| 1. |
- Beech, Jason P., et al.
(författare)
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Separation of pathogenic bacteria by chain length
- 2018
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Ingår i: Analytica Chimica Acta. - : Elsevier BV. - 0003-2670 .- 1873-4324. ; 1000, s. 223-231
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Tidskriftsartikel (refereegranskat)abstract
- Using Deterministic Lateral Displacement devices optimized for sensitivity to particle length, we separate subpopulations of bacteria depending on known properties that affect their capability to cause disease (virulence). For the human bacterial pathogen Streptococcus pneumoniae, bacterial chain length and the presence of a capsule are known virulence factors contributing to its ability to cause severe disease. Separation of cultured pneumococci into subpopulations based on morphological type (single cocci, diplococci and chains) will enable more detailed studies of the role they play in virulence. Moreover, we present separation of mixed populations of almost genetically identical encapsulated and non-encapsulated pneumococcal strains in our device.
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| 2. |
- Beech, Jason P., et al.
(författare)
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Sorting bacteria by chain length - A factor of virulence?
- 2016
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Ingår i: 20th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2016. - 9780979806490 ; , s. 250-251
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Konferensbidrag (refereegranskat)abstract
- Using Deterministic Lateral Displacement (DLD), we are able to separate bacteria by their size and their chain length. This separation enables the study of these properties as factors of virulence.
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| 3. |
- Codemo, Mario, et al.
(författare)
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Immunomodulatory Effects of Pneumococcal Extracellular Vesicles on Cellular and Humoral Host Defenses
- 2018
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Ingår i: mBio. - : American Society for Microbiology. - 2161-2129 .- 2150-7511. ; 9:2
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Tidskriftsartikel (refereegranskat)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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| 4. |
- Dou, Dan, et al.
(författare)
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Analysis of IAV Replication and Co-infection Dynamics by a Versatile RNA Viral Genome Labeling Method
- 2017
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Ingår i: Cell Reports. - : Elsevier BV. - 2211-1247. ; 20:1, s. 251-263
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Tidskriftsartikel (refereegranskat)abstract
- Genome delivery to the proper cellular compartment for transcription and replication is a primary goal of viruses. However, methods for analyzing viral genome localization and differentiating genomes with high identity are lacking, making it difficult to investigate entry-related processes and co-examine heterogeneous RNA viral populations. Here, we present an RNA labeling approach for single-cell analysis of RNA viral replication and co-infection dynamics in situ, which uses the versatility of padlock probes. We applied this method to identify influenza A virus (IAV) infections in cells and lung tissue with single-nucleotide specificity and to classify entry and replication stages by gene segment localization. Extending the classification strategy to co-infections of IAVs with single-nucleotide variations, we found that the dependence on intracellular trafficking places a time restriction on secondary co-infections necessary for genome reassortment. Altogether, these data demonstrate how RNA viral genome labeling can help dissect entry and co-infections.
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| 5. |
- Henning, Dorian F., et al.
(författare)
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Luminescent CeO2:Eu3+ nanocrystals for robust in situ H2O2 real-time detection in bacterial cell cultures
- 2019
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Ingår i: Biosensors & bioelectronics. - : ELSEVIER ADVANCED TECHNOLOGY. - 0956-5663 .- 1873-4235. ; 132, s. 286-293
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Tidskriftsartikel (refereegranskat)abstract
- Hydrogen peroxide (H2O2) quantification in biomedicine is valuable as inflammation biomarker but also in assays employing enzymes that generate or consume H2O2 linked to a specific biomarker. Optical H2O2 detection is typically performed through peroxidase-coupled reactions utilizing organic dyes that suffer, however, from poor stability/reproducibility and also cannot be employed in situ in dynamic complex cell cultures to monitor H2O2 levels in real-time. Here, we utilize enzyme-mimetic CeO2 nanocrystals that are sensitive to H2O2 and study the effect of H2O2 presence on their electronic and luminescent properties. We produce and dope with Eu3+ these particles in a single-step by flame synthesis and directly deposit them on Si and glass substrates to fabricate nanoparticle layers to monitor in real-time and in situ the H2O2 concentrations generated by Streptococcus pneumoniae clinical isolates. Furthermore, the small CeO2:Eu3+ nanocrystals are combined in a single-step with larger, non-responsive Y2O3:Tb3+ nanoparticles during their double-nozzle flame synthesis to engineer hybrid luminescent nanoaggregates as ratiometric robust biosensors. We demonstrate the functionality of these biosensors by monitoring their response in the presence of a broad range of H2O2 concentrations in vitro from S. pneumoniae, highlighting their potential for facile real-time H2O2 detection in vitro in cell cultures.
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| 6. |
- Iovino, Federico, et al.
