| 1. |
- Aherne, Olivia, et al.
(författare)
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A novel multiplex fluorescent-labeling method for the visualization of mixed-species biofilms in vitro
- 2024
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Ingår i: Microbiology Spectrum. - : American Society for Microbiology. - 2165-0497. ; 12:7
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Tidskriftsartikel (refereegranskat)abstract
- In nature, bacteria usually exist as mixed-species biofilms, where they engage in a range of synergistic and antagonistic interactions that increase their resistance to environmental challenges. Biofilms are a major cause of persistent infections, and dispersal from initial foci can cause new infections at distal sites thus warranting further investigation. Studies of development and spatial interactions in mixed-species biofilms can be challenging due to difficulties in identifying the different bacterial species in situ. Here, we apply CellTrace dyes to studies of biofilm bacteria and present a novel application for multiplex labeling, allowing identification of different bacteria in mixed-species, in vitro biofilm models. Oral bacteria labeled with CellTrace dyes (far red, yellow, violet, and CFSE [green]) were used to create single- and mixed-species biofilms, which were analyzed with confocal spinning disk microscopy (CSDM). Biofilm supernatants were studied with flow cytometry (FC). Both Gram-positive and Gram-negative bacteria were well labeled and CSDM revealed biofilms with clear morphology and stable staining for up to 4 days. Analysis of CellTrace labeled cells in supernatants using FC showed differences in the biofilm dispersal between bacterial species. Multiplexing with different colored dyes allowed visualization of spatial relationships between bacteria in mixed-species biofilms and relative coverage by the different species was revealed through segmentation of the CSDM images. This novel application, thus, offers a powerful tool for studying structure and composition of mixed-species biofilms in vitro. IMPORTANCE Although most chronic infections are caused by mixed-species biofilms, much of our knowledge still comes from planktonic cultures of single bacterial species. Studies of formation and development of mixed-species biofilms are, therefore, required. This work describes a method applicable to labeling of bacteria for in vitro studies of biofilm structure and dispersal. Critically, labeled bacteria can be multiplexed for identification of different species in mixed-species biofilms using confocal spinning disk microscopy, facilitating investigation of biofilm development and spatial interactions under different environmental conditions. The study is an important step in increasing the tools available for such complex and challenging studies. IMPORTANCE Although most chronic infections are caused by mixed-species biofilms, much of our knowledge still comes from planktonic cultures of single bacterial species. Studies of formation and development of mixed-species biofilms are, therefore, required. This work describes a method applicable to labeling of bacteria for in vitro studies of biofilm structure and dispersal. Critically, labeled bacteria can be multiplexed for identification of different species in mixed-species biofilms using confocal spinning disk microscopy, facilitating investigation of biofilm development and spatial interactions under different environmental conditions. The study is an important step in increasing the tools available for such complex and challenging studies.
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| 2. |
- Bahnan, Wael, et al.
(författare)
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A human monoclonal antibody bivalently binding two different epitopes in streptococcal M protein mediates immune function
- 2023
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Ingår i: EMBO Molecular Medicine. - : EMBO. - 1757-4684 .- 1757-4676. ; 15:2, s. 1-21
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Tidskriftsartikel (refereegranskat)abstract
- Group A streptococci have evolved multiple strategies to evade human antibodies, making it challenging to create effective vaccines or antibody treatments. Here, we have generated antibodies derived from the memory B cells of an individual who had successfully cleared a group A streptococcal infection. The antibodies bind with high affinity in the central region of the surface-bound M protein. Such antibodies are typically non-opsonic. However, one antibody could effectively promote vital immune functions, including phagocytosis and in vivo protection. Remarkably, this antibody primarily interacts through a bivalent dual-Fab cis mode, where the Fabs bind to two distinct epitopes in the M protein. The dual-Fab cis-binding phenomenon is conserved across different groups of M types. In contrast, other antibodies binding with normal single-Fab mode to the same region cannot bypass the M protein's virulent effects. A broadly binding, protective monoclonal antibody could be a candidate for anti-streptococcal therapy. Our findings highlight the concept of dual-Fab cis binding as a means to access conserved, and normally non-opsonic regions, regions for protective antibody targeting.
