| 1. |
- Aherne, Olivia, et al.
(author)
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A novel multiplex fluorescent-labeling method for the visualization of mixed-species biofilms in vitro
- 2024
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In: Microbiology Spectrum. - : American Society for Microbiology. - 2165-0497. ; 12:7
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Journal article (peer-reviewed)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.
(author)
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A human monoclonal antibody bivalently binding two different epitopes in streptococcal M protein mediates immune function
- 2023
-
In: EMBO Molecular Medicine. - : EMBO. - 1757-4684 .- 1757-4676. ; 15:2, s. 1-21
-
Journal article (peer-reviewed)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.
(author)
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Acoustic trapping based high throughput isolation and characterization of pathogen activated platelet derived extracellular vesicles from plasma
- 2023
-
Conference paper (peer-reviewed)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.
(author)
-
Rapid multinodal acoustic trapping of extracellular vesicles for downstream mass spectrometry analysis
- 2020
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In: MicroTAS 2020 - 24th International Conference on Miniaturized Systems for Chemistry and Life Sciences. - 9781733419017 ; , s. 148-149
-
Conference paper (peer-reviewed)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.
(author)
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Biofilm-dispersed pneumococci induce elevated leukocyte and platelet activation
- 2024
-
In: Frontiers in cellular and infection microbiology. - 2235-2988. ; 14, s. 1-14
-
Journal article (peer-reviewed)abstract
- INTRODUCTION: Streptococcus pneumoniae (the pneumococcus) effectively colonizes the human nasopharynx, but can migrate to other host sites, causing infections such as pneumonia and sepsis. Previous studies indicate that pneumococci grown as biofilms have phenotypes of bacteria associated with colonization whereas bacteria released from biofilms in response to changes in the local environment (i.e., dispersed bacteria) represent populations with phenotypes associated with disease. How these niche-adapted populations interact with immune cells upon reaching the vascular compartment has not previously been studied. Here, we investigated neutrophil, monocyte, and platelet activation using ex vivo stimulation of whole blood and platelet-rich plasma with pneumococcal populations representing distinct stages of the infectious process (biofilm bacteria and dispersed bacteria) as well as conventional broth-grown culture (planktonic bacteria). METHODS: Flow cytometry and ELISA were used to assess surface and soluble activation markers for neutrophil and monocyte activation, platelet-neutrophil complex and platelet-monocyte complex formation, and platelet activation and responsiveness.RESULTS: Overall, we found that biofilm-derived bacteria (biofilm bacteria and dispersed bacteria) induced significant activation of neutrophils, monocytes, and platelets. In contrast, little to no activation was induced by planktonic bacteria. Platelets remained functional after stimulation with bacterial populations and the degree of responsiveness was inversely related to initial activation. Bacterial association with immune cells followed a similar pattern as activation.DISCUSSION: Differences in activation of and association with immune cells by biofilm-derived populations could be an important consideration for other pathogens that have a biofilm state. Gaining insight into how these bacterial populations interact with the host immune response may reveal immunomodulatory targets to interfere with disease development.
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| 7. |
- Chao, Yashuan, et al.
(author)
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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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In: Platelets. - : Informa UK Limited. - 0953-7104 .- 1369-1635. ; 32:8, s. 1092-1102
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Journal article (peer-reviewed)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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| 8. |
- Collin, Mattias, et al.
(author)
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IgG glycan hydrolysis by a bacterial enzyme as a therapy against autoimmune conditions.
- 2008
-
In: 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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Journal article (peer-reviewed)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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| 9. |
- Collins, James, et al.
(author)
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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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In: Molecular Microbiology. - : Wiley. - 1365-2958 .- 0950-382X. ; 85:5, s. 862-877
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Journal article (peer-reviewed)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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| 10. |
- Frick, Inga-Maria, et al.
(author)
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Antibacterial activity of the contact and complement systems is blocked by SIC, a protein secreted by Streptococcus pyogenes.
- 2011
-
In: Journal of Biological Chemistry. - 1083-351X. ; 286, s. 1331-1340
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Journal article (peer-reviewed)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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| 11. |
- Frick, Inga Maria, et al.
(author)
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Streptococcal inhibitor of complement (SIC) modulates fibrinolysis and enhances bacterial survival within fibrin clots
- 2018
-
In: Journal of Biological Chemistry. - 0021-9258. ; 293:35, s. 13578-13591
-
Journal article (peer-reviewed)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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| 12. |
- Hovold, Gisela, et al.
(author)
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BPI-ANCA is expressed in the airways of cystic fibrosis patients and correlates to platelet numbers and Pseudomonas aeruginosa colonization
- 2020
-
In: Respiratory Medicine. - : Elsevier BV. - 0954-6111. ; 170
-
Journal article (peer-reviewed)abstract
- Background: Autoantibodies to bactericidal/permeability-increasing protein (BPI), BPI-ANCA, are often present in serum of patients with cystic fibrosis (CF), and correlate to airway colonization with Pseudomonas aeruginosa. The aim of the study was to investigate if BPI-ANCA IgA is also present in the airways of CF patients, and if its presence correlates with neutrophil counts, platelets, and P. aeruginosa DNA in sputum. Methods: BPI-ANCA IgA was quantified in serum and sputum samples from adult CF patients (n = 45) by ELISA. Sputum neutrophil counts, platelets, and platelet-neutrophil complexes were assessed by flow cytometry, and P. aeruginosa DNA was analysed with RT-PCR. Results: Serum BPI-ANCA IgA was present in 44% of the study participants, and this group also had significantly enhanced BPI-ANCA levels in sputum compared to serum negative patients. Sputum levels of BPI-ANCA IgA correlated with P. aeruginosa DNA (r = 0.63, p = 0.0003) and platelet counts in sputum (r = 0.60, p = 0.0002). Conclusions: BPI-ANCA is expressed in the airways of CF patients and correlates with P. aeruginosa load and platelet counts, suggesting a link to airway inflammation and mucosal immunity.
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| 13. |
- Hurley, Sinead M., et al.
(author)
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The dynamics of platelet activation during the progression of streptococcal sepsis
- 2016
-
In: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 11:9
-
Journal article (peer-reviewed)abstract
- Platelets contribute to inflammation however, the role of platelet activation during the pathophysiological response to invasive bacterial infection and sepsis is not clear. Herein, we have investigated platelet activation in a mouse model of invasive Streptococcus pyogenes infection at 5,12, and 18 hours post infection and correlated this to parameters of infection. The platelet population in ex-vivo blood samples showed no increased integrin activation or surface presentation of CD62P, however platelet-neutrophil complex formation and plasma levels of CD62P were increased during bacterial dissemination and the progression of sepsis, indicating that platelet activation had occurred in vivo. Platelet-neutrophil complex formation was the most discriminatory marker of platelet activation. Platelet-neutrophil complexes were increased above baseline levels during early sepsis but decreased to significantly lower levels than baseline during late sepsis. The removal of these complexes from the circulation coincided with a significant increase in organ damage and the accumulation of platelets in the liver sinusoids, suggesting that platelet activation in the circulation precedes accumulation of platelets in damaged organs. The results demonstrate that monitoring platelet activation using complementary methods may provide prognostic information during the pathogenesis of invasive S. pyogenes infection.
