| 1. |
- Godaly, Gabriela, et al.
(författare)
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Mycobacterium bovis bacille Calmette Guerin infection of human neutrophils induces CXCL8 secretion by MyD88-dependent TLR2 and TLR4 activation
- 2005
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Ingår i: Cellular Microbiology. - : Hindawi Limited. - 1462-5814 .- 1462-5822. ; 7:4, s. 591-601
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Tidskriftsartikel (refereegranskat)abstract
- To investigate the potential role of neutrophils in initiation of immune responses to mycobacteria, we have characterized the response of human neutrophils to infection with Mycobacterium bovis bacille Calmette Guerin, the BCG vaccine. BCG induced transcription and secretion of the chemokine CXCL8, by signalling through Toll-like receptors TLR2 and TLR4, in conjunction with the adaptor protein myeloid differentiation factor 88 (MyD88). Blocking of responses with antibodies revealed a difference in the kinetics of signalling through the different TLRs. Anti-TLR2 antibody blocked the early phase of CXCL8 and MyD88 induction. Anti-TLR4 antibody blocked the late phase of induction occurring 2 h after infection. The existence of a TLR/MyD88 pathway for recognition and response to mycobacterial ligands provides neutrophils with the ability to drive the recruitment and activation of inflammatory cells during the early phase of mycobacterial infection and immunization.
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| 2. |
- Paz, Irit, et al.
(författare)
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Galectin-3, a marker for vacuole lysis by invasive pathogens
- 2010
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Ingår i: Cellular Microbiology. - : Hindawi Limited. - 1462-5814 .- 1462-5822. ; 12:4, s. 530-544
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Tidskriftsartikel (refereegranskat)abstract
- P>Shigella bacteria invade macrophages and epithelial cells and following internalization lyse the phagosome and escape to the cytoplasm. Galectin-3, an abundant protein in macrophages and epithelial cells, belongs to a family of beta-galactoside-binding proteins, the galectins, with many proposed functions in immune response, development, differentiation, cancer and infection. Galectins are synthesized as cytosolic proteins and following non-classical secretion bind extracellular beta-galactosides. Here we analysed the localization of galectin-3 following entry of Shigella into the cytosol and detected a striking phenomenon. Very shortly after bacterial invasion, intracellular galectin-3 accumulated in structures in vicinity to internalized bacteria. By using immuno-electron microscopy analysis we identified galectin-3 in membranes localized in the phagosome and in tubules and vesicles that derive from the endocytic pathway. We also demonstrated that the binding of galectin-3 to host N-acetyllactosamine-containing glycans, was required for forming the structures. Accumulation of the structures was a type three secretion system-dependent process. More specifically, existence of structures was strictly dependent upon lysis of the phagocytic vacuole and could be shown also by Gram-positive Listeria and Salmonella sifA mutant. We suggest that galectin-3-containing structures may serve as a potential novel tool to spot vacuole lysis.
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| 3. |
- Påhlman, Lisa, et al.
(författare)
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Soluble M1 protein of Streptococcus pyogenes triggers potent T cell activation.
- 2008
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Ingår i: Cellular Microbiology. - : Hindawi Limited. - 1462-5814 .- 1462-5822. ; 10:2, s. 404-414
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Tidskriftsartikel (refereegranskat)abstract
- Streptococcus pyogenes of the M1 serotype is commonly associated with large outbreaks of invasive streptococcal infections and development of streptococcal toxic shock syndrome (STSS). The pathogenesis behind these infections is believed to involve bacterial superantigens that induce potent inflammatory responses, but the reason why strains of the M1 serotype are over-represented in STSS is still not understood. In the present investigation, we show that a highly purified soluble form of the M1 protein from S. pyogenes, which lacks the membrane-spanning region, is a potent inducer of T cell proliferation and release of Th1 type cytokines. M1 protein-evoked T cell proliferation was HLA class II-dependent but not MHC-restricted, did not require intracellular processing and was Vβ-restricted. Extensive mass spectrometry studies indicated that there were no other detectable proteins in the preparation. Taken together, our data demonstrate that soluble M1 protein is a novel streptococcal superantigen, which likely contributes to the excessive T cell activation and hyperinflammatory response seen in severe invasive streptococcal infections.
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| 4. |
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| 5. |
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| 6. |
- Wullt, Björn, et al.
