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      Immunomodulatory Effects of Pneumococcal Extracellular Vesicles on Cellular and Humoral Host Defenses

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          ABSTRACT

          Gram-positive bacteria, including the major respiratory pathogen Streptococcus pneumoniae, were recently shown to produce extracellular vesicles (EVs) that likely originate from the plasma membrane and are released into the extracellular environment. EVs may function as cargo for many bacterial proteins, however, their involvement in cellular processes and their interactions with the innate immune system are poorly understood. Here, EVs from pneumococci were characterized and their immunomodulatory effects investigated. Pneumococcal EVs were protruding from the bacterial surface and released into the medium as 25 to 250 nm lipid stained vesicles containing a large number of cytosolic, membrane, and surface-associated proteins. The cytosolic pore-forming toxin pneumolysin was significantly enriched in EVs compared to a total bacterial lysate but was not required for EV formation. Pneumococcal EVs were internalized into A549 lung epithelial cells and human monocyte-derived dendritic cells and induced proinflammatory cytokine responses irrespective of pneumolysin content. EVs from encapsulated pneumococci were recognized by serum proteins, resulting in C3b deposition and formation of C5b-9 membrane attack complexes as well as factor H recruitment, depending on the presence of the choline binding protein PspC. Addition of EVs to human serum decreased opsonophagocytic killing of encapsulated pneumococci. Our data suggest that EVs may act in an immunomodulatory manner by allowing delivery of vesicle-associated proteins and other macromolecules into host cells. In addition, EVs expose targets for complement factors in serum, promoting pneumococcal evasion of humoral host defense.

          IMPORTANCE

          Streptococcus pneumoniae is a major contributor to morbidity and mortality worldwide, being the major cause of milder respiratory tract infections such as otitis and sinusitis and of severe infections such as community-acquired pneumonia, with or without septicemia, and meningitis. More knowledge is needed on how pneumococci interact with the host, deliver virulence factors, and activate immune defenses. Here we show that pneumococci form extracellular vesicles that emanate from the plasma membrane and contain virulence properties, including enrichment of pneumolysin. We found that pneumococcal vesicles can be internalized into epithelial and dendritic cells and bind complement proteins, thereby promoting pneumococcal evasion of complement-mediated opsonophagocytosis. They also induce pneumolysin-independent proinflammatory responses. We suggest that these vesicles can function as a mechanism for delivery of pneumococcal proteins and other immunomodulatory components into host cells and help pneumococci to avoid complement deposition and phagocytosis-mediated killing, thereby possibly contributing to the symptoms found in pneumococcal infections.

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          Most cited references20

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          Prediction of lipoprotein signal peptides in Gram-negative bacteria.

          A method to predict lipoprotein signal peptides in Gram-negative Eubacteria, LipoP, has been developed. The hidden Markov model (HMM) was able to distinguish between lipoproteins (SPaseII-cleaved proteins), SPaseI-cleaved proteins, cytoplasmic proteins, and transmembrane proteins. This predictor was able to predict 96.8% of the lipoproteins correctly with only 0.3% false positives in a set of SPaseI-cleaved, cytoplasmic, and transmembrane proteins. The results obtained were significantly better than those of previously developed methods. Even though Gram-positive lipoprotein signal peptides differ from Gram-negatives, the HMM was able to identify 92.9% of the lipoproteins included in a Gram-positive test set. A genome search was carried out for 12 Gram-negative genomes and one Gram-positive genome. The results for Escherichia coli K12 were compared with new experimental data, and the predictions by the HMM agree well with the experimentally verified lipoproteins. A neural network-based predictor was developed for comparison, and it gave very similar results. LipoP is available as a Web server at www.cbs.dtu.dk/services/LipoP/.
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            Gram-positive bacteria produce membrane vesicles: proteomics-based characterization of Staphylococcus aureus-derived membrane vesicles.

            Although archaea, Gram-negative bacteria, and mammalian cells constitutively secrete membrane vesicles (MVs) as a mechanism for cell-free intercellular communication, this cellular process has been overlooked in Gram-positive bacteria. Here, we found for the first time that Gram-positive bacteria naturally produce MVs into the extracellular milieu. Further characterizations showed that the density and size of Staphylococcus aureus-derived MVs are both similar to those of Gram-negative bacteria. With a proteomics approach, we identified with high confidence a total of 90 protein components of S. aureus-derived MVs. In the group of identified proteins, the highly enriched extracellular proteins suggested that a specific sorting mechanism for vesicular proteins exists. We also identified proteins that facilitate the transfer of proteins to other bacteria, as well to eliminate competing organisms, antibiotic resistance, pathological functions in systemic infections, and MV biogenesis. Taken together, these observations suggest that the secretion of MVs is an evolutionally conserved, universal process that occurs from simple organisms to complex multicellular organisms. This information will help us not only to elucidate the biogenesis and functions of MVs, but also to develop therapeutic tools for vaccines, diagnosis, and antibiotics effective against pathogenic strains of Gram-positive bacteria.
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              The pneumococcus: epidemiology, microbiology, and pathogenesis.

              The pneumococcus is the classic Gram-positive extracellular pathogen. The medical burden of diseases it causes is amongst the greatest in the world. Intense study for more than 100 years has yielded an understanding of fundamental aspects of its physiology, pathogenesis, and immunity. Efforts to control infection have led to the deployment of polysaccharide vaccines and an understanding of antibiotic resistance. The inflammatory response to pneumococci, one of the most potent in medicine, has revealed the double-edged sword of clearance of infection but at a cost of damage to host cells. In virtually every aspect of the infectious process, the pneumococcus has set the rules of the Gram-positive pathogenesis game.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                mBio
                MBio
                mbio
                mbio
                mBio
                mBio
                American Society for Microbiology (1752 N St., N.W., Washington, DC )
                2150-7511
                10 April 2018
                Mar-Apr 2018
                : 9
                : 2
                : e00559-18
                Affiliations
                [a ]Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet and Department of Clinical Microbiology, Karolinska University Hospital, Stockholm, Sweden
                [b ]Department of Molecular Biology and the Umeå Centre for Microbial Research (UCMR), Umeå University, Umeå, Sweden
                [c ]Singapore Centre on Environmental Life Sciences Engineering and Lee Kong Chian School of Medicine (LKC), Nanyang Technological University (NTU), Singapore, Singapore
                GSK Vaccines
                Author notes
                Address correspondence to Birgitta Henriques-Normark, birgitta.henriques@ 123456ki.se .

                This article is a direct contribution from a Fellow of the American Academy of Microbiology. Solicited external reviewers: Marco Rinaldo Oggioni, University of Leicester; Mariagrazia Pizza, GSK Vaccines.

                Article
                mBio00559-18
                10.1128/mBio.00559-18
                5893880
                29636428
                afddf13b-da4d-4760-9fe9-1e9c8221a87b
                Copyright © 2018 Codemo et al.

                This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

                History
                : 10 March 2018
                : 14 March 2018
                Page count
                supplementary-material: 10, Figures: 7, Tables: 0, Equations: 0, References: 36, Pages: 15, Words: 9969
                Funding
                Funded by: The Swedish Research Council;
                Award Recipient :
                Funded by: Stockholm County Council;
                Award Recipient :
                Funded by: The Swedish Foundation for Strategic research (SSF);
                Award Recipient :
                Funded by: Knut and Alice Wallenberg Foundation;
                Award Recipient :
                Categories
                Research Article
                Custom metadata
                March/April 2018

                Life sciences
                streptococcus pneumoniae,cellular and humoral defense,complement,extracellular vesicles,pneumococcal evasion of immune response,pneumococci,pneumolysin

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