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| 14. |
- Hurley, Sinead, et al.
(author)
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Platelet-dependent neutrophil function is dysregulated by M protein from Streptococcus pyogenes.
- 2015
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In: Infection and Immunity. - 1098-5522. ; 83:9, s. 3515-3525
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Journal article (peer-reviewed)abstract
- Platelets are rapidly responsive sentinel cells that patrol the bloodstream and contribute to the host response to infection. Platelets have been reported to form heterotypic aggregates with leukocytes and may modulate their function. Herein we have investigated platelet-neutrophil complex formation and neutrophil function in response to distinct agonists. The endogenous platelet activator thrombin gave rise to platelet-dependent neutrophil activation, resulting in enhanced phagocytosis and bacterial killing. Streptococcus pyogenes is an important causative agent of severe infectious disease, which can manifest as sepsis and septic shock. M1 protein from S. pyogenes also mediated platelet-neutrophil complex formation, however these neutrophils were dysfunctional and exhibited diminished chemotactic ability and bacterial killing. This reveals an important agonist dependent neutrophil dysfunction during platelet-neutrophil complex formation, and highlights the role of platelets during the immune response to streptococcal infection.
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| 15. |
- Izadi, Arman, et al.
(author)
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The hinge-engineered IgG1-IgG3 hybrid subclass IgGh47 potently enhances Fc-mediated function of anti-streptococcal and SARS-CoV-2 antibodies
- 2024
-
In: Nature Communications. - : Springer Nature. - 2041-1723. ; 15, s. 1-22
-
Journal article (peer-reviewed)abstract
- Streptococcus pyogenes can cause invasive disease with high mortality despite adequate antibiotic treatments. To address this unmet need, we have previously generated an opsonic IgG1 monoclonal antibody, Ab25, targeting the bacterial M protein. Here, we engineer the IgG2-4 subclasses of Ab25. Despite having reduced binding, the IgG3 version promotes stronger phagocytosis of bacteria. Using atomic simulations, we show that IgG3’s Fc tail has extensive movement in 3D space due to its extended hinge region, possibly facilitating interactions with immune cells. We replaced the hinge of IgG1 with four different IgG3-hinge segment subclasses, IgGhxx. Hinge-engineering does not diminish binding as with IgG3 but enhances opsonic function, where a 47 amino acid hinge is comparable to IgG3 in function. IgGh47 shows improved protection against S. pyogenes in a systemic infection mouse model, suggesting that IgGh47 has promise as a preclinical therapeutic candidate. Importantly, the enhanced opsonic function of IgGh47 is generalizable to diverse S. pyogenes strains from clinical isolates. We generated IgGh47 versions of anti-SARS-CoV-2 mAbs to broaden the biological applicability, and these also exhibit strongly enhanced opsonic function compared to the IgG1 subclass. The improved function of the IgGh47 subclass in two distant biological systems provides new insights into antibody function.
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| 16. |
- Johansson, Björn, et al.
(author)
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IdeS: A Bacterial Proteolytic Enzyme with Therapeutic Potential.
- 2008
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In: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 3:2
-
Journal article (peer-reviewed)abstract
- BACKGROUND: IdeS, a proteinase from Streptococcus pyogenes, cleaves immunoglobulin (Ig)G antibodies with a unique degree of specificity. Pathogenic IgG antibodies constitute an important clinical problem contributing to the pathogenesis of a number of autoimmune conditions and acute transplant rejection. To be able to effectively remove such antibodies is therefore an important clinical challenge. METHODOLOGY/PRINCIPAL FINDINGS: IdeS was found to specifically and efficiently cleave IgG in human blood in vitro (20 microg of IdeS caused a complete degradation of IgG in one ml of human whole blood in 15 minutes) and to clear IgG from the blood stream of rabbits in vivo (no IgG was detected six hours following an intravenous injection of 5 mg of IdeS) without any side effects. In a mouse model of immune thrombocytopenic purpura (ITP), polyclonal IgG antibodies against platelet surface antigens were used to induce a lethal disease. These profoundly thrombocytopenic animals were treated and cured by a single injection of IdeS. CONCLUSIONS/SIGNIFICANCE: Novel information is provided concerning the IgG-cleaving activity of IdeS in vitro and in vivo. The highly specific and rapid elimination of IgG in vivo, the dramatic effect in a mouse model of ITP, and the lack of side effects in the treated animals, indicate that IdeS could also be used to treat IgG-driven diseases in humans.
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| 17. |
- Johansson, Daniel, et al.
(author)
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Platelet and neutrophil responses to gram positive pathogens in patients with bacteremic infection.
- 2011
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In: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 6:11
-
Journal article (peer-reviewed)abstract
- BACKGROUND: Many Gram-positive pathogens aggregate and activate platelets in vitro and this has been proposed to contribute to virulence. Platelets can also form complexes with neutrophils but little is however known about platelet and platelet-neutrophil responses in bacterial infection. METHODOLOGY/PRINCIPAL FINDINGS: We added isolates of Gram-positive bacteria from 38 patients with a bacteremic infection to blood drawn from the same patient. Aggregometry and flow cytometry were used to assess platelet aggregation and to quantify activation of platelets, neutrophils, and platelet-neutrophils complexes (PNCs) induced by the bacteria. Fifteen healthy persons served as controls. Most isolates of Staphylococcus aureus, beta hemolytic streptococci, and Enterococcus faecalis induced aggregation of platelets from their respective hosts, whereas pneumococci failed to do so. S. aureus isolates induced platelet aggregation more rapidly in patients than in controls, whereas platelet activation by S. aureus was lower in patients than in controls. PNCs were more abundant in baseline samples from patients than in healthy controls and most bacterial isolates induced additional PNC formation and neutrophil activation. CONCLUSION/SIGNIFICANCE: We have demonstrated for the first time that bacteria isolated from patients with Gram-positive bacteremia can induce platelet activation and aggregation, PNC formation, and neutrophil activation in the same infected host. This underlines the significance of these interactions during infection, which could be a target for future therapies in sepsis.
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| 18. |
- Kahn, Fredrik, et al.
(author)
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Antibodies against a surface protein of Streptococcus pyogenes promote a pathological inflammatory response.
- 2008
-
In: PLoS Pathogens. - : Public Library of Science (PLoS). - 1553-7366 .- 1553-7374. ; 4:9
-
Journal article (peer-reviewed)abstract
- Streptococcal toxic shock syndrome (STSS) caused by Streptococcus pyogenes is a clinical condition with a high mortality rate despite modern intensive care. A key feature of STSS is excessive plasma leakage leading to hypovolemic hypotension, disturbed microcirculation and multiorgan failure. Previous work has identified a virulence mechanism in STSS where M1 protein of S. pyogenes forms complexes with fibrinogen that activate neutrophils to release heparin-binding protein (HBP), an inducer of vascular leakage. Here, we report a marked inter-individual difference in the response to M1 protein-induced HBP release, a difference found to be related to IgG antibodies directed against the central region of the M1 protein. To elicit massive HBP release, such antibodies need to be part of the M1 protein-fibrinogen complexes. The data add a novel aspect to bacterial pathogenesis where antibodies contribute to the severity of disease by promoting a pathologic inflammatory response.
