| 1. |
- Ben Nasr, Abdelhakim, et al.
(author)
-
Absorption of kininogen from human plasma by Streptococcus pyogenes is followed by the release of bradykinin
- 1997
-
In: Biochemical Journal. - : Portland Press Ltd.. - 0264-6021 .- 1470-8728. ; 326:3, s. 657-660
-
Journal article (peer-reviewed)abstract
- H-kininogen (high-molecular-mass kininogen, HK) is the precursor of the vasoactive peptide hormone bradykinin (BK). Previous work has demonstrated that HK binds to Streptococcus pyogenes through M-proteins, fibrous surface proteins and important virulence factors of these bacteria. Here we find that M-protein-expressing bacteria absorb HK from human plasma. The HK bound to the bacteria was found to be cleaved, and analysis of the degradation pattern suggested that the cleavage of HK at the bacterial surface is associated with the release of BK. Moreover, addition of activated plasma prekallikrein to bacteria preincubated with human plasma, resulted in BK release. This mechanism, by which a potent vasoactive and proinflammatory peptide is generated at the site of infection, should influence the host-parasite relationship during S. pyogenes infections.
|
|
| 2. |
- Ben Nasr, Abdelhakim, et al.
(author)
-
Assembly of human contact phase factors and release of bradykinin at the surface of curli-expressing Escherichia coli
- 1996
-
In: Molecular Microbiology. - 1365-2958. ; 20:5, s. 35-927
-
Journal article (peer-reviewed)abstract
- Previous work has demonstrated that most strains of the human pathogen Streptococcus pyogenes bind kininogens through M protein, a fibrous surface protein and virulence determinant. Here we find that strains of several other pathogenic bacterial species, both Gram-positive and Gram-negative, isolated from patients with sepsis, also bind kininogens, especially kininogen (HK). The most pronounced interaction was seen between HK and Escherichia coli. Among clinical isolates of E. coli, the majority of the enterohaemorrhagic, enterotoxigenic, and sepsis strains, but none of the enteroinvasive and enteropathogenic strains, bound HK. Binding of HK to E. coli correlated with the expression of curli, another fibrous bacterial surface protein, and the binding of HK to purified curli was specific, saturable, and of high affinity; Ka = 9 x 10(7) M-1. Other contact phase proteins such as factor XI, factor XII, and prekallikrein bound to curliated E. coli, but not to an isogenic curli-deficient mutant strain, suggesting that contact phase activation may occur at the surface of curliated bacteria. Kininogens are also precursor molecules of the vasoactive kinins. When incubated with human plasma, curli-expressing bacteria absorbed HK. Addition of purified plasma kallikrein to the HK-loaded bacteria resulted in a rapid and efficient release of bradykinin from surface-bound HK. The assembly of contact phase factors at the surface of pathogenic bacteria and the release of the potent proinflammatory and vasoactive peptide bradykinin, should have a major impact on the host-microbe relationship and may contribute to bacterial pathogenicity and virulence.
|
|
| 3. |
- Ben Nasr, Abdelhakim, et al.
(author)
-
Streptokinase activates plasminogen bound to human group C and group G streptococci through M-like proteins
- 1994
-
In: European Journal of Biochemistry. - 0014-2956. ; 222:2, s. 76-267
-
Journal article (peer-reviewed)abstract
- An ability to interact with plasminogen or plasmin could provide micro-organisms with a mechanism for invasion. Thus, group A, C and G streptococci secrete streptokinase which binds and activates plasminogen. Some streptococci also express surface structures which bind plasminogen without causing its activation. Plasminogen-binding surface proteins were extracted from one group C and one group G streptococcal isolate. Both proteins were found to bind plasmin, fibrinogen and serum albumin in addition to plasminogen. Gene fragments encoding the streptococcal proteins were amplified by PCR and were subsequently cloned and expressed in Escherichia coli. DNA sequence determination revealed for both genes open reading frames encoding proteins which contained repetitive domains and a carboxyl-terminal unrepeated region that were typical of M and M-like proteins. Though the amino-terminal regions of the group C and G streptococcal proteins demonstrated a rather high overall similarity between themselves, they were not similar to the variable regions of other M-like proteins with one exception: there was a 46% identity between the first 22 amino acids of the group G streptococcal protein and the corresponding sequence of PAM, the plasminogen-binding M-like protein of type M53 group A streptococci. Like the proteins extracted from the streptococci, the recombinant proteins bound plasminogen, fibrinogen and albumin. The three plasma proteins bound to separate sites on the streptococcal M-like proteins. Plasminogen bound by the group C and G streptococcal proteins was readily activated by streptokinase, providing evidence for a functional link between the secreted plasminogen-activator and proteins exposed on the bacterial surface.