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| 3. |
- Broman, Axel, et al.
(författare)
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Acoustic trapping based high throughput isolation and characterization of pathogen activated platelet derived extracellular vesicles from plasma
- 2023
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Konferensbidrag (refereegranskat)abstract
- We present the use of a high capacity and high throughput acoustic trapping platform for phenotypic characterization and functional studies of extracellular vesicles (EVs) from pathogen activated platelets. Platelet rich plasma was stimulated with bacterial M1 protein isolated from S. Pyogenes, which is known to activate platelets. The subsequently released platelet EVs were isolated from 400 μL plasma by acoustic trapping at a flowrate of 500 μL/min. We have previously reported on the acoustic trapping platform, which can process milliliter sized samples in minutes1. The EVs were then compared to EVs released by platelets stimulated with endogenous platelet activator (Thrombin) and negative control (HEPES buffer).A schematic of the sample processing can be seen in Fig. 1. Human plasma from healthy donors was incubated with HEPES buffer, Thrombin or M1 protein to stimulate platelets and induce EV release. The platelets were then removed by centrifugation, leaving EVs in plasma. The EVs were isolated and enriched by acoustic trapping and the protein content was analyzed using mass spectrometry. The EVs were also analyzed by immunoblotting, as well as immunogold labelling against CD42b and M1 protein and imaged by transmission electron microscopy (TEM). Additionally, isolated EVs were incubated with whole blood from healthy donors to investigate functional immunomodulatory effects compared to known platelet agonists (Thrombin, M1).The mass spectrometry data showed a clear distinction between isolated vesicles and plasma, Fig. 2A, with one protein cluster enriched for vesicles and one enriched for plasma samples. There was also a clear distinction between EVs from activated platelets (Thrombin, M1) and resting platelets (HEPES), Fig. 2B. Interestingly, the bacterial M1 protein was enriched in the vesicle fraction, Fig. 3A, suggesting that M1 protein binds to platelet EVs.To confirm that M1 binds to EVs, trapped samples and centrifuged samples (both pellet and supernatant) were analyzed with immunoblot against M1 protein, Fig. 3B. Clear bands are present around 54 kDa, in accordance with M1 mass, in samples containing enriched EVs. This further confirms that M1 protein binds to EVs. The TEM images showed isolated EVs for all samples, Fig. 3C. Vesicles from platelets stimulated with thrombin were found positive for CD42b. Pathogen activated platelet EVs were found positive for both CD42b and M1 protein, showing that the bacterial protein binds to platelet EVs. Although no CD42b positive EVs were found in HEPES stimulated samples in the TEM analysis, we observed a wealth of them in cytometry data.The whole blood assay showed that isolated platelet EVs stimulated platelet-neutrophil complex formation, compared to resting state, Fig. 4A. Additionally, platelet EVs stimulated IL-8 cytokine release from monocytes, Fig. 4B, suggesting functionally intact vesicles.We have demonstrated rapid isolation and enrichment of platelet EVs from plasma samples by acoustic trapping. The isolated vesicles were functionally intact, and it was possible to perform several downstream analyses, including whole blood stimulation. We found that bacterial M1 protein from S. Pyogenes binds to platelet EVs and is transported with them, a mechanism which could contribute to the rapid infectious progress in sepsis.
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| 4. |
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| 5. |
- Broman, Axel, et al.
(författare)
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Rapid multinodal acoustic trapping of extracellular vesicles for downstream mass spectrometry analysis
- 2020
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Ingår i: MicroTAS 2020 - 24th International Conference on Miniaturized Systems for Chemistry and Life Sciences. - 9781733419017 ; , s. 148-149
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Konferensbidrag (refereegranskat)abstract
- We report the use of multinodal acoustic trapping for high throughput and high capacity capturing of EV's (extracellular vesicles) for quantitative mass spectrometry analysis. The multinode trapping unit was shown to isolate sufficient amount of EV's from dilute biological samples (urine and cell culture supernatant) at flow rates of 500 ul/min within minutes, enabling EV proteome profiling. This was shown by differential protein expression analysis of urine and the urine EV fraction. Differential protein profiling of trapped EVs from stimulated versus non-stimulated platelets also demonstrated an easy access to differential expression in the EV-proteome.