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| 19. |
- Kahn, Fredrik, et al.
(author)
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Platelets promote bacterial dissemination in a mouse model of streptococcal sepsis.
- 2013
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In: Microbes and Infection. - : Elsevier BV. - 1769-714X .- 1286-4579. ; 15:10-11, s. 669-676
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Journal article (peer-reviewed)abstract
- Platelets have been reported to contribute to inflammation and inflammatory disorders. In the present study, we demonstrate that platelets contribute to the acute response to bacterial infection in a mouse model of invasive Streptococcus pyogenes infection. Thrombocytopenia occurred rapidly in infected animals and this was associated with platelet activation, formation of platelet-neutrophil complexes and neutrophil activation. In order to assess the role of platelets during infection, platelets were depleted prior to infection. Platelet-depleted animals had significantly decreased platelet-neutrophil complex formation and neutrophil activation in response to infection. Importantly, significantly fewer bacteria disseminated to the blood, lungs, and spleen of platelet-depleted animals. Platelet-depleted animals did not decrease as significantly in weight as the infected control animals. The results demonstrate a previously unappreciated role for platelets during the pathophysiological response to infection, whereby S. pyogenes bacteria bind to platelets and platelets facilitate bacterial dissemination.
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| 20. |
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| 21. |
- Karlsson Söbirk, Sara, et al.
(author)
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Human Chemokines as Antimicrobial Peptides with Direct Parasiticidal Effect on Leishmania mexicana In Vitro.
- 2013
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In: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 8:3
-
Journal article (peer-reviewed)abstract
- Chemokines and chemokine receptor-mediated effects are important mediators of the immunological response and cure in human leishmaniasis. However, in addition to their signalling properties for leukocytes, many chemokines have also been shown to act directly as antimicrobial peptides on bacteria and fungi. We screened ten human chemokines (CXCL2, CXCL6, CXCL8, CXCL9, CXCL10, CCL2, CCL3, CCL20, CCL27, CCL28) for antimicrobial effects on the promastigote form of the protozoan parasite Leishmania mexicana, and observed direct parasiticidal effects of several, CCL28 being the most potent. Damage to the plasma membrane integrity could be visualised by entrance of propidium iodide, as measured with flow cytometry, and by scanning electron microscopy, which showed morphological changes and aggregation of cells. The findings were in concordance with parasiticidal activity, measured by decreased mitochondrial activity in an MTT-assay. This is the first report of direct antimicrobial activity by chemokines on parasites. This component of immunity against Leishmania parasites identified here warrants further investigation that might lead to new insight in the mechanisms of human infection and/or new therapeutic approaches.
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| 22. |
- Lindberg, Ulrika, et al.
(author)
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Increased platelet activation occurs in cystic fibrosis patients and correlates to clinical status
- 2018
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In: Thrombosis Research. - : Elsevier BV. - 0049-3848 .- 1879-2472. ; 162, s. 32-37
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Journal article (peer-reviewed)abstract
- Cystic fibrosis (CF) is an inflammatory lung disease. Platelets have an emerging role in inflammation, however previous studies of platelet activation in CF have generated conflicting results. In this study, we determined platelet function in CF patients and correlated platelet activation to establish clinical and laboratory parameters. Twenty-two patients, aged 20.7 to 54.4 (mean 34.0, SD 9.45) years and with a mean FEV1%pred (forced expiratory volume in one second, % of predicted) of 72 (SD 21.4, range 32–110) were recruited. A combination of platelet assays was used: platelet aggregation, platelet activation and platelet-leukocyte complex formation. Platelets from CF patients exhibited significantly increased aggregation when stimulated ex-vivo, a tendency towards increased platelet upregulation of CD62P, but no increase of GPIIb/IIIa activation (PAC-1). Platelet-monocyte complex (PMC) formation was significantly increased in CF patients compared to controls, while platelet-neutrophil complex formation was not. In the CF group, platelet aggregation correlates with levels of anti-neutrophil cytoplasmic antibodies (ANCA) with specificity for bactericidal/permeability-increasing protein (BPI), BPI-ANCA (r = 0.56). The formation of PMCs correlates with lung function decline (1-FEV1%), CRP and BPI-ANCA (r = 0.61, 0.55, 0.5). We therefore confirm the presence of increased platelet activation in CF patients, and determine that further evaluation of platelet activation in relation to prognostic factors in CF is warranted.
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| 23. |
- Malmström, Erik, et al.
(author)
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Protein C inhibitor - a novel antimicrobial agent
- 2009
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In: PLoS Pathogens. - : Public Library of Science (PLoS). - 1553-7366 .- 1553-7374. ; 5:12, s. e1000698-
-
Journal article (peer-reviewed)abstract
- Protein C inhibitor (PCI) is a heparin-binding serine proteinase inhibitor belonging to the family of serpin proteins. Here we describe that PCI exerts broad antimicrobial activity against bacterial pathogens. This ability is mediated by the interaction of PCI with lipid membranes, which subsequently leads to their permeabilization. As shown by negative staining electron microscopy, treatment of Escherichia coli or Streptococcus pyogenes bacteria with PCI triggers membrane disruption followed by the efflux of bacterial cytosolic contents and bacterial killing. The antimicrobial activity of PCI is located to the heparin-binding site of the protein and a peptide spanning this region was found to mimic the antimicrobial activity of PCI, without causing lysis or membrane destruction of eukaryotic cells. Finally, we show that platelets can assemble PCI on their surface upon activation. As platelets are recruited to the site of a bacterial infection, these results may explain our finding that PCI levels are increased in tissue biopsies from patients suffering from necrotizing fasciitis caused by S. pyogenes. Taken together, our data describe a new function for PCI in innate immunity.
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| 24. |
- Malmström, Erik, et al.
(author)
-
Targeted mass spectrometry analysis of neutrophil-derived proteins released during sepsis progression.
- 2014
-
In: Thrombosis and Haemostasis. - 0340-6245. ; 112:6, s. 1230-1243
-
Journal article (peer-reviewed)abstract
- Early diagnosis of severe infectious diseases is essential for timely implementation of lifesaving therapies. In a search for novel biomarkers in sepsis diagnosis we focused on polymorphonuclear neutrophils (PMNs). Notably, PMNs have their protein cargo readily stored in granules and following systemic stimulation an immediate increase of neutrophil-borne proteins can be observed into the circulation of sepsis patients. We applied a combination of mass spectrometry (MS) based approaches, LC-MS/MS and selected reaction monitoring (SRM), to characterise and quantify the neutrophil proteome in healthy or disease conditions. With this approach we identified a neutrophil-derived protein abundance pattern in blood plasma consisting of 20 proteins that can be used as a protein signature for severe infectious diseases. Our results also show that SRM is highly sensitive, specific, and reproducible and, thus, a promising technology to study a complex, dynamic and multifactorial disease such as sepsis.
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| 25. |
- Mohanty, Tirthankar, et al.