(författare)
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pIgR and PEC AM-1 bind to pneumococcal adhesins RrgA and PspC mediating bacterial brain invasion
- 2017
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Ingår i: Journal of Experimental Medicine. - : Rockefeller University Press. - 0022-1007 .- 1540-9538. ; 214:6, s. 1619-1630
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Tidskriftsartikel (refereegranskat)abstract
- Streptococcus pneumoniae is the main cause of bacterial meningitis, a life-threating disease with a high case fatality rate despite treatment with antibiotics. Pneumococci cause meningitis by invading the blood and penetrating the blood-brain barrier (BBB). Using stimulated emission depletion (STED) super-resolution microscopy of brain biopsies from patients who died of pneumococcal meningitis, we observe that pneumococci colocalize with the two BBB endothelial receptors: polymeric immunoglobulin receptor (pIgR) and platelet endothelial cell adhesion molecule (PECAM-1). We show that the major adhesin of the pneumococcal pilus-1, RrgA, binds both receptors, whereas the choline binding protein PspC binds, but to a lower extent, only pIgR. Using a bacteremia-derived meningitis model and mutant mice, as well as antibodies against the two receptors, we prevent pneumococcal entry into the brain and meningitis development. By adding antibodies to antibiotic (ceftriaxone)-treated mice, we further reduce the bacterial burden in the brain. Our data suggest that inhibition of pIgR and PECAM-1 has the potential to prevent pneumococcal meningitis.
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| 7. |
- Iovino, Federico, et al.
(författare)
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Pneumococcal meningitis is promoted by single cocci expressing pilus adhesin RrgA
- 2016
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Ingår i: Journal of Clinical Investigation. - 0021-9738 .- 1558-8238. ; 126:8, s. 2821-2826
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Tidskriftsartikel (refereegranskat)abstract
- Streptococcus pneumoniae (pneumococcus) is the primary cause of bacterial meningitis. Pneumococcal bacteria penetrates the blood-brain barrier (BBB), but the bacterial factors that enable this process are not known. Here, we determined that expression of pneumococcal pilus-1, which includes the pilus adhesin RrgA, promotes bacterial penetration through the BBB in a mouse model. S. pneumoniae that colonized the respiratory epithelium and grew in the bloodstream were chains of variable lengths; however, the pneumococci that entered the brain were division-competent, spherical, single cocci that expressed adhesive RrgA–containing pili. The cell division protein DivIVA, which is required for an ovoid shape, was localized at the poles and septum of pneumococcal chains of ovoid, nonseparated bacteria, but was absent in spherical, single cocci. In the bloodstream, a small percentage of pneumococci appeared as piliated, RrgA-expressing, DivIVA-negative single cocci, suggesting that only a minority of S. pneumoniae are poised to cross the BBB. Together, our data indicate that small bacterial cell size, which is signified by the absence of DivIVA, and the presence of an adhesive RrgA-containing pilus-1 mediate pneumococcal passage from the bloodstream through the BBB into the brain to cause lethal meningitis.
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| 8. |
- Lindstrand, Ann, et al.
(författare)
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Pneumococcal Carriage in Children under Five Years in Uganda-Will Present Pneumococcal Conjugate Vaccines Be Appropriate?
- 2016
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 11:11
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Pneumonia is the major cause of death in children globally, with more than 900,000 deaths annually in children under five years of age. Streptococcus pneumoniae causes most deaths, most often in the form of community acquired pneumonia. Pneumococcal conjugate vaccines (PCVs) are currently being implemented in many low-income countries. PCVs decrease vaccine-type pneumococcal carriage, a prerequisite for invasive pneumococcal disease, and thereby affects pneumococcal disease and transmission. In Uganda, PCV was launched in 2014, but baseline data is lacking for pneumococcal serotypes in carriage.OBJECTIVES: To study pneumococcal nasopharyngeal carriage and serotype distribution in children under 5 years of age prior to PCV introduction in Uganda.METHODS: Three cross-sectional pneumococcal carriage surveys were conducted in 2008, 2009 and 2011, comprising respectively 150, 587 and 1024 randomly selected children aged less than five years from the Iganga/Mayuge Health and Demographic Surveillance Site. The caretakers were interviewed about illness history of the child and 1723 nasopharyngeal specimens were collected. From these, 927 isolates of S. pneumoniae were serotyped.RESULTS: Overall, the carriage rate of S. pneumoniae was 56% (957/1723). Pneumococcal carriage was associated with illness on the day of the interview (OR = 1.50, p = 0.04). The most common pneumococcal serotypes were in descending order 19F (16%), 23F (9%), 6A (8%), 29 (7%) and 6B (7%). One percent of the strains were non-typeable. The potential serotype coverage rate for PCV10 was 42% and 54% for PCV13.CONCLUSION: About half of circulating pneumococcal serotypes in carriage in the Ugandan under-five population studied was covered by available PCVs.
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| 9. |
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| 10. |
- Pathak, Anuj, et al.
(författare)
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Factor H binding proteins protect division septa on encapsulated Streptococcus pneumoniae against complement C3b deposition and amplification
- 2018
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Ingår i: Nature Communications. - : Nature Publishing Group. - 2041-1723. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- Streptococcus pneumoniae evades C3-mediated opsonization and effector functions by expressing an immuno-protective polysaccharide capsule and Factor H (FH)-binding proteins. Here we use super-resolution microscopy, mutants and functional analysis to show how these two defense mechanisms are functionally and spatially coordinated on the bacterial cell surface. We show that the pneumococcal capsule is less abundant at the cell wall septum, providing C3/C3b entry to underlying nucleophilic targets. Evasion of C3b deposition at division septa and lateral amplification underneath the capsule requires localization of the FH-binding protein PspC at division sites. Most pneumococcal strains have one PspC protein, but successful lineages in colonization and disease may have two, PspC1 and PspC2, that we show affect virulence differently. We find that spatial localization of these FH-recruiting proteins relative to division septa and capsular layer is instrumental for pneumococci to resist complement-mediated opsonophagocytosis, formation of membrane-attack complexes, and for the function as adhesins.
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