|
|
| 4. |
|
|
| 5. |
- Frick, Inga-Maria, et al.
(author)
-
Interactions between M proteins of Streptococcus pyogenes and glycosaminoglycans promote bacterial adhesion to host cells.
- 2003
-
In: European Journal of Biochemistry. - : Wiley. - 0014-2956. ; 270:10, s. 2303-2311
-
Journal article (peer-reviewed)abstract
- Several microbial pathogens have been reported to interact with glycosaminoglycans (GAGs) on cell surfaces and in the extracellular matrix. Here we demonstrate that M protein, a major surface-expressed virulence factor of the human bacterial pathogen, Streptococcus pyogenes, mediates binding to various forms of GAGs. Hence, S. pyogenes strains expressing a large number of different types of M proteins bound to dermatan sulfate (DS), highly sulfated fractions of heparan sulfate (HS) and heparin, whereas strains deficient in M protein surface expression failed to interact with these GAGs. Soluble M protein bound DS directly and could also inhibit the interaction between DS and S. pyogenes. Experiments with M protein fragments and with streptococci expressing deletion constructs of M protein, showed that determinants located in the NH2-terminal part as well as in the C-repeat region of the streptococcal proteins are required for full binding to GAGs. Treatment with ABC-chondroitinase and HS lyase that specifically remove DS and HS chains from cell surfaces, resulted in significantly reduced adhesion of S. pyogenes bacteria to human epithelial cells and skin fibroblasts. Together with the finding that exogenous DS and HS could inhibit streptococcal adhesion, these data suggest that GAGs function as receptors in M protein-mediated adhesion of S. pyogenes.
|
|
| 6. |
- Frick, Inga-Maria, et al.
(author)
-
Protein H--a surface protein of Streptococcus pyogenes with separate binding sites for IgG and albumin
- 1994
-
In: Molecular Microbiology. - : Wiley. - 1365-2958 .- 0950-382X. ; 12:1, s. 143-151
-
Journal article (peer-reviewed)abstract
- Protein H, a molecule expressed at the surface of some strains of Streptococcus pyogenes, has affinity for the constant (IgGFc) region of immunoglobulin (Ig) G. In absorption experiments with human plasma, protein H-sepharose could absorb not only IgG but also albumin from plasma. The affinity constant for the reaction between albumin and protein H was 7.8 x 10(9) M-1, which is higher than the affinity between IgG and protein H (Ka = 1.6 x 10(9) M-1). Fragments of protein H were generated with deletion plasmids and polymerase chain reaction (PCR) technology. Using these fragments in various protein-protein interaction assays, the binding of albumin was mapped to three repeats (C1-C3) in the C-terminal half of protein H. On the albumin molecule, the binding site for protein H was found to overlap the site for protein G, another albumin- and IgGFc-binding bacterial surface protein. Also IgGFc-binding could be mapped with the protein H fragments and the region was found N-terminally of the C repeats. A synthetic peptide (25 amino acid residues long) based on a sequence in this region was shown to inhibit the binding of protein H to immobilized IgG or IgGFc. This sequence was not found in previously described IgGFc-binding proteins. However, two other cell surface proteins of S. pyogenes exhibited highly homologous regions. The results identify IgGFc- and albumin-binding regions of protein H and further define and emphasize the convergent evolution among bacterial surface proteins interacting with human plasma proteins.
|
|
| 7. |
- Herwald, Heiko, et al.
