the Caribbean and in particular Brazil have led to concern for the spread of the disease and potential to cause Guillain-Barré syndrome and microcephaly. Although ZIKV has been known of for over 60 years there is very little in the way of knowledge of the virus with few publications and no crystal structures. No antivirals have been tested against it either in vitro or in vivo. ZIKV therefore epitomizes a neglected disease. Several suggested steps have been proposed which could be taken to initiate ZIKV antiviral drug discovery using both high throughput screens as well as structure-based design based on homology models for the key proteins. We now describe preliminary homology models created for NS5, FtsJ, NS4B, NS4A, HELICc, DEXDc, peptidase S7, NS2B, NS2A, NS1, E stem, glycoprotein M, propeptide, capsid and glycoprotein E using SWISS-MODEL. Eleven out of 15 models pass our model quality criteria for their further use. While a ZIKV glycoprotein E homology model was initially described in the immature conformation as a trimer, we now describe the mature dimer conformer which allowed the construction of an illustration of the complete virion. By comparing illustrations of ZIKV based on this new homology model and the dengue virus crystal structure we propose potential differences that could be exploited for antiviral and vaccine design. The prediction of sites for glycosylation on this protein may also be useful in this regard. While we await a cryo-EM structure of ZIKV and eventual crystal structures of the individual proteins, these homology models provide the community with a starting point for structure-based design of drugs and vaccines as well as a for computational virtual screening

Proteome-minimized outer membrane vesicles from Escherichia coli as a generalized vaccine platform

Because of their potent adjuvanticity, ease of manipulation and simplicity of production Gram-negative Outer Membrane Vesicles OMVs have the potential to become a highly effective vaccine platform. However, some optimization is required, including the reduction of the number of endogenous proteins, the increase of the loading capacity with respect to heterologous antigens, the enhancement of productivity in terms of number of vesicles per culture volume. In this work we describe the use of Synthetic Biology to create Escherichia coli BL21(DE3)Δ60, a strain releasing OMVs (OMVsΔ60) deprived of 59 endogenous proteins. The strain produces large quantities of vesicles (> 40 mg/L under laboratory conditions), which can accommodate recombinant proteins to a level ranging from 5% to 30% of total OMV proteins. Moreover, also thanks to the absence of immune responses toward the inactivated endogenous proteins, OMVsΔ60 decorated with heterologous antigens/epitopes elicit elevated antigens/epitopes-specific antibody titers and high frequencies of epitope-specific IFN-γ-producing CD8+ T cells. Altogether, we believe that E. coli BL21(DE3)Δ60 have the potential to become a workhorse factory for novel OMV-based vaccines