(author)
-
A pharmacoproteomic landscape of organotypic intervention responses in Gram-negative sepsis
- 2023
-
In: Nature Communications. - 2041-1723. ; 14, s. 1-17
-
Journal article (peer-reviewed)abstract
- Sepsis is the major cause of mortality across intensive care units globally, yet details of accompanying pathological molecular events remain unclear. This knowledge gap has resulted in ineffective biomarker development and suboptimal treatment regimens to prevent and manage organ dysfunction/damage. Here, we used pharmacoproteomics to score time-dependent treatment impact in a murine Escherichia coli sepsis model after administering beta-lactam antibiotic meropenem (Mem) and/or the immunomodulatory glucocorticoid methylprednisolone (Gcc). Three distinct proteome response patterns were identified, which depended on the underlying proteotype for each organ. Gcc enhanced some positive proteome responses of Mem, including superior reduction of the inflammatory response in kidneys and partial restoration of sepsis-induced metabolic dysfunction. Mem introduced sepsis-independent perturbations in the mitochondrial proteome that Gcc counteracted. We provide a strategy for the quantitative and organotypic assessment of treatment effects of candidate therapies in relationship to dosing, timing, and potential synergistic intervention combinations during sepsis.
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| 26. |
- Naudin, Clément, et al.
(author)
-
Active but inoperable thrombin is accumulated in a plasma protein layer surrounding Streptococcus pyogenes.
- 2015
-
In: Thrombosis and Haemostasis. - 0340-6245. ; 114:4, s. 717-726
-
Journal article (peer-reviewed)abstract
- Activation of thrombin is a critical determinant in many physiological and pathological processes including haemostasis and inflammation. Under physiological conditions many of these functions are involved in wound healing or eradication of an invading pathogen. However, when activated systemically, thrombin can contribute to severe and life-threatening conditions by causing complications such as multiple multi-organ failure and disseminated intravascular coagulation. In the present study we investigated how the activity of thrombin is modulated when it is bound to the surface of Streptococcus pyogenes. Our data show that S. pyogenes bacteria become covered with a proteinaceous layer when incubated with human plasma, and that thrombin is a constituent of this layer. Though the coagulation factor is found attached to the bacteria with a functional active site, thrombin has lost its capacity to interact with its natural substrates and inhibitors. Thus, the interaction of bacteria with human plasma renders thrombin completely inoperable at the streptococcal surface. This could represent a host defense mechanism to avoid systemic activation of coagulation which could be otherwise induced when bacteria enter the circulation and cause systemic infection.
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| 27. |
- Nägeli, Andreas, et al.
(author)
-
Streptococcus pyogenes evades adaptive immunity through specific IgG glycan hydrolysis
- 2019
-
In: Journal of Experimental Medicine. - : Rockefeller University Press. - 1540-9538 .- 0022-1007. ; 216:7, s. 1615-1629
-
Journal article (peer-reviewed)abstract
- Streptococcus pyogenes (Group A streptococcus; GAS) is a human pathogen causing diseases from uncomplicated tonsillitis to life-threatening invasive infections. GAS secretes EndoS, an endoglycosidase that specifically cleaves the conserved N-glycan on IgG antibodies. In vitro, removal of this glycan impairs IgG effector functions, but its relevance to GAS infection in vivo is unclear. Using targeted mass spectrometry, we characterized the effects of EndoS on host IgG glycosylation during the course of infections in humans. Substantial IgG glycan hydrolysis occurred at the site of infection and systemically in the severe cases. We demonstrated decreased resistance to phagocytic killing of GAS lacking EndoS in vitro and decreased virulence in a mouse model of invasive infection. This is the first described example of specific bacterial IgG glycan hydrolysis during infection and thereby verifies the hypothesis that EndoS modifies antibodies in vivo. This mechanisms of immune evasion could have implications for treatment of severe GAS infections and for future efforts at vaccine development.
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| 28. |
- Oehmcke, Sonja, et al.
(author)
-
Streptococcal M proteins and their role as virulence determinants.
- 2010
-
In: Clinica Chimica Acta. - : Elsevier BV. - 0009-8981. ; 411, s. 1172-1180
-
Journal article (peer-reviewed)abstract
- Group A streptococci (GAS, Streptococcus pyogenes) are exclusive human pathogens that have been extensively studied for many decades. The spectrum of diseases caused by these bacteria ranges from uncomplicated and superficial to severe and invasive infections. In order to give rise to these complications, GAS have evolved a number of surface-bound and secreted virulence factors, of which the M proteins are probably the best characterized. Evidence has emerged that M proteins are multifunctional pathogenic determinants, and over the years many interactions between M proteins and the human host have been reported. The present review article aims to present a state-of-the-art overview of the most important virulence mechanisms employed by M proteins to trigger disease.
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| 29. |
- Oehmcke, Sonja, et al.
(author)
-
Treatment of invasive streptococcal infection with a peptide derived from human high molecular weight kininogen.
- 2009
-
In: Blood. - : American Society of Hematology. - 1528-0020 .- 0006-4971. ; 114:2, s. 444-451
-
Journal article (peer-reviewed)abstract
- Sepsis and septic shock remain an important medical problem, emphasizing the need to identify novel therapeutic opportunities. Hypovolemic hypotension, coagulation dysfunction, disturbed microcirculation, and multiorgan failure due to vascular leakage are often observed in these severe conditions. In the present study, we find that HKH20, a peptide derived from human high molecular weight kininogen (HK), down-regulates inflammatory reactions caused by Streptococcus pyogenes in a mouse model of sepsis. HK is a component of the pro-inflammatory and pro-coagulant contact system. Activation of the contact system in the bloodstream by S. pyogenes leads to massive tissue damage in the lungs of the infected mice, which eventually results in the death of the animals. HKH20 inhibits activation of the contact system and protects mice with invasive S. pyogenes infection from lung damage. In combination with clindamycin treatment, the peptide also significantly prolongs the survival of infected mice.
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| 30. |
- Palm, Frida, et al.
(author)
-
Complement Activation Occurs at the Surface of Platelets Activated by Streptococcal M1 Protein and This Results in Phagocytosis of Platelets
- 2019
-
In: Journal of immunology. - : The American Association of Immunologists. - 1550-6606 .- 0022-1767. ; 202:2, s. 503-513
-
Journal article (peer-reviewed)abstract
- Platelets circulate the bloodstream and principally maintain hemostasis. Disturbed hemostasis, a dysregulated inflammatory state, and a decreased platelet count are all hallmarks of severe invasive Streptococcus pyogenes infection, sepsis. We have previously demonstrated that the released M1 protein from S. pyogenes activates platelets, and this activation is dependent on the binding of M1 protein, fibrinogen, and M1-specific IgG to platelets in susceptible donors. In this study, we characterize the M1-associated protein interactions in human plasma and investigate the acquisition of proteins to the surface of activated platelets and the consequences for platelet immune function. Using quantitative mass spectrometry, M1 protein was determined to form a protein complex in plasma with statistically significant enrichment of fibrinogen, IgG3, and complement components, especially C1q. Using flow cytometry, these plasma proteins were also confirmed to be acquired to the platelet surface, resulting in complement activation on M1-activated human platelets. Furthermore, we demonstrated an increased phagocytosis of M1-activated platelets by monocytes, which was not observed with other physiological platelet agonists. This reveals a novel mechanism of complement activation during streptococcal sepsis, which contributes to the platelet consumption that occurs in sepsis.