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| 6. |
- Chao, Yashuan, et al.
(författare)
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Distinct phenotypes of platelet, monocyte, and neutrophil activation occur during the acute and convalescent phase of COVID-19
- 2021
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Ingår i: Platelets. - : Informa UK Limited. - 0953-7104 .- 1369-1635. ; 32:8, s. 1092-1102
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Tidskriftsartikel (refereegranskat)abstract
- SARS-CoV-2 has spread rapidly worldwide, causing the COVID-19 pandemic. Platelet activation and platelet-leukocyte complex formation are proposed to contribute to disease progression. Here, we report platelet and leukocyte activation during acute and convalescent COVID-19 in patients recruited between May-July 2020. Blood samples were analyzed by flow cytometry and ELISA using paired comparison between inclusion (day 0) and 28 days later. The majority of patients were mildly or moderately ill with significantly higher cytokine levels (IL-6 and IL-10) on day 0 as compared with day 28. Platelet activation and granule release were significantly higher on day 0 compared with day 28, as determined by ADP- or thrombin-induced surface CD62P expression, baseline released CD62P, and thrombin-induced platelet-monocyte complex formation. Monocyte activation and procoagulant status at baseline and post activation were heterogeneous but generally lower on day 0 compared with day 28. Baseline and thrombin- or fMLF-induced neutrophil activation and procoagulant status were significantly lower on day 0 compared with day 28. We demonstrate that during the acute phase of COVID-19 compared with the convalescent phase, platelets are more responsive while neutrophils are less responsive. COVID-19 is associated with thromboembolic events where platelet activation and interaction with leukocytes may play an important role.
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| 7. |
- Collin, Mattias, et al.
(författare)
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IgG glycan hydrolysis by a bacterial enzyme as a therapy against autoimmune conditions.
- 2008
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Ingår i: Proceedings of the National Academy of Sciences. - : Proceedings of the National Academy of Sciences. - 1091-6490 .- 0027-8424. ; 105:11, s. 4265-4270
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Tidskriftsartikel (refereegranskat)abstract
- EndoS from Streptococcus pyogenes efficiently hydrolyzes the functionally important and conserved N-linked glycan of IgG in human blood. Repeated i.v. administration of EndoS in rabbits completely hydrolyzes the glycans of the whole IgG pool, despite the generation of anti-EndoS antibodies. EndoS administration had no apparent effects on the health of the animals. EndoS hydrolysis of the IgG glycan has profound effects on IgG effector functions, such as complement activation and Fc receptor binding, suggesting that the enzyme could be used as an immunomodulatory therapeutic agent against IgG-mediated diseases. We demonstrate here that EndoS indeed has a protective effect in a mouse model of lethal IgG-driven immune (or idiopathic) thrombocytopenic purpura. EndoS pretreatment of pathogenic antibodies inhibits the development of disease, and the enzyme also rescues mice from already established disease when severe thrombocytopenia and s.c. bleeding have developed. These results identify EndoS as a potential therapeutic agent against diseases where pathogenic IgG antibodies are important and further emphasize antibody glycans as possible targets in future therapies against antibody-mediated autoimmune conditions.
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| 8. |
- Collins, James, et al.
(författare)
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Fibrinogen-binding and platelet-aggregation activities of a Lactobacillus salivarius septicaemia isolate are mediated by a novel fibrinogen-binding protein.