(författare)
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P-fimbriae trigger mucosal responses to Escherichia coli in the human urinary tract
- 2001
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Ingår i: Cellular Microbiology. - : Hindawi Limited. - 1462-5814 .- 1462-5822. ; 3:4, s. 255-264
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Tidskriftsartikel (refereegranskat)abstract
- Uropathogenic Escherichia coli elicit a host response that determines the severity of urinary tract infection (UTI). Specific adherence mechanisms allow the bacteria to initiate this process by targeting epithelial cells in the urinary tract mucosa. Epidemiological studies show a strong association of P-fimbriae with disease severity, suggesting that adherence mediated by these organelles has a direct effect on mucosal inflammation in vivo. The present study examined the ability of P-fimbriae to induce inflammation in the human urinary tract. Patients were subjected to intravesical inoculation with a non-fimbriated E. coli strain or transformants of this strain expressing P-fimbriae. The inflammatory response was analysed as a function of P-fimbrial expression. The P-fimbriated transformants invariably caused higher interleukin (IL)-8, IL-6 and neutrophil responses in the urinary tract than the ABU strain. Furthermore, loss of P-fimbrial expression in vivo was accompanied by a return to background levels of neutrophils, IL-6 and IL-8 in individual patients. The results demonstrate that the pap sequences confer on a non-fimbriated, avirulent strain the ability to induce a host response in the human urinary tract. P-fimbriae thus fulfil the 'molecular Koch-Henle postulates' linking a single virulence factor to host response induction.
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| 7. |
- Areschoug, Thomas, et al.
(författare)
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Scavenger receptors: role in innate immunity and microbial pathogenesis.
- 2009
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Ingår i: Cellular Microbiology. - : Hindawi Limited. - 1462-5814 .- 1462-5822. ; 11, s. 1160-1169
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Tidskriftsartikel (refereegranskat)abstract
- Summary Accumulating evidence shows that many scavenger receptors (SR), including SR-A, MARCO and CD36, represent an important part of the innate immune defence by acting as pattern-recognition receptors (PRR), in particular against bacterial pathogens. Several SR are expressed on macrophages and dendritic cells, where they act as phagocytic receptors mediating non-opsonic phagocytosis of pathogenic microbes. Another important function of some SR is to act as co-receptors to TLRs, modulating the inflammatory response to TLR agonists. On bacteria, the SR ligands have commonly been reported to be LPS and LTA, but recent advances in the field indicate that bacterial surface proteins play a more important role as target molecules for SR than previously thought. Interestingly, recent data show that major pathogens, including Streptococcus pyogenes and the group B streptococcus (GBS), have evolved mechanisms to evade SR-mediated recognition. Moreover, intracellular pathogens, such as hepatitis C virus (HCV) and Plasmodium falciparum, utilize the SR to gain entry into host cells, focusing interest on the importance of SR also in the molecular pathogenesis of infectious diseases. This review highlights the complex interactions between SR and pathogenic microbes, and discusses the role of these interactions in host defence and microbial pathogenesis.
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| 8. |
- Bergsten, Göran, et al.
(författare)
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Do type 1 fimbriae promote inflammation in the human urinary tract?
- 2007
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Ingår i: Cellular Microbiology. - : Hindawi Limited. - 1462-5814 .- 1462-5822. ; 9:7, s. 1766-1781
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Tidskriftsartikel (refereegranskat)abstract
- Type 1 fimbriae have been implicated as virulence factors in animal models of urinary tract infection (UTI), but the function in human disease remains unclear. This study used a human challenge model to examine if type 1 fimbriae trigger inflammation in the urinary tract. The asymptomatic bacteriuria strain Escherichia coli 83972, which fails to express type 1 fimbriae, due to a 4.25 kb fimB-fimD deletion, was reconstituted with a functional fim gene cluster and fimbrial expression was monitored through a gfp reporter. Each patient was inoculated with the fim+ or fim- variants on separate occasions, and the host response to type 1 fimbriae was quantified by intraindividual comparisons of the responses to the fim+ or fim- isogens, using cytokines and neutrophils as end-points. Type 1 fimbriae did not promote inflammation and adherence was poor, as examined on exfoliated cells in urine. This was unexpected, as type 1 fimbriae enhanced the inflammatory response to the same strain in the murine urinary tract and as P fimbrial expression by E. coli 83972 enhances adherence and inflammation in challenged patients. We conclude that type 1 fimbriae do not contribute to the mucosal inflammatory response in the human urinary tract.
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| 9. |
- Fischer, Hans, et al.
(författare)
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Ceramide as a TLR4 agonist; a putative signalling intermediate between sphingolipid receptors for microbial ligands and TLR4.
- 2007
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Ingår i: Cellular Microbiology. - : Hindawi Limited. - 1462-5814 .- 1462-5822. ; 9:5, s. 1239-1251
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Tidskriftsartikel (refereegranskat)abstract
- Mucosal Toll-like receptors (TLRs) respond to pathogens, but remain inert to the indigenous flora, suggesting that the TLRs can receive pathogen-specific signals. For example, TLR4 signalling is activated in CD14-negative epithelial cells by P-fimbriated, uropathogenic Escherichia coli, but not by lipopolysaccharide. The fimbriae use glycosphingolipids as recognition receptors and there is release of ceramide, which is the membrane-anchoring domain of the receptors. In this study, ceramide was identified as a TLR4 agonist and as a putative signalling intermediate between the glycosphingolipid recognition receptors and TLR4. Exogenous ceramide activated a TLR4-dependent epithelial cell response, as shown by exposing stably transfected TLR4-positive or -negative human embryonal kidney cells to C2 and C6 ceramide. A similar, TLR4-dependent response occurred after deliberate release of endogenous long-chained ceramide with sphingomyelinase. Microbial ligands with glycosphingolipid specificity (P fimbriae or the B subunit of Shiga toxin) were shown to increase the levels of ceramide and to trigger a TLR4-dependent response in epithelial cells. The results show that ceramide activates TLR4 signalling and suggest that this mechanism might allow pathogens to elicit mucosal TLR4 responses by perturbing sphingolipid receptors for virulence ligands like P fimbriae.