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| 31. |
- Palm, Frida, et al.
(author)
-
Distinct Serotypes of Streptococcal M Proteins Mediate Fibrinogen-Dependent Platelet Activation and Proinflammatory Effects
- 2022
-
In: Infection and Immunity. - 1098-5522. ; 90:2, s. 1-12
-
Journal article (peer-reviewed)abstract
- Sepsis is a life-threatening complication of infection that is characterised by a dysregulated inflammatory state and disturbed hemostasis. Platelets are the main regulators of hemostasis, and they also respond to inflammation. The human pathogen Streptococcus pyogenes can cause local infection that may progress to sepsis. There are more than 200 different serotypes of S. pyogenes defined according to sequence variations in the M protein. The M1 serotype is among ten serotypes that are predominant in invasive infection. M1 protein can be released from the surface and has previously been shown to generate platelet, neutrophil and monocyte activation. The platelet dependent pro-inflammatory effects of other serotypes of M protein associated with invasive infection (M3, M5, M28, M49 and M89) is now investigated using a combination of multiparameter flow cytometry, ELISA, aggregometry and quantitative mass spectrometry. We demonstrate that only M1-, M3- and M5 protein serotypes can bind fibrinogen in plasma and mediate fibrinogen and IgG dependent platelet activation and aggregation, release of granule proteins, upregulation of CD62P to the platelet surface, and complex formation with neutrophils and monocytes. Neutrophil and monocyte activation, determined as upregulation of surface CD11b, is also mediated by M1-, M3- and M5 protein serotypes, while M28-, M49- or M89 proteins failed to mediate activation of platelets or leukocytes. Collectively, our findings reveal novel aspects of the immunomodulatory role of fibrinogen acquisition and platelet activation during streptococcal infections.
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| 32. |
- Palm, Frida, et al.
(author)
-
Phenotypic characterization of acoustically enriched extracellular vesicles from pathogen-activated platelets
- 2023
-
In: Journal of Innate Immunity. - : S. Karger. - 1662-811X .- 1662-8128. ; 15:1, s. 599-613
-
Journal article (peer-reviewed)abstract
- Extracellular vesicles (EVs) are derived from the membrane of platelets and released in the circulation upon activation or injury. Analogous to the parent cell, platelet derived EVs play an important role in hemostasis and immune responses by transfer of bioactive cargo from the parent cells. Platelet activation and release of EVs increases in several pathological inflammatory diseases, such as sepsis. We have previously reported that the M1 protein released from the bacterial pathogen Streptococcus pyogenes directly mediates platelet activation. In this study, EVs were isolated from these pathogen-activated platelets using acoustic trapping and their inflammation phenotype was characterized using quantitative mass spectrometry-based proteomics and cell-based models of inflammation. We determined that M1 protein mediated release of platelet derived EVs that contained the M1 protein. The isolated EVs derived from pathogen-activated platelets contained a similar protein cargo to those from physiologically activated platelets (thrombin), and included platelet membrane proteins, granule proteins and cytoskeletal proteins, coagulation factors and immune mediators. Immunomodulatory cargo, complement proteins and IgG3, were significantly enriched in EVs isolated from M1 protein-stimulated platelets. Acoustically enriched EVs were functionally intact and exhibited proinflammatory effects on addition to blood, including platelet-neutrophil complex formation, neutrophil activation, and cytokine release. Collectively, our findings reveal novel aspects of pathogen-mediated platelet activation during invasive streptococcal infection.
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| 33. |
- Pernow, Gustav, et al.
(author)
-
Platelet Activation and Aggregation Induced by Streptococcus bovis/Streptococcus equinus Complex
- 2022
-
In: Microbiology spectrum. - : American Society for Microbiology. - 2165-0497. ; 10:6
-
Journal article (peer-reviewed)abstract
- Streptococcus bovis/Streptococcus equinus complex (SBSEC) is a common cause of infective endocarditis (IE). For IE-pathogens, the capacity to activate and aggregate platelets is believed to be an important virulence mechanism. While the interactions between bacteria and platelets have been described in detail for many Gram-positive pathogens, little research has been carried out with SBSEC in this respect. Twenty-six isolates of the four most common species and subspecies of SBSEC identified in bacteremia were collected, and interactions with platelets were investigated in platelet rich plasma (PRP) from three donors. Aggregation was studied using light-transmission aggregometry and platelet activation using flow cytometry detecting surface upregulation of CD62P. Platelets and serum were treated with different inhibitors to determine mechanisms involved in platelet aggregation and activation. Twenty-two of 26 isolates induced aggregation in at least one donor, and four isolates induced aggregation in all three donors. In PRP from donor 1, isolate SL1 induced a rapid aggregation with a median time of 70 s to reach 50% aggregation. Blockade of the platelet Fc-receptor or enzymatic cleavage of IgG abolished platelet activation and aggregation. The capacity for bacteria-induced platelet aggregation was also shown to be transferable between donors through serum. SBSEC mediates platelet aggregation in an IgG and IgG-Fc-receptor dependent manner. Bacterial activation of platelets through this pathway is common for many bacteria causing IE and could be a potential therapeutic target for the prevention and treatment of this infection. IMPORTANCE The capacity of bacteria to activate and aggregate platelets is believed to contribute to the pathogenesis of IE. The Streptococcus bovis/Streptococcus equinus complex (SBSEC) contains known IE-pathogens, but there is limited research on the different subspecies ability to interact with platelets and what signaling pathways are involved. This study reports that 22 of 26 tested isolates of different subspecies within SBSEC can induce aggregation, and that aggregation is host dependent. The Fc-IgG-receptor pathway was shown essential for platelet activation and aggregation. To the best of our knowledge, this is the first study that reports on platelet interactions of SBSEC-isolates other than Streptococcus gallolyticus subspecies gallolyticus as well as the first study to report of mechanisms of platelet interaction of SBSEC-isolates. It adds SBSEC to a group of bacteria that activate and aggregate platelets via the platelet Fc-receptor. This could be a potential therapeutic target for prevention of IE.
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| 34. |
- Petersson, Frida, et al.
(author)
-
Platelet activation and aggregation by the opportunistic pathogen Cutibacterium (Propionibacterium) acnes
- 2018
-
In: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 13:1
-
Journal article (peer-reviewed)abstract
- Cutibacterium (Propionibacterium) acnes, considered a part of the skin microbiota, is one of the most commonly isolated anaerobic bacteria from medical implants in contact with plasma. However, the precise interaction of C. acnes with blood cells and plasma proteins has not been fully elucidated. Herein, we have investigated the molecular interaction of C. acnes with platelets and plasma proteins. We report that the ability of C. acnes to aggregate platelets is dependent on phylotype, with a significantly lower ability amongst type IB isolates, and the interaction of specific donor-dependent plasma proteins (or concentrations thereof) with C. acnes. Pretreatment of C. acnes with plasma reduces the lag time before aggregation demonstrating that pre-deposition of plasma proteins on C. acnes is an important step in platelet aggregation. Using mass spectrometry we identified several plasma proteins deposited on C. acnes, including IgG, fibrinogen and complement factors. Inhibition of IgG, fibrinogen or complement decreased C. acnes-mediated platelet aggregation, demonstrating the importance of these plasma proteins for aggregation. The interaction of C. acnes and platelets was visualized using fluorescence microscopy, verifying the presence of IgG and fibrinogen as components of the aggregates, and co-localization of C. acnes and platelets in the aggregates. Here, we have demonstrated the ability of C. acnes to activate and aggregate platelets in a bacterium and donor-specific fashion, as well as added mechanistic insights into this interaction.