- 2012
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Ingår i: Molecular Microbiology. - : Wiley. - 1365-2958 .- 0950-382X. ; 85:5, s. 862-877
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Tidskriftsartikel (refereegranskat)abstract
- The marketplace for probiotic foods is burgeoning, measured in billions of euro per annum. It is imperative, however, that all bacterial strains are fully assessed for human safety. The ability to bind fibrinogen is considered a potential pathogenicity trait that can lead to platelet aggregation, serious medical complications, and in some instances, death. Here we examined strains from species frequently used as probiotics for their ability to bind human fibrinogen. Only one strain (CCUG 47825), a Lactobacillus salivarius isolate from a case of septicaemia, was found to strongly adhere to fibrinogen. Furthermore, this strain was found to aggregate human platelets at a level comparable to the human pathogen Staphylococcus aureus. By sequencing the genome of CCUG 47825, we were able to identify candidate genes responsible for fibrinogen binding. Complementing the genetic analysis with traditional molecular microbiological techniques enabled the identification of the novel fibrinogen receptor, CCUG_2371. Although only strain CCUG 47825 bound fibrinogen under laboratory conditions, homologues of the novel fibrinogen binding gene CCUG_2371 are widespread among L. salivarius strains, maintaining their potential to bind fibrinogen if expressed. We highlight the fact that without a full genetic analysis of strains for human consumption, potential pathogenicity traits may go undetected. © 2012 Blackwell Publishing Ltd.
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| 9. |
- Frick, Inga-Maria, et al.
(författare)
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Antibacterial activity of the contact and complement systems is blocked by SIC, a protein secreted by Streptococcus pyogenes.
- 2011
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 286, s. 1331-1340
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Tidskriftsartikel (refereegranskat)abstract
- Recent studies have shown that activation of the complement and contact systems results in the generation of antibacterial peptides. Streptococcus pyogenes, a major bacterial pathogen in humans, exists in more than one hundred different serotypes due to sequence variation in the surface-associated M protein. Cases of invasive and life-threatening S. pyogenes infections are commonly associated with isolates of the M1 serotype, and in contrast to the large majority of M serotypes, M1 isolates all secrete the SIC protein. Here we show that SIC interferes with the activation of the contact system, and blocks the activity of antibacterial peptides generated through complement and contact activation. This effect promotes the growth of S. pyogenes in human plasma, and in a mouse model of S. pyogenes sepsis, SIC enhances bacterial dissemination, results which help to explain the high frequency of severe S. pyogenes infections caused by isolates of the M1 serotype.
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| 10. |
- Frick, Inga Maria, et al.
(författare)
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Streptococcal inhibitor of complement (SIC) modulates fibrinolysis and enhances bacterial survival within fibrin clots
- 2018
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Ingår i: Journal of Biological Chemistry. - 0021-9258. ; 293:35, s. 13578-13591
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Tidskriftsartikel (refereegranskat)abstract
- Some strains of the bacterial pathogen Streptococcus pyogenes secrete protein SIC (streptococcal inhibitor of complement), including strains of the clinically relevant M1 serotype. SIC neutralizes the effect of a number of antimicrobial proteins/peptides and interferes with the function of the host complement system. Previous studies have shown that some S. pyogenes proteins bind and modulate coagulation and fibrinolysis factors, raising the possibility that SIC also may interfere with the activity of these factors. Here we show that SIC interacts with both human thrombin and plasminogen, key components of coagulation and fibrinolysis. We found that during clot formation, SIC binds fibrin through its central region and that SIC inhibits fibrinolysis by interacting with plasminogen. Flow cytometry results indicated that SIC and plasminogen bind simultaneously to S. pyogenes bacteria, and fluorescence microscopy revealed co-localization of the two proteins at the bacterial surface. As a consequence, SIC-expressing bacteria entrapped in clots inhibit fibrinolysis, leading to delayed bacterial escape from the clots as compared with mutant bacteria lacking SIC. Moreover, within the clots SIC-expressing bacteria were protected against killing. In an animal model of subcutaneous infection, SIC-expressing bacteria exhibited a delayed systemic spread. These results demonstrate that the bacterial protein SIC interferes with coagulation and fibrinolysis and thereby enhances bacterial survival, a finding that has significant implications for S. pyogenes virulence.
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