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| 10. |
- John, Constance M, et al.
(författare)
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Galectin-3 binds lactosaminylated lipooligosaccharides from Neisseria gonorrhoeae and is selectively expressed by mucosal epithelial cells that are infected
- 2002
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Ingår i: Cellular Microbiology. - : Hindawi Limited. - 1462-5814 .- 1462-5822. ; 4:10, s. 649-661
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Tidskriftsartikel (refereegranskat)abstract
- Galectins are a family of beta-galactoside binding proteins that have been proposed as host receptors for bacteria because beta-galactoside carbohydrates are common in bacterial membrane glycolipid lipooligosaccharides (LOS) and lipopolysaccharides. We investigated the interaction of galectin-3 with gonococcal LOS that make lactosyl (Lc(2) or Lac), paraglobosyl (nLc(4) ; LNnT; lacto-N -neotetraose), gangliosyl (IV3 GalNAcnLc(4) ), and neolactohexaosyl (nLc(6) , lactonorhexaosyl) oligosaccharides. All but gangliosyl LOS terminate in beta-galactoside. Galectin-3 had the highest affinity for the nLc(6) LOS, which is made by a strain that is highly infectious for the male urethra, but also bound nLc(4) LOS and to a Lac LOS. The lacto-N -neotetraose tetrasaccharide was a more potent inhibitor of galectin-3 binding to LOS than either lactose or N -acetyllactosamine. The relative affinity of galectin-3 for gonococci mirrored its affinity for purified LOS. Western blot analysis revealed expression of galectin-3 by human endometrial adenocarcinoma and prostatic epithelial cells that can be invaded by gonococci. Immunohistochemistry of human fallopian tube epithelium showed localized expression of galectin-3 by non-ciliated cells, the specific cell gonococci invade in this tissue. We conclude that because of its location and affinity for gonococcal LOS galectin-3 could play a role in gonococcal infection.
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| 11. |
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| 12. |
- Aili, Margareta, et al.
(författare)
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Functional analysis of the YopE GTPase-activating protein (GAP) activity of Yersinia pseudotuberculosis
- 2006
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Ingår i: Cellular Microbiology. - : Wiley. - 1462-5814 .- 1462-5822. ; 8:6, s. 1020-1033
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Tidskriftsartikel (refereegranskat)abstract
- YopE of Yersinia pseudotuberculosis inactivates three members of the small RhoGTPase family (RhoA, Rac1 and Cdc42) in vitro and mutation of a critical arginine abolishes both in vitro GTPase-activating protein (GAP) activity and cytotoxicity towards HeLa cells, and renders the pathogen avirulent in a mouse model. To understand the functional role of YopE, in vivo studies of the GAP activity in infected eukaryotic cells were conducted. Wild-type YopE inactivated Rac1 as early as 5 min after infection whereas RhoA was down regulated about 30 min after infection. No effect of YopE was found on the activation state of Cdc42 in Yersinia-infected cells. Single-amino-acid substitution mutants of YopE revealed two different phenotypes: (i) mutants with significantly lowered in vivo GAP activity towards RhoA and Rac1 displaying full virulence in mice, and (ii) avirulent mutants with wild-type in vivo GAP activity towards RhoA and Rac1. Our results show that Cdc42 is not an in vivo target for YopE and that YopE interacts preferentially with Rac1, and to a lesser extent with RhoA, during in vivo conditions. Surprisingly, we present results suggesting that these interactions are not a prerequisite to establish infection in mice. Finally, we show that avirulent yopE mutants translocate YopE in about sixfold higher amount compared with wild type. This raises the question whether YopE's primary function is to sense the level of translocation rather than being directly involved in downregulation of the host defence.
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| 13. |
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| 14. |
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| 15. |
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| 16. |
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| 17. |
- Bassères, Eugénie, et al.
(författare)
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The ubiquitin C-terminal hydrolase UCH-L1 promotes bacterial invasion by altering the dynamics of the actin cytoskeleton
- 2010
-
Ingår i: Cellular Microbiology. - : Wiley-Blackwell. - 1462-5814 .- 1462-5822. ; 12:11, s. 1622-1633
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Tidskriftsartikel (refereegranskat)abstract
- Invasion of eukaryotic target cells by pathogenic bacteria requires extensive remodelling of the membrane and actin cytoskeleton. Here we show that the remodelling process is regulated by the ubiquitin C-terminal hydrolase UCH-L1 that promotes the invasion of epithelial cells by Listeria monocytogenes and Salmonella enterica. Knockdown of UCH-L1 reduced the uptake of both bacteria, while expression of the catalytically active enzyme promoted efficient internalization in the UCH-L1-negative HeLa cell line. The entry of L. monocytogenes involves binding to the receptor tyrosine kinase Met, which leads to receptor phosphorylation and ubiquitination. UCH-L1 controls the early membrane-associated events of this triggering cascade since knockdown was associated with altered phosphorylation of the c-cbl docking site on Tyr1003, reduced ubiquitination of the receptor and altered activation of downstream ERK1/2- and AKT-dependent signalling in response to the natural ligand Hepatocyte Growth Factor (HGF). The regulation of cytoskeleton dynamics was further confirmed by the induction of actin stress fibres in HeLa expressing the active enzyme but not the catalytic mutant UCH-L1(C90S). These findings highlight a previously unrecognized involvement of the ubiquitin cycle in bacterial entry. UCH-L1 is highly expressed in malignant cells that may therefore be particularly susceptible to invasion by bacteria-based drug delivery systems.