(author)
-
Zinc-dependent conformational changes in domain D5 of high molecular mass kininogen modulate contact activation
- 2001
-
In: European Journal of Biochemistry. - : Wiley. - 0014-2956. ; 268:2, s. 396-404
-
Journal article (peer-reviewed)abstract
- Human high molecular mass kininogen (HK) participates as nonenzymatic cofactor in the contact system. Here, we show that recombinant domain D5 of HK (rD5) prolongs the clotting time of the intrinsic pathway of coagulation and attenuates the generation of bradykinin. Further studies indicate that a correct fold of domain D5 within HK is required for the activation of the contact system. The folding of rD5 seems to be modulated by the metal ions Zn2+, Ni2+, and Cu2+ as a specific antibody directed against the zinc-binding site in HK binds to HK and rD5 in a metal ion concentration dependent manner. The finding that these three metal ions specifically affect contact activation suggests that they regulate the accessibility of rD5 for negatively charged surfaces. Support for the assumption that the observed phenomena are due to conformational changes was obtained by fluorescence spectroscopy of rD5, demonstrating that its fluorescence spectrum was changed in the presence of ZnCl2. Moreover, negative staining electron microscopy experiments suggest that the zinc-induced changes in D5 also affect the conformation of the entire HK protein. The present data emphasize the role of zinc and other metal ions in the regulation of contact activation.
|
|
| 8. |
- McArthur, Jason D, et al.
(author)
-
Allelic variants of streptokinase from Streptococcus pyogenes display functional differences in plasminogen activation.
- 2008
-
In: The FASEB journal : official publication of the Federation of American Societies for Experimental Biology. - : Wiley. - 1530-6860. ; 22:9, s. 3146-3153
-
Journal article (peer-reviewed)abstract
- A common mammalian defense mechanism employed to prevent systemic dissemination of invasive bacteria involves occlusion of local microvasculature and encapsulation of bacteria within fibrin networks. Acquisition of plasmin activity at the bacterial cell surface circumvents this defense mechanism, allowing invasive disease initiation. To facilitate this process, S. pyogenes secretes streptokinase, a plasminogen-activating protein. Streptokinase polymorphism exhibited by S. pyogenes isolates is well characterized. However, the functional differences displayed by these variants and the biological significance of this variation has not been elucidated. Phylogenetic analysis of ska sequences from 28 S. pyogenes isolates revealed 2 main sequence clusters (clusters 1 and 2). All strains secreted streptokinase, as determined by Western blotting, and were capable of acquiring cell surface plasmin activity after incubation in human plasma. Whereas culture supernatants from strains containing cluster 1 ska alleles also displayed soluble plasminogen activation activity, supernatants from strains containing cluster 2 ska alleles did not. Furthermore, plasminogen activation activity in culture supernatants from strains containing cluster 2 ska alleles could only be detected when plasminogen was prebound with fibrinogen. This study indicates that variant streptokinase proteins secreted by S. pyogenes isolates display differing plasminogen activation characteristics and may therefore play distinct roles in disease pathogenesis.-McArthur, J. D., McKay, F. C., Ramachandran, V., Shyam, P., Cork, A. J., Sanderson-Smith, M. L., Cole, J. N., Ringdahl, U., Sjöbring, U., Ranson, M., Walker, M. J. Allelic variants of streptokinase from Streptococcus pyogenes display functional differences in plasminogen activation.
|
|
| 9. |
|
|
| 10. |
|
|
| 11. |
- Ringdahl, Ulrika, et al.
(author)
-
A role for the fibrinogen-binding regions of streptococcal M proteins in phagocytosis resistance
- 2000
-
In: Molecular Microbiology. - : Wiley. - 1365-2958 .- 0950-382X. ; 37:6, s. 1318-1326
-
Journal article (peer-reviewed)abstract
- All virulent group A streptococcal isolates bind fibrinogen, a property that is closely linked to expression of type-specific antiphagocytic surface molecules designated M proteins. Here we show that although the M proteins from two different strains, M1 and M5, both bind fibrinogen with high affinity, they interact with different regions in the ligand. Moreover, mapping experiments demonstrated that the fibrinogen-binding regions in the M1 and M5 proteins are quite dissimilar at the amino acid sequence level and that they bind to different regions in the plasma protein. In spite of these differences, the fibrinogen-binding regions of M1 and M5 could both be shown to contribute to streptococcal survival in human blood, providing evidence for the distinct function of a plasma protein interaction in bacterial pathogenesis.
|
|
| 12. |
- Ringdahl, Ulrika, et al.