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| 35. |
- Rasmussen, Magnus, et al.
(author)
-
Clinical isolates of Enterococcus faecalis aggregate human platelets.
- 2010
-
In: Microbes and Infection. - : Elsevier BV. - 1769-714X .- 1286-4579. ; 12, s. 295-301
-
Journal article (peer-reviewed)abstract
- Many endocarditis pathogens activate human platelets and this has been proposed to contribute to virulence. Here we report for the first time that many clinical isolates of Enterococcus faecalis, a common pathogen in infective endocarditis, aggregate human platelets. 84 isolates from human blood and urine were screened for their ability to aggregate platelets from four different donors. Platelet aggregation occurred for between 11 and 65 % of isolates depending on the donor. In one donor, a significantly larger proportion of isolates from blood than from urine caused platelet aggregation. Median time to aggregation was 11minutes and had a tendency to be shorter for blood isolates as compared to urine isolates. Immunoglobulin G (IgG) was shown to be essential in mediating activation and aggregation. Platelet aggregation could be abolished by an IgG-specific proteinase (IdeS), by an antibody blocking FcRgammaIIa on platelets, or by preabsorption of plasma with an E. faecalis isolate. Fibrinogen binding to bacteria or platelets does not contribute to platelet activation or aggregation under our experimental conditions. These results indicate that platelet activation and aggregation by E. faecalis is dependent on both host and bacterial factors and that it may be involved in the pathogenesis of invasive disease with this organism.
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| 36. |
- Rydengård, Victoria, et al.
(author)
-
Histidine-rich glycoprotein protects from systemic Candida infection
- 2008
-
In: PLoS pathogens. - : Public Library of Science (PLoS). - 1553-7374 .- 1553-7366. ; 4:8, s. e1000116-
-
Journal article (peer-reviewed)abstract
- Fungi, such as Candida spp., are commonly found on the skin and at mucosal surfaces. Yet, they rarely cause invasive infections in immunocompetent individuals, an observation reflecting the ability of our innate immune system to control potentially invasive microbes found at biological boundaries. Antimicrobial proteins and peptides are becoming increasingly recognized as important effectors of innate immunity. This is illustrated further by the present investigation, demonstrating a novel antifungal role of histidine-rich glycoprotein (HRG), an abundant and multimodular plasma protein. HRG bound to Candida cells, and induced breaks in the cell walls of the organisms. Correspondingly, HRG preferentially lysed ergosterol-containing liposomes but not cholesterol-containing ones, indicating a specificity for fungal versus other types of eukaryotic membranes. Both antifungal and membrane-rupturing activities of HRG were enhanced at low pH, and mapped to the histidine-rich region of the protein. Ex vivo, HRG-containing plasma as well as fibrin clots exerted antifungal effects. In vivo, Hrg(-/-) mice were susceptible to infection by C. albicans, in contrast to wild-type mice, which were highly resistant to infection. The results demonstrate a key and previously unknown antifungal role of HRG in innate immunity.
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| 37. |
- Sahl, Cecilia, et al.
(author)
-
Exoproducts of the Most Common Achromobacter Species in Cystic Fibrosis Evoke Similar Inflammatory Responses In Vitro
- 2023
-
In: Microbiology spectrum. - 2165-0497. ; 11:4
-
Journal article (peer-reviewed)abstract
- Achromobacter is a genus of Gram-negative rods, which can cause persistent airway infections in people with cystic fibrosis (CF). The knowledge about virulence and clinical implications of Achromobacter is still limited, and it is not fully established whether Achromobacter infections contribute to disease progression or if it is a marker of poor lung function. The most commonly reported Achromobacter species in CF is A. xylosoxidans. While other Achromobacter spp. are also identified in CF airways, the currently used Matrix-Assisted Laser Desorption/Ionization Time Of Flight Mass Spectrometry (MALDI-TOF MS) method in routine diagnostics cannot distinguish between species. Differences in virulence between Achromobacter species have consequently not been well studied. In this study, we compare phenotypes and proinflammatory properties of A. xylosoxidans, A. dolens, A. insuavis, and A. ruhlandii using in vitro models. Bacterial supernatants were used to stimulate CF bronchial epithelial cells and whole blood from healthy individuals. Supernatants from the well-characterized CF-pathogen Pseudomonas aeruginosa were included for comparison. Inflammatory mediators were analyzed with ELISA and leukocyte activation was assessed using flow cytometry. The four Achromobacter species differed in morphology seen in scanning electron microscopy (SEM), but there were no observed differences in swimming motility or biofilm formation. Exoproducts from all Achromobacter species except A. insuavis caused significant IL-6 and IL-8 secretion from CF lung epithelium. The cytokine release was equivalent or stronger than the response induced by P. aeruginosa. All Achromobacter species activated neutrophils and monocytes ex vivo in a lipopolysaccharide (LPS)-independent manner. Our results indicate that exoproducts of the four included Achromobacter species do not differ consistently in causing inflammatory responses, but they are equally or even more capable of inducing inflammation compared with the classical CF pathogen P. aeruginosa.IMPORTANCE Achromobacter xylosoxidans is an emerging pathogen among people with cystic fibrosis (CF). Current routine diagnostic methods are often unable to distinguish A. xylosoxidans from other Achromobacter species, and the clinical relevance of different species is still unknown. In this work, we show that four different Achromobacter species relevant to CF evoke similar inflammatory responses from airway epithelium and leukocytes in vitro, but they are all equally or even more proinflammatory compared to the classic CF-pathogen Pseudomonas aeruginosa. The results suggest that Achromobacter species are important airway pathogens in CF, and that all Achromobacter species are relevant to treat.
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| 38. |
- Sandrini, Michael, et al.
(author)
-
Deoxyribonucleoside kinases activate nucleoside antibiotics in severe pathogenic bacteria.
- 2007
-
In: Antimicrobial Agents and Chemotherapy. - 1098-6596. ; 51:8, s. 2726-2732
-
Journal article (peer-reviewed)abstract
- Common bacterial pathogens are becoming progressively more resistant to traditional antibiotics, representing a major public-health crisis. Therefore, there is a need for a variety of antibiotics with alternative modes of action. In our study, several nucleoside analogs were tested against pathogenic staphylococci and streptococci. We show that pyrimidine-based nucleoside analogs, like 3'-azido-3'-deoxythymidine (AZT) and 2',2'-difluoro-2'deoxycytidine (gemcitabine), are specifically activated by the endogenous bacterial deoxyribonucleoside kinases, leading to cell death. Deoxyribonucleoside kinase-deficient Escherichia coli strains become highly susceptible to nucleoside analogs when they express recombinant kinases from Staphylococcus aureus or Streptococcus pyogenes. We further demonstrate that recombinant S. aureus deoxyadenosine kinase efficiently phosphorylates the anticancer drug gemcitabine in vitro and is therefore the key enzyme in the activation pathway. When adult mice were infected intraperitoneally with a fatal dose of S. pyogenes strain AP1 and afterwards received gemcitabine, they failed to develop a systemic infection. Nucleoside analogs may therefore represent a promising alternative for combating pathogenic bacteria.