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| 18. |
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| 19. |
- Bäckhed, Fredrik, 1973, et al.
(författare)
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TLR4-dependent lipopolysaccharide signalling in epithelial cells is independent of extracellular protease activity.
- 2002
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Ingår i: Cellular microbiology. - : Hindawi Limited. - 1462-5814 .- 1462-5822. ; 4:5, s. 297-303
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Tidskriftsartikel (refereegranskat)abstract
- Epithelial cells are the first cells that encounter infecting bacteria and, as such, they have developed several mechanisms for microbial protection. We have shown previously that bladder epithelial cells express the lipopolysaccharide (LPS) receptor Toll-like receptor (TLR) 4 that enables a rapid cellular interleukin (IL)-8 response when exposed to Escherichia coli and LPS. TLR4 belongs to a family of receptors that was initially identified in Drosophila, in which Toll is required for the immune response against fungi. Fungal exposure activates a series of serine proteases that process the protein Spaetzle to a cytokine-like form that acts as a ligand for Toll. Here, we investigated whether a similar proteolytic cascade is required for human TLR activation. When screening a set of 18 protease inhibitors, three serine protease inhibitors (TPCK, TLCK and Pefabloc) were shown to inhibit LPS- and peptidoglycan-induced IL-8 production in TLR2- and TLR4-positive bladder epithelial cells. However, they were equally effective inhibitors of IL-1beta-induced signalling, indicating that their target(s) is/are located downstream of the TLRs. Further characterization showed that these inhibitors blocked I kappa B degradation but not phosphorylation in LPS-stimulated cells, which suggests that the serine protease inhibitors target the 26S proteasome. Identical results were obtained on LPS-stimulated monocytes. Based on these data, we find no evidence for the involvement of proteases upstream of TLRs in either epithelial cells or cells of the monocytic lineage.
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| 20. |
- Corleis, Björn, et al.
(författare)
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Escape of Mycobacterium tuberculosis from oxidative killing by neutrophils.
- 2012
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Ingår i: Cellular microbiology. - : Hindawi Limited. - 1462-5822 .- 1462-5814. ; 14:7, s. 1109-1121
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Tidskriftsartikel (refereegranskat)abstract
- Neutrophils enter sites of infection, where they can eliminate pathogenic bacteria in an oxidative manner. Despite their predominance in active tuberculosis lesions, the function of neutrophils in this important human infection is still highly controversial. We observed that virulent Mycobacterium tuberculosis survived inside human neutrophils despite prompt activation of these defence cells' microbicidal effectors. Survival of M.tuberculosis was accompanied by necrotic cell death of infected neutrophils. Necrotic cell death entirely depended on radical oxygen species production since chronic granulomatous disease neutrophils were protected from M.tuberculosis-triggered necrosis. More, importantly, the M. tuberculosisΔRD1 mutant failed to induce neutrophil necrosis rendering this strain susceptible to radical oxygen species-mediated killing. We conclude that this virulence function is instrumental for M.tuberculosis to escape killing by neutrophils and contributes to pathogenesis in tuberculosis.
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| 21. |
- Deleuil, Fabienne, et al.
(författare)
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Interaction between the Yersinia protein tyrosine phosphatase YopH and eukaryotic Cas/Fyb is an important virulence mechanism
- 2003
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Ingår i: Cellular Microbiology. - : Blackwell Publishing. - 1462-5814 .- 1462-5822. ; 5:1, s. 53-64
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Tidskriftsartikel (refereegranskat)abstract
- The tyrosine phosphatase YopH is an essential virulence factor produced by pathogenic Yersinia species. YopH is translocated into host cells via a type III secretion system and its dephosphorylating activity causes disruption of focal complex structures and blockage of the phagocytic process. Among the host cell targets of YopH are the focal adhesion proteins Crk-associated substrate (p130Cas) and focal adhesion kinase (FAK) in epithelial cells, and p130Cas and Fyn-binding protein (Fyb) in macrophages. Previous studies have shown that the N-terminal domain of YopH acts as a substrate-binding domain. In this study, the mechanism and biological importance of the targeting of YopH to focal complexes relative to its interaction with p130Cas/Fyb was elucidated. Mutants of YopH that were defective in p130Cas/Fyb binding but otherwise indistinguishable from wild type were constructed. Mutants unable to bind p130Cas did not localize to focal complex structures in infected cells, indicating that the association with p130Cas is critical for appropriate subcellular localization of YopH. These yopH mutants were also clearly attenuated in virulence, showing that binding to p130Cas and/or Fyb is biologically relevant in Yersinia infections.