(author)
-
Molecular co-operation between protein PAM and streptokinase for plasmin acquisition by Streptococcus pyogenes.
- 1998
-
In: Journal of Biological Chemistry. - 1083-351X. ; 273:11, s. 6424-6430
-
Journal article (peer-reviewed)abstract
- Bacterial surface-associated plasmin formation is believed to contribute to invasion, although the underlying molecular mechanisms are poorly understood. To define the components necessary for plasmin generation on group A streptococci we used strain AP53 which exposes an M-like protein ("PAM") that contains a plasminogen-binding sequence with two 13-amino acid residues long tandem repeats (a1 and a2). Utilizing an Escherichia coli-streptococcal shuttle vector, we replaced a 29-residue long sequence segment of Arp4, an M-like protein that does not bind plasminogen, with a single (a1) or the combined a1a2 repeats of PAM. When expressed in E. coli, the purified chimeric Arp/PAM proteins both bound plasminogen, as well as plasmin, and when used to transform group A streptococcal strains lacking the plasminogen-binding ability, transformants with the Arp/PAM constructs efficiently bound plasminogen. Moreover, when grown in the presence of plasminogen, both Arp/PAM- and PAM-expressing streptococci acquired surface-bound plasmin. In contrast, plasminogen activation failed to occur on PAM- and Arp/PAM-expressing streptococci carrying an inactivated streptokinase gene: this block was overcome by exogenous streptokinase. Together, these results provide evidence for an unusual co-operation between a surface-bound protein, PAM, and a secreted protein, streptokinase, resulting in bacterial acquisition of a host protease that is likely to spur parasite invasion of host tissues.
|
|
| 13. |
- Roupé, Markus, et al.
(author)
-
Injury Is a Major Inducer of Epidermal Innate Immune Responses during Wound Healing.
- 2010
-
In: Journal of Investigative Dermatology. - : Elsevier BV. - 1523-1747 .- 0022-202X. ; 130, s. 1167-1177
-
Journal article (peer-reviewed)abstract
- We examined the importance of injury for the epidermal innate immune response in human skin wounds. We found that injury, independent of infiltrating inflammatory cells, generated prominent chemotactic activity toward neutrophils in injured skin because of IL-8 production. Furthermore, injury was a major inducer of the expression of antimicrobial (poly)peptides (AMPs) in skin wounds. In human skin, these injury-induced innate immune responses were mediated by activation of the epidermal growth factor receptor (EGFR). Consequently, inhibition of the EGFR blocked both the chemotactic activity generated in injured skin and the expression of the majority of the AMPs. The importance of injury was confirmed in mouse experiments in vivo, in which injury independent of infection was a potent inducer of AMPs in skin wounds. To our knowledge, these data thereby provide a previously unreported molecular link between injury and neutrophil accumulation and identify the molecular background for the vast expression of IL-8 and AMPs in wounded epidermis. Conceptually, these data show that the growth factor response elicited by injury is important for the recruitment of neutrophils in skin wounds.Journal of Investigative Dermatology advance online publication, 3 September 2009; doi:10.1038/jid.2009.284.
|
|
| 14. |
- Savic, B, et al.
(author)
-
Evaluation of polymerase chain reaction, tuberculostearic acid analysis, and direct microscopy for the detection of Mycobacterium tuberculosis in sputum
- 1992
-
In: Journal of Infectious Diseases. - 1537-6613. ; 166:5, s. 1177-1180
-
Journal article (peer-reviewed)abstract
- Tuberculosis remains a major global cause of morbidity and mortality. There is an urgent need for improved bacteriologic diagnosis of Mycobacterium tuberculosis infection. Three methods for rapid identification of M. tuberculosis in sputum samples (direct microscopy, gas chromatography-mass spectrometry [GC-MS], and polymerase chain reaction [PCR]), were compared with culture on Lowenstein-Jensen medium. Growth of M. tuberculosis was observed in 38 of 145 sputum samples. Detection of acid-fast bacilli by direct microscopy gave a sensitivity of 66% and a specificity of 100%. Detection of tuberculostearic acid by GC-MS gave a sensitivity of 55% and a specificity of 87%. Amplification by PCR of a fragment of the insertion sequence IS6110 gave a sensitivity of 95% and a specificity of 93% compared with culture and a corrected specificity of 99% compared with both culture and clinical data. This study indicates that PCR can be adapted for clinical use and is the method of choice for rapid diagnosis of pulmonary tuberculosis.