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| 39. |
- Shannon, Oonagh
(author)
-
Biological effects of extracellular fibrinogen binding protein (Efb) in staphylococcus aureus infection
- 2005
-
Doctoral thesis (other academic/artistic)abstract
- Staphylococcus aureus is a leading cause of human and animal infection. The increasing incidence of antibiotic resistance among strains has complicated treatment of these infections. In order to develop new treatment strategies, it is important to identify and characterise bacterial factors that contribute to infection. S. aureus produces a diverse array of virulence factors, among these Extracellular fibrinogen binding protein, Efb. Efb is one of several fibrinogen-binding proteins produced by S. aureus. Efb is produced in vivo during infection and contributes to the pathogenesis of severe S. aureus wound infection in an animal model. This thesis has been focussed on characterising the biological function of Efb in S. aureus infection. We have shown that Efb interacts with the Aalpha chain of fibrinogen, at a site of functional importance for the interaction between fibrinogen and platelets. We have studied the effect of Efb on platelet function in vitro and in vivo. Efb bound specifically to a receptor on activated platelets, however fibrinogen was not involved in mediating this binding. Efb stimulated a novel type of fibrinogen binding to the platelet, which did not involve the normal platelet fibrinogen receptor, GPIIb/IIIa. We propose that fibrinogen can also bind to the platelet via Efb and this generates the novel fibrinogen binding mediated by Efb. In the presence of Efb, platelet activation was diminished and Efb also inhibited platelet aggregation in response to various platelet agonists. This antiplatelet effect of Efb was confirmed in vivo. Intravenous Efb significantly prolonged bleeding time but had no effect on the coagulation system. This confirms that Efb specifically inhibits platelet function. Moreover, in a mouse model, intravenous Efb rescued 100% of animals from death due to acute thrombosis. This reflects the inability of the platelets to aggregate in the presence of Efb, an effect so powerful that Efb can counteract the massive thrombosis generated in this model. We have also studied the immune response against Efb and characterised a protective antibody response. Hyperimmune IgG against Efb neutralised Efb and blocked the various biological effects of Efb in vitro. IgG against Efb blocked Efb binding to fibrinogen, neutralised Efb and returned platelet aggregation in the presence of Efb to normal. Furthermore, immunization with Efb protected against the development of severe infection in an animal model of S. aureus infection. The animals in the vaccinated group developed high titre, specific antibodies against Efb and had significantly less severe infection than those in the unvaccinated group. This establishes Efb as a worthy vaccine candidate for S. aureus infection. Collectively, in this thesis we have elucidated the biological function of Efb and confirmed its importance in S. aureus wound infection. Efb is a powerful antiplatelet agent, which impairs haemostasis and wound healing. Efb also represents a worthwhile vaccine candidate for certain S. aureus infections.
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| 40. |
- Shannon, Oonagh
(author)
-
Determining platelet activation and aggregation in response to bacteria
- 2017
-
In: Methods in Molecular Biology. - New York, NY : Springer New York. - 1064-3745. ; 1535, s. 267-273
-
Book chapter (peer-reviewed)abstract
- Many pathogenic bacteria have been reported to interact with human platelets to mediate platelet activation and aggregation. The importance of these interactions to the immune response or pathogenesis of bacterial infection has not been clarified. It may therefore be valuable to assess platelet responses mediated by diverse strains of bacteria. Here, I describe a method to study platelet integrin activation and granule release using flow cytometry, and a complementary method to study platelet aggregation using a dedicated platelet aggregometer. The combination of these methods represents a rapid and cost-effective strategy to provide mechanistic insight on the type of platelet response mediated by the bacteria.
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| 41. |
- Shannon, Oonagh, et al.
(author)
-
Histidine-rich glycoprotein promotes bacterial entrapment in clots and decreases mortality in a mouse model of sepsis.
- 2010
-
In: Blood. - : American Society of Hematology. - 1528-0020 .- 0006-4971. ; 116, s. 2365-2372
-
Journal article (peer-reviewed)abstract
- Streptococcus pyogenes is a significant bacterial pathogen in humans. In this study, Histidine-rich glycoprotein (HRG), an abundant plasma protein, was found to kill S. pyogenes. Furthermore, S. pyogenes grew more efficiently in HRG deficient plasma, and clots formed in this plasma were significantly less effective at bacterial entrapment and killing. HRG deficient mice were strikingly more susceptible to S. pyogenes infection. These animals failed to control the infection at the local subcutaneous site, and abscess formation and inflammation was diminished as compared with control animals. As a result, bacterial dissemination occurred more rapidly in HRG deficient mice and they succumbed earlier and with a significantly higher mortality rate than control animals. HRG deficient mice supplemented with purified HRG gave the same phenotype as control animals, demonstrating that the lack of HRG was responsible for the increased susceptibility. The results demonstrate a previously unappreciated role for HRG as a regulator of inflammation and in the defence at the local site of bacterial infection.
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| 42. |
- Shannon, Oonagh, et al.
(author)
-
Measuring Antibody Orientation at the Bacterial Surface
- 2017
-
In: Bacterial Pathogenesis : Methods and Protocols - Methods and Protocols. - New York, NY : Springer New York. - 1064-3745. - 9781493966738 - 9781493966714 ; 1535, s. 331-337
-
Book chapter (peer-reviewed)abstract
- Many bacteria have the ability to interact with antibodies as a means to circumvent the immune response. This includes binding to the Fc portion of antibodies, effectively reversing the antibody orientation and thus decreasing the Fc-mediated immune signaling. Since antibody orientation at the bacterial surface has been shown to be important in human disease, it is valuable to be able to assess how antibodies are interacting with bacterial pathogens. Here, we describe a method to measure the proportion of human IgG that are bound via their Fc or Fabs to a bacterial surface. This is achieved by treating antibody-coated bacteria with the bacterial enzyme IdeS - which will cleave IgG into Fc and Fab fragments - and subsequently detect remaining fragments with fluorescent Fabs. The method is easy and fast, and the principle is most likely also applicable to other systems where distinguishing between antibody Fc and Fab binding is important.
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| 43. |
- Shannon, Oonagh, et al.
(author)
-
Modulation of the Coagulation System During Severe Streptococcal Disease.
- 2012
-
In: Current Topics in Microbiology and Immunology. - Berlin, Heidelberg : Springer Berlin Heidelberg. - 0070-217X.
-
Journal article (peer-reviewed)abstract
- Haemostasis is maintained by a tightly regulated coagulation system that comprises platelets, procoagulant proteins, and anticoagulant proteins. During the local and systemic response to bacterial infection, the coagulation system becomes activated, and contributes to the pathophysiological response to infection. The significant human pathogen, Streptococcus pyogenes has multiple strategies to modulate coagulation. This can range from systemic activation of the intrinsic and extrinsic pathway of coagulation to local stimulation of fibrinolysis. Such diverse effects on this host system imply a finely tuned host-bacteria interaction. The molecular mechanisms that underlie this modulation of the coagulation system are discussed in this review.