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| 22. |
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| 23. |
- Fahlgren, Anna, et al.
(författare)
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Cell type-specific effects of Yersinia pseudotuberculosis virulence effectors
- 2009
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Ingår i: Cellular Microbiology. - : Hindawi Limited. - 1462-5814 .- 1462-5822. ; 11:12, s. 1750-1767
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Tidskriftsartikel (refereegranskat)abstract
- One important feature of Yersinia pseudotuberculosis that enables resistance against the host immune defence is delivery of the antiphagocytic effectors YopH and YopE into phagocytic cells. The tyrosine phosphatase YopH influences integrin signalling, and YopE impairs cytoskeletal dynamics by inactivating Rho GTPases. Here, we report the impact of these effectors on internalization by dendritic cells (DCs), which internalize antigens to orchestrate host immune responses. We found that this pathogen resists internalization by DCs via YopE. YopH that is important for blocking phagocytosis by macrophages and neutrophils and which is also present inside the DCs does not contribute to the resistance. However, the YopH targets Fyb and p130Cas show higher expression levels in macrophages than in DCs. Furthermore, live cell microscopy revealed that the cells internalize Y. pseudotuberculosis in different ways: the macrophages utilize a locally restricted receptor-mediated zipper mechanism, whereas DCs utilize macropinocytosis involving constitutive ruffling that randomly catches bacteria into membrane folds. We conclude that YopH impacts early phagocytic signalling from the integrin receptor to which the bacterium binds and that this tight receptor-mediated stimulation is absent in DC macropinocytosis. Inactivation of cytoskeletal dynamics by YopE affects ruffling activity and hence also internalization. The different modes of internalization can be coupled to the major functions of these respective cell types: elimination by phagocytosis and antigen sampling.
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| 24. |
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| 25. |
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| 26. |
- Gekara, Nelson O, et al.
(författare)
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Mast cells initiate early anti-Listeria host defences.
- 2008
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Ingår i: Cellular Microbiology. - : Hindawi Limited. - 1462-5814 .- 1462-5822. ; 10:1, s. 225-236
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Tidskriftsartikel (refereegranskat)abstract
- The Gram-positive bacterium Listeria monocytogenes (L. m.) is the aetiological agent of listeriosis. The early phase listeriosis is characterized by strong innate host responses that play a major role in bacterial clearance. This is emphasized by the fact that mice deficient in T and B cells have a remarkable ability to control infection. Mast cells, among the principal effectors of innate immunity, have largely been studied in the context of hyper-reactive conditions such as allergy and autoimmune diseases. In the present study, we evaluated the significance of mast cells during the early phase of listeriosis. Compared with controls, mice depleted of mast cells showed hundred-fold higher bacterial burden in spleen and liver and were significantly impaired in neutrophil mobilization. Although L. m. interacts with and triggers mast cell degranulation, bacteria were hardly found within such cells. Mainly neutrophils and macrophages phagozytosed L. m. Thus, mast cells control infection not via direct bacterial uptake, but by initiating neutrophils influx to the site of infection. We show that this is initiated by pre-synthesized TNF-alpha, rapidly secreted by mast cell upon activation by L. m. We also show that upon recruitment, neutrophils also become activated and additionally secrete TNF-alpha thus amplifying the anti-L. m. inflammatory response.
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| 27. |
- Gekara, Nelson O, et al.
(författare)
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The cholesterol-dependent cytolysin listeriolysin O aggregates rafts via oligomerization.
- 2005
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Ingår i: Cellular Microbiology. - : Hindawi Limited. - 1462-5814 .- 1462-5822. ; 7:9, s. 1345-1356
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Tidskriftsartikel (refereegranskat)abstract
- The pore-forming toxin listeriolysin O (LLO) is the main virulence factor of Listeria monocytogenes. LLO is known to act as a pseudo cytokine/chemokine, which induces a broad spectrum of host responses that ultimately influences the outcome of listeriosis. In the present study we demonstrate that LLO is a potent aggregator of lipid rafts. LLO was found to aggregate the raft associated molecules GM1, the GPI-anchored proteins CD14 and CD16 as well as the tyrosine kinase Lyn. Abrogation of the cytolytic activity of LLO by cholesterol pretreatment was found not to interfere with LLO's ability to aggregate rafts or trigger tyrosine phosphorylation in cells. However, a monoclonal antibody that blocks the oligomerization of LLO was found to inhibit rafts' aggregation as well as the induction of tyrosine phosphorylation. This implies that rafts aggregation by LLO which is independent of cytolytic activity, is due to the oligomerization of its membrane bound toxin monomers. Thus, LLO most likely induces signalling through the coaggregation of rafts' associated receptors, kinases and adaptors.