|
|
| 15. |
- 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
-
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.
|
|
| 16. |
- Sjöbring, Ulf, et al.
(author)
-
Analysis of plasminogen-binding M proteins of Streptococcus pyogenes.
- 2000
-
In: Methods. - 1095-9130. ; 21:2, s. 143-150
-
Journal article (peer-reviewed)abstract
- Group A streptococci are common human pathogens that cause a variety of infections. They express M proteins which are important cell wall-bound type-specific virulence factors. We have found that a set of strains, associated primarily with skin infections, express M proteins that bind plasminogen and plasmin with high affinity. The binding is mediated by a 13-amino-acid internal repeated sequence located in the N-terminal surface-exposed portion of these M proteins. This sequence binds to kringle 2 in plasminogen, a domain that is not involved in the interaction with streptokinase, a potent group A streptococcal activator of plasminogen. It could be demonstrated that plasminogen, absorbed from plasma by growing group A streptococci expressing the plasminogen-binding M proteins, could be activated by exogenous and endogenous streptokinase, thereby providing the bacteria with a surface-associated enzyme that could act on the tissue barriers in the infected host.
|
|
| 17. |
- Sjöbring, Ulf, et al.
(author)
-
Induction of platelet thrombi by bacteria and antibodies
- 2002
-
In: Blood. - : American Society of Hematology. - 1528-0020 .- 0006-4971. ; 100:13, s. 4470-4477
-
Journal article (peer-reviewed)abstract
- We have characterized 2 distinct mechanisms through which infectious agents may promote platelet adhesion and thrombus formation in flowing blood, thus contributing to the progression of disease. In one case, the process initiates when the integrin alpha(IIb)beta(3) mediates platelet arrest onto immobilized bacterial constituents that have bound plasma fibrinogen. If blood contains antibodies against the bacteria, immunoglobulin (Ig) G may cluster on the same surface and activate adherent platelets through the FcgammaRIIA receptor, leading to thrombus growth. As an alternative, bacteria that cannot bind fibrinogen may attach to substrates, such as immobilized plasma proteins or components of the extracellular matrix, which also support platelet adhesion. As a result of this colocalization, IgG bound to bacteria can activate neighboring platelets and induce thrombus growth regard-less of their ability to initiate platelet-surface contact. Our results demonstrate that intrinsic constituents of infectious agents and host proteins play distinct but complementary roles in recruiting platelets into thrombi, possibly contributing to complications of acute and chronic infections.
|
|
| 18. |
|
|
| 19. |
- Sjöbring, Ulf, et al.
(author)
-
Polymerase chain reaction for detection of Mycobacterium tuberculosis
- 1990
-
In: Journal of Clinical Microbiology. - 1098-660X. ; 28:10, s. 2200-2204
-
Journal article (peer-reviewed)abstract
- A polymerase chain reaction for the specific detection of mycobacteria belonging to the Mycobacterium tuberculosis complex was developed. Using a single primer pair derived from the nucleotide sequence of protein antigen b of M. tuberculosis, we achieved specific amplification of a 419-base-pair DNA fragment in M. tuberculosis and M. bovis. After DNA was extracted from mycobacteria by using a simple, safe lysis procedure, we detected the 419-base-pair sequence in samples containing few mycobacteria. Preliminary data suggested that this technique could be applied to clinical specimens for early and specific diagnosis of tuberculosis.
|
|
| 20. |
- Sun, HM, et al.