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| 44. |
- Shannon, Oonagh, et al.
(author)
-
Platelet activation and biofilm formation by Aerococcus urinae, an endocarditis causing pathogen.
- 2010
-
In: Infection and Immunity. - 1098-5522. ; 78:10, s. 4268-4275
-
Journal article (peer-reviewed)abstract
- The Gram-positive bacterium Aerococcus urinae can cause infectious endocarditis (IE) in older persons. Biofilm formation and platelet aggregation is believed to contribute to bacterial virulence in IE. Five A. urinae isolates from human blood were shown to form biofilms in vitro and biofilm formation was enhanced by the presence of human plasma. Four of the A. urinae isolates caused platelet aggregation in platelet rich plasma from healthy donors. The Au3 isolate, which induced platelet aggregation in all donors, also activated platelets as determined by flow cytometry. Platelet aggregation was dependent on bacterial protein structures and on platelet activation since it was sensitive to both trypsin and Prostaglandin E1. Plasma proteins at the bacterial surface were needed for platelet aggregation and a role for the complement system, fibrinogen, and immunoglobulin G was demonstrated. Complement depleted serum was unable to support platelet aggregation by Au3 and complement blockade using compstatin inhibited platelet activation. Platelet activation by Au3 was inhibited by blocking of the platelet fibrinogen receptor and this isolate was also shown to bind radiolabeled fibrinogen. Removal of IgG from platelet rich plasma by a specific protease inhibited the platelet aggregation induced by A. urinae and blockade of the platelet FcRgammaIIa hindered platelet activation induced by Au3. Convalescent serum from a patient with A. urinae IE transferred the ability of the bacterium to aggregate platelets in an otherwise non-responsive donor. Our results show that A. urinae exhibits virulence strategies of importance for IE.
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| 45. |
- Shannon, Oonagh
(author)
-
Platelet interaction with bacterial toxins and secreted products.
- 2015
-
In: Platelets. - : Informa UK Limited. - 1369-1635 .- 0953-7104. ; 26:4, s. 302-308
-
Research review (peer-reviewed)abstract
- Bacteria that enter the bloodstream will encounter components of the cellular and soluble immune response. Platelets contribute to this response and have emerged as an important target for bacterial pathogens. Bacteria produce diverse extracellular proteins and toxins that have been reported to modulate platelet function. These interactions can result in complete or incomplete platelet activation or inhibition of platelet activation, depending on the bacteria and bacterial product. The nature of the platelet response may be highly relevant to disease pathogenesis.
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| 46. |
|
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| 47. |
- Shannon, Oonagh, et al.
(author)
-
Severe streptococcal infection is associated with M protein-induced platelet activation and thrombus formation.
- 2007
-
In: Molecular Microbiology. - : Wiley. - 1365-2958 .- 0950-382X. ; 65:5, s. 1147-1157
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Journal article (peer-reviewed)abstract
- Disturbed haemostasis is a central finding in severe Streptococcus pyogenes infection. In particular, microthrombi are found both at the local site of infection and at distant sites. Platelets are responsible for maintaining vascular function and haemostasis. We report here that M1 protein of S. pyogenes triggers immune-mediated platelet activation and thrombus formation. M1 protein is released from the bacterial surface and forms complexes with plasma fibrinogen. These complexes bind to the fibrinogen receptor on resting platelets. When these complexes also contain immunoglobulin G (IgG) against M1 protein, this will engage the Fc receptor on the platelets and activation will occur. Activation of the platelets leads to platelet aggregation and the generation of platelet-rich thrombi. Neutrophils and monocytes are in turn activated by the platelets. Platelet thrombi are deposited in the microvasculature, and aggregated platelets, IgG and M1 protein colocalize in biopsies from patients diagnosed with S. pyogenes toxic shock syndrome. This chain of events results in a procoagulant and pro-inflammatory state typical of severe S. pyogenes infection.
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| 48. |
- Shannon, Oonagh
(author)
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The role of platelets in sepsis
- 2021
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In: Research and practice in thrombosis and haemostasis. - : Elsevier BV. - 2475-0379. ; 5:1, s. 27-37
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Journal article (peer-reviewed)abstract
- A State of the Art lecture titled “The role of platelets in sepsis” was presented at the ISTH congress in 2020. Sepsis is a life-threatening organ dysfunction caused by a dysregulated and multifaceted host response to infection. Platelets play a significant role in the coordinated immune response to infection and therefore in the inflammation and coagulation dysfunction that contributes to organ damage in sepsis. Thrombocytopenia has a high incidence in sepsis, and it is a marker of poor prognosis. The genesis of thrombocytopenia is likely multifactorial, and unraveling the involved molecular mechanisms will allow development of biomarkers of platelet function in sepsis. Such platelet biomarkers can facilitate study of antiplatelet interventions as immunomodulatory treatment in sepsis. Finally, relevant new data on this topic presented during the 2020 ISTH virtual congress are reviewed.
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| 49. |
- Svensson, Lisbeth, et al.
(author)
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Group G streptococci mediate fibrinogendependent platelet aggregation leading to transient entrapment in platelet aggregates
- 2016
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In: Microbiology. - : Microbiology Society. - 1350-0872 .- 1465-2080. ; 162:1, s. 117-126
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Journal article (peer-reviewed)abstract
- Platelets have been reported to become activated in response to bacteria and this is proposed to contribute to the acute response to bacterial infection. In the present study, we investigated platelet aggregation in response to group G streptococci (GGS) in vitro in healthy human donors and in vivo in a mouse model of streptococcal sepsis. Platelet aggregation by GGS was dependent on the bacterial surface protein FOG and engagement of the platelet fibrinogen receptor; however, it was independent of IgG and the platelet Fc receptor. Platelets exerted no antibacterial effects on the bacteria, and aggregates formed were markedly unstable, allowing bacteria to rapidly return to the plasma and grow post-aggregation. Thrombocytopenia and platelet activation occurred during invasive infection with GGS, and platelets were demonstrated to contribute to bacterial dissemination during infection. These findings reveal an important role for bacteria–platelet interactions during the pathogenesis of streptococcal infection.
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| 50. |
- Svensson, Lisbeth, et al.
(author)
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Platelet activation by Streptococcus pyogenes leads to entrapment in platelet aggregates from which bacteria subsequently escape.
- 2014
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In: Infection and Immunity. - 1098-5522. ; 82:10, s. 4307-4314
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Journal article (peer-reviewed)abstract
- Platelet activation and aggregation has been reported to occur in response to a number of Gram-positive pathogens. Here we show that platelet aggregates induced by Streptococcus pyogenes were unstable and viable bacteria escaped from the aggregates over time. This was not due to a differential activation in response to the bacteria as compared with physiological activators. All the bacteria isolates induced significant platelet activation, including integrin activation, alpha and dense granule release, at equivalent levels to potent physiological platelet activators that induced stable aggregates. The ability to escape the aggregates and resist antibacterial effects of platelets was dependent on active protein synthesis by the bacteria within the aggregate. We conclude that S. pyogenes can temporarily cover themselves with activated platelets and we propose that this may facilitate survival of bacteria in the presence of platelets.
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