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| 28. |
- Gekara, Nelson O, et al.
(författare)
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The multiple mechanisms of Ca2+ signalling by listeriolysin O, the cholesterol-dependent cytolysin of Listeria monocytogenes.
- 2007
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Ingår i: Cellular Microbiology. - : Hindawi Limited. - 1462-5814 .- 1462-5822. ; 9:8, s. 2008-2021
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Tidskriftsartikel (refereegranskat)abstract
- Cholesterol-dependent cytolysins (CDCs) represent a large family of conserved pore-forming toxins produced by several Gram-positive bacteria such as Listeria monocytogenes, Streptococcus pyrogenes and Bacillus anthracis. These toxins trigger a broad range of cellular responses that greatly influence pathogenesis. Using mast cells, we demonstrate that listeriolysin O (LLO), a prototype of CDCs produced by L. monocytogenes, triggers cellular responses such as degranulation and cytokine synthesis in a Ca(2+)-dependent manner. Ca(2+) signalling by LLO is due to Ca(2+) influx from extracellular milieu and release of from intracellular stores. We show that LLO-induced release of Ca(2+) from intracellular stores occurs via at least two mechanisms: (i) activation of intracellular Ca(2+) channels and (ii) a Ca(2+) channels independent mechanism. The former involves PLC-IP(3)R operated Ca(2+) channels activated via G-proteins and protein tyrosine kinases. For the latter, we propose a novel mechanism of intracellular Ca(2+) release involving injury of intracellular Ca(2+) stores such as the endoplasmic reticulum. In addition to Ca(2+) signalling, the discovery that LLO causes damage to an intracellular organelle provides a new perspective in our understanding of how CDCs affect target cells during infection by the respective bacterial pathogens.
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| 29. |
- Genisset, Christophe, et al.
(författare)
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The concerted action of the Helicobacter pylori cytotoxin VacA and of the v-ATPase proton pump induces swelling of isolated endosomes
- 2007
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Ingår i: Cellular Microbiology. - : Hindawi Limited. - 1462-5814 .- 1462-5822. ; 9:6, s. 1481-1490
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Tidskriftsartikel (refereegranskat)abstract
- The vacuolating cytotoxin (VacA) is a major virulence factor of Helicobacter pylori, the bacterium associated to gastroduodenal ulcers and stomach cancers. VacA induces formation of cellular vacuoles that originate from late endosomal compartments. VacA forms an anion-selective channel and its activity has been suggested to increase the osmotic pressure in the lumen of these acidic compartments, driving their swelling to vacuoles. Here, we have tested this proposal on isolated endosomes that allow one to manipulate at will the medium. We have found that VacA enhances the v-ATPase proton pump activity and the acidification of isolated endosomes in a Cl- dependent manner. Other counter-anions such as pyruvate, Br-, I- and SCN- can be transported by VacA with stimulation of the v-ATPase. The VacA action on isolated endosomes is associated with their increase in size. Single amino acid substituted VacA with no channel-forming and vacuolating activity is unable to induce swelling of endosomes. These data provide a direct evidence that the transmembrane VacA channel mediates an influx of anions into endosomes that stimulates the electrogenic v-ATPase proton pump, leading to their osmotic swelling and transformation into vacuoles.
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| 30. |
- Guerra, Lina, et al.
(författare)
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Cellular internalization of cytolethal distending toxin : a new end to a known pathway
- 2005
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Ingår i: Cellular Microbiology. - : Hindawi Limited. - 1462-5814 .- 1462-5822. ; 7:7, s. 921-34
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Tidskriftsartikel (refereegranskat)abstract
- The cytolethal distending toxins (CDTs) are unique in their ability to induce DNA damage, activate checkpoint responses and cause cell cycle arrest or apoptosis in intoxicated cells. However, little is known about their cellular internalization pathway. We demonstrate that binding of the Haemophilus ducreyi CDT (HdCDT) on the plasma membrane of sensitive cells was abolished by cholesterol extraction with methyl-beta-cyclodextrin. The toxin was internalized via the Golgi complex, and retrogradely transported to the endoplasmic reticulum (ER), as assessed by N-linked glycosylation. Further translocation from the ER did not require the ER-associated degradation (ERAD) pathway, and was Derlin-1 independent. The genotoxic activity of HdCDT was dependent on its internalization and its DNase activity, as induction of DNA double-stranded breaks was prevented in Brefeldin A-treated cells and in cells exposed to a catalytically inactive toxin. Our data contribute to a better understanding of the CDT mode of action and highlight two important aspects of the biology of this bacterial toxin family: (i) HdCDT translocation from the ER to the nucleus does not involve the classical pathways followed by other retrogradely transported toxins and (ii) toxin internalization is crucial for execution of its genotoxic activity.
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| 31. |
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| 32. |
- Holm, Åsa, et al.
(författare)
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Leishmania donovani lipophosphoglycan causes periphagosomal actin accumulation : correlation with impaired translocation of PKCα and defective phagosome maturation
- 2001
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Ingår i: Cellular Microbiology. - : Hindawi Limited. - 1462-5814 .- 1462-5822. ; 3:7, s. 439-447
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Tidskriftsartikel (refereegranskat)abstract
- Lipophosphoglycan (LPG) is the major surface glycoconjugate of Leishmania donovani promastigotes. The repeating disaccharide–phosphate units of LPG are crucial for promastigote survival inside macrophages and establishment of infection. LPG has a number of effects on the host cell, including inhibition of PKC activity, inhibition of nitric oxide production and altered expression of cytokines. LPG also inhibits phagosomal maturation, a process requiring depolymerization of periphagosomal F-actin. In the present study, we have characterized the dynamics of F-actin during the phagocytosis of L. donovani promastigotes in J774 macrophages. We observed that F-actin accumulated progressively around phagosomes containing wild-type L. donovani promastigotes during the first hour of phagocytosis. Using LPG-defective mutants and yeast particles coated with purified LPG, we obtained evidence that this effect could be attributed to the repeating units of LPG. LPG also disturbed cortical actin turnover during phagocytosis. The LPG-dependent accumulation of periphagosomal F-actin correlated with an impaired recruitment of the lysosomal marker LAMP1 and PKCα to the phagosome. Accumulation of periphagosomal F-actin during phagocytosis of L. donovani promastigotes may contribute to the inhibition of phagosomal maturation by physically preventing vesicular trafficking to and from the phagosome.
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- Lycke, Nils Y, 1954
(författare)
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From toxin to adjuvant: the rational design of a vaccine adjuvant vector, CTA1-DD/ISCOM.
- 2004
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Ingår i: Cellular microbiology. - : Hindawi Limited. - 1462-5814 .- 1462-5822. ; 6:1, s. 23-32
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Forskningsöversikt (refereegranskat)abstract
- Mucosally active vaccine adjuvants which will prime a full range of local and systemic immune responses against defined antigenic epitopes are much needed. Cholera toxin (CT) and lipophilic immune stimulating complexes (ISCOMs) containing Quil A can both act as adjuvants for orally administered antigens, but through separate pathways, as evidenced by the dependence on IL-12 for the effect of ISCOMs, whereas CT is independent of this cytokine. Unfortunately the toxicity of CT and recent findings of accumulation of CT in the olfactory nerve and bulb after intranasal administration precludes the clinical use of CT. However, we have been successful in separating the adjuvant and toxic effects of CT, by constructing a gene fusion protein, CTA1-DD, that combines the enzymatically active CTA1-subunit with a B cell targeting moiety, D, derived from Staphylococcus aureus protein A. The present review gives a background to mucosal immunization and the use of -adjuvants in general, followed by a description of a strategy to rationally design a vaccine adjuvant vector that fulfils the criteria of targeting and immunomodulating innate immunity in order to boost a strong adaptive immune response. We have combined CTA1-DD and ISCOMs into a new highly promising vaccine adjuvant vector, CTA1-DD/ISCOMs. The combined vector is immunogenic when given by the subcutaneous, oral or nasal routes, inducing strong cell--mediated and humoral immune responses, including local mucosal IgA. It requires the ADP ribosylating property of the CTA1-enzyme and the effect of the combined vector greatly exceeded the effect of either ISCOMs or CT used alone. Antigens could be incorporated into or just admixed with the new vector. Thus, we have demonstrated that rationally designed vectors consisting of CTA1-DD and ISCOMS may provide a novel strategy for the generation of potent and safe mucosal vaccines.
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| 44. |
- Melican, K, et al.
(författare)
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Bacterial infection-mediated mucosal signalling induces local renal ischaemia as a defence against sepsis
- 2008
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Ingår i: Cellular Microbiology. - : Hindawi Limited. - 1462-5814 .- 1462-5822. ; 10:10, s. 1987-1998
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Tidskriftsartikel (refereegranskat)abstract
- Ascending urinary tract infections can cause extensive damage to kidney structure and function. We have used a number of advanced techniques including multiphoton microscopy to investigate the crucial early phases of uropathogenic Escherichia coli induced pyelonephritis within a living animal. Our results reveal a previously undescribed innate vascular response to mucosal infection, allowing isolation and eradication of the pathogen. The extremely rapid host response to mucosal infection was highlighted by the triggering of a cascade of events within 3-4 h. Epithelial signalling produced an increase in cellular O-2 consumption and affected microvascular flow by clotting, causing localized ischaemia. Subsequent ischaemic damage affected pathophysiology with actin re-arrangement and epithelial sloughing leading to paracellular bacterial movement. A denuded tubular basement membrane is shown to hinder immediate dissemination of bacteria, giving the host time to isolate the infection by clotting. Suppression of clotting by heparin treatment caused fatal urosepsis. Clinically these findings may be relevant in antibiotics delivery in pyelonephritis patients and to the use of anticoagulants in sepsis.
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| 47. |
- Plant, Laura, et al.
(författare)
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Non-lipooligosaccharide-mediated signalling via Toll-like receptor 4 causes fatal meningococcal sepsis in a mouse model
- 2007
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Ingår i: Cellular Microbiology. - : Hindawi Limited. - 1462-5814 .- 1462-5822. ; 9:3, s. 657-669
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Tidskriftsartikel (refereegranskat)abstract
- Meningococcal lipooligosaccharide (LOS) is a major inflammatory mediator of fulminant meningococcal sepsis and meningitis with disease severity correlating with circulating concentrations of LOS and proinflammatory cytokines. In this study we show that the proinflammatory response to live meningococci in a mouse model of sepsis involves TLR4, but can develop independently of the expression of LOS. This is supported by data showing that in vivo an isogenic LOS-deficient strain, lpxA, induced equivalent disease severity and similar proinflammatory responses as the serogroup C wild-type parent strain FAM20. This response was abolished in TLR4–/– mice, and neither the wild-type strain of meningococci nor the LOS-deficient mutant was able to cause fatal sepsis in these mice. Mouse survival correlated with low levels of cytokines and chemokines, the chemotactic complement factor C5a and neutrophil levels in blood at 24 h post infection. These data suggest that during meningococcal sepsis the recognition of one or more unidentified non-LOS component(s) by TLR4 is important in stimulating proinflammatory responses, and that fatality associated with meningococcal sepsis in mice is induced by the proinflammatory host response.
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| 48. |
- Raabe, Andreas C, et al.
(författare)
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Multiple roles for Plasmodium berghei phosphoinositide-specific phospholipase C in regulating gametocyte activation and differentiation
- 2011
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Ingår i: Cellular Microbiology. - : Blackwell Publishing Ltd. - 1462-5814 .- 1462-5822. ; 13:7, s. 955-966
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Tidskriftsartikel (refereegranskat)abstract
- Critical events in the life cycle of malaria parasites are controlled by calcium-dependent signalling cascades, yet the molecular mechanisms of calcium release remain poorly understood. The synchronized development of Plasmodium berghei gametocytes relies on rapid calcium release from internal stores within 10 s of gametocytes being exposed to mosquito-derived xanthurenic acid (XA). Here we addressed the function of phosphoinositide-specific phospholipase C (PI-PLC) for regulating gametocyte activation. XA triggered the hydrolysis of PIP(2) and the production of the secondary messenger IP(3) in gametocytes. Both processes were selectively blocked by a PI-PLC inhibitor, which also reduced the early Ca(2+) signal. However, microgametocyte differentiation into microgametes was blocked even when the inhibitor was added up to 5 min after activation, suggesting a requirement for PI-PLC beyond the early mobilization of calcium. In contrast, inhibitors of calcium release through ryanodine receptor channels were active only during the first minute of gametocyte activation. Biochemical determination of PI-PLC activity was confirmed using transgenic parasites expressing a fluorescent PIP(2) /IP(3) probe that translocates from the parasite plasmalemma to the cytosol upon cell activation. Our study revealed a complex interdependency of Ca(2+) and PI-PLC activity, with PI-PLC being essential throughout gamete formation, possibly explaining the irreversibility of this process.
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| 50. |
- Söderblom, Tomas, et al.
(författare)
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Effects of the Escherichia coli toxin cytolysin A on mucosal immunostimulation via epithelial Ca2+ signalling and Toll-like receptor 4.
- 2005
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Ingår i: Cell Microbiol. - : Hindawi Limited. - 1462-5814 .- 1462-5822. ; 7:6, s. 779-88
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Tidskriftsartikel (refereegranskat)abstract
- Epithelial cells are vital to sense the presence of bacteria, thereby initiating a proper innate immune response. This occurs via different mechanisms, e.g. recognition by pattern recognition receptors (TLR), or alteration of the cellular Ca2+ homeostasis. The Escherichia coli toxin cytolysin A (ClyA) is naturally delivered to target cells as active pore assemblies within outer membrane vesicles (OMVs), and we here investigate a possible role of ClyA-containing OMVs (ClyA+ (OMV)) for induction of proinflammatory responses via the above-mentioned mechanisms. We report that low, sublytic concentrations of ClyA+ (OMV) affect the Ca2+ homeostasis in epithelial cells by induction of slow, intracellular Ca2+ oscillations, while increased concentrations act cytolytically. Thus, ClyA belongs to the novel group of pore-forming toxins shown to elicit such biphasic responses. Ca2+ waves in the minute range have been shown to regulate gene transcription of, e.g. interleukin (IL)-6 and -8. While the periodicity of ClyA+ (OMV)-induced Ca2+ waves (22.9 +/- 0.9 min) fail to induce an IL-8 response, our data fit to the general concept of frequency-specific gene expression. Molecular investigations of the signal transduction pathway reveals that ClyA+ (OMV) utilize a different one as compared with those previously reported for other toxins causing Ca2+ waves. The ClyA protein per se and ClyA pore assemblies are non-immunogenic, while lipopolysaccharide present on the OMVs induces a TLR4-dependent proinflammatory response as expected. Additional membrane components of the OMV, e.g. OmpW, was also found to elicit proinflammatory responses that was independent of TLR4 and Ca2+ signalling.
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