(author)
-
Plasminogen is a critical host pathogenicity factor for group A streptococcal infection
- 2004
-
In: Science. - : American Association for the Advancement of Science (AAAS). - 1095-9203 .- 0036-8075. ; 305:5688, s. 1283-1286
-
Journal article (peer-reviewed)abstract
- Group A streptococci, a common human pathogen, secrete streptokinase, which activates the host's blood clot-dissolving protein, plasminogen. Streptokinase is highly specific for human plasminogen, exhibiting little or no activity against other mammalian species, including mouse. Here, a transgene expressing human plasminogen markedly increased mortality in mice infected with streptococci, and this susceptibility was dependent on bacterial streptokinase expression. Thus, streptokinase is a key pathogenicity factor and the primary determinant of host species specificity for group A streptococcal infection. In addition, local fibrin clot formation may be implicated in host defense against microbial pathogens.
|
|
| 21. |
|
|
| 22. |
- Svensson, Mik, et al.
(author)
-
Roles of the plasminogen activator streptokinase and the plasminogen-associated M protein in an experimental model for streptococcal impetigo
- 2002
-
In: Microbiology. - 1465-2080. ; 148:12, s. 3933-3945
-
Journal article (peer-reviewed)abstract
- Primary infection by group A streptococci (GAS) takes place at either the throat or skin of the human host, often leading to pharyngitis or impetigo, respectively. Many GAS strains differ in their preference for throat and skin tissue sites. Previous epidemiological findings show that many of the strains displaying strong tropism for the skin have a high-affinity binding site for plasminogen, located within M protein (PAM), a prominent surface fibril. Plasminogen bound by PAM interacts with streptokinase, a plasminogen activator secreted by GAS, to yield bacterial-bound plasmin activity. In this study, PAM and streptokinase were tested for their roles in infection using an experimental model that closely mimics human impetigo. Inactivation of genes encoding either PAM or streptokinase led to a partial, but significant, loss of virulence in vivo, as measured by net growth of the bacteria and pathological alterations. The relative loss in virulence in vivo was greater for the streptokinase mutant than for the PAM mutant. However, the PAM mutant, but not the streptokinase mutant, displayed a partial loss in resistance to phagocytosis in vitro. The combined experimental and epidemiological data provide evidence that PAM and streptokinase play a key role in mediating skin-specific infection by GAS. In addition, secreted cysteine proteinase activity due to SpeB leads to degradation of streptokinase in stationary phase broth cultures. Since SpeB is also a determinant of tissue-specific GAS infection at the skin, direct interactions between these two proteolytic pathways may constitute an important pathogenic mechanism. An integrated model for superficial infection at the skin is presented.
|
|
| 23. |
|
|
| 24. |
- Weineisen, Maria, et al.
(author)
-
Streptococcal M5 protein prevents neutrophil phagocytosis by interfering with CD11b/CD18 receptor-mediated association and signaling.
- 2004
-
In: J Immunol. - 0022-1767. ; 172:6, s. 3798-3807
-
Journal article (peer-reviewed)abstract
- Group A streptococci (GAS) are common human pathogens that express major surface-associated virulence factors designated M proteins. In this study, we explored directly the cellular mechanisms behind their supposed ability to prevent phagocytosis. Isolated human neutrophils killed an M-negative GAS mutant (DeltaM5), but not the wild-type parent strain (M5). After 3 h, 3-4 times as many DeltaM5 as M5 bacteria were associated with the neutrophils, and more DeltaM5 than M5 bacteria were ingested. However, there was no statistically significant difference between DeltaM5 and M5 bacteria in regard to the percentage of the neutrophil-associated bacteria that were ingested, indicating that M5 protein prevents an adhesion receptor-dependent association with neutrophils and not the phagocytic machinery per se. Different Abs against CD11b/CD18 (CR3) blocked adhesion and killing of DeltaM5 bacteria, whereas the blocking of two other complement receptors, CD11c/CD18 (CR4) and CD35 (CR1), did not. The CD11b/CD18-mediated killing of DeltaM5 bacteria resulted in protein tyrosine phosphorylations and Cdc42 activation. Furthermore, inhibition of CD11b/CD18 receptor engagement or tyrosine kinase activity blocked the DeltaM5-induced activation of Cdc42 as well as the killing of these bacteria. We conclude that M5 protein interferes with the CD11b/CD18-dependent association between GAS and neutrophils, and thereby blocks subsequent ingestion of the bacteria.
|
|
| 25. |
- Wetterö, Jonas, 1972-
(author)
-
Acute inflammation on model biomaterial surfaces : studies on proteins, neutrophils and platelets
- 2002
-
Doctoral thesis (other academic/artistic)abstract
- Although most biomedical devices are non-toxic, disturbed acute and chronic inflammation and the lack of integration in tissues is a concern. At the time of biomaterial insertion, protein adsorption onto material surfaces precedes cell adhesion and is believed to alter unfavorably the acute inflammatory response and the subsequent tissue healing. The wound healing may encapsulate the biomaterial in a fibrous tissue. The process depends probably on the surface physical and chemical characteristics, and the accumulation of blood plasma proteins such as fibrinogen, immunoglobulins (Ig:s) and complement. Platelets and neutrophil granulocytes, which both possess inflammatory capabilities, are the first cells to appear at a surface during contact with blood. In the present thesis, model biomaterial surfaces were prepared, and the in vitro deposition of plasma proteins and the subsequent behavior of neutrophils and platelets evaluated.Complement activation at artificial surfaces during contact with blood is generally believed to proceed via the alternative pathway, i.e. through a direct covalent binding of the factor 3 (C3) thioester to nucleophilic surface groups (e.g. -OH and -NH2). The serum protein deposition onto a hydroxylated potent complement activator surface, mercaptoglycerol on gold, was studied by a combination of null-ellipsometry and polyclonal antibodies. It was observed that deposited C3 did not withstand elution with sodium dodecyl sulfate (SDS), and the binding was unaffected by reduction with hydroxylamine. Opposite results have been reported for biological surfaces and our findings call for a revision of the current activation model at artificial surfaces where instead the classical pathway of complement may be highly relevant.The effects of immobilized and partially denatured IgG on the neutrophil respiratory burst at hydrophilic and hydrophobic model surfaces were studied by lurninol-arnplitied chemiluminescence in serum containing media. IgG supported frustrated phagocytosis and generation of extracellular reactive oxygen species (ROS) on both types of surfaces, although the kinetics were different. The response was particularly potent on IgG at hydrophobic surfaces, and the finding that the respiratory burst was only moderately quenched by the blocking of complement receptors (CR:s) or Fcγ (IgG) receptors, indicates a role for intracellular cross-talk. The IgG-triggered response depended on the presence of both C3 and C1q in serum and was inhibited by disruption of the intracellular actin dynamics. Classical complement activation may also be initiated by immobilized IgM. When the activation by spontaneously adsorbed IgG and IgM on methylated hydrophobic silicon was compared, both Ig:s deposited C3 from serum, but only the activation at IgG was C1q- and Ca2+-dependent. Depletion of C1q from serum lowered the neutrophil respiratory burst and the formation of intracellular filamentous (F) actin upon adhesion to IgG-surfaces. Hence, IgG- but not IgM-coated hydrophobic surfaces activate the classical pathway via the C1 complex.Surface-bound IgG is also a potent platelet agonist via the Fcγ receptor. Neutrophil and platdet ROS generation, aggregation, and release of adenosine triphosphate in response to spontaneously adsorbed and covalendy immobilized IgG show that platelets enhance the neutrophil respiratory burst under both stirred and non-stirred serum free conditions. Blocking of the neutrophil Fcγ receptors was not sufficient to inhibit the amplification. Platelets supported neutrophil adhesion in a contact-dependent way, and the effect was mediated by intact platelets or platelet-derived fragments/microparticles. The response was, in contrast to complement dependent activation in serum, unaffected by the disruption of the actin cytoskeleton, or by blocking of neutrophil CR3 or platelet glycoprotein IIb/IIIa, suggesting an integrin- and fibrinogen-independent mechanism. Antibodies against platelet P-selectin (CD62) and P-selectin glycoprotein ligand-1 (PSGL-1 or CD162), but not L-selectin (CD62L), inhibited partly the neutrophil-platelet interaction, especially under shear. Accordingly, we suggest that during stimulation of the cells with immobilized IgG, platdet P-selectin interacts with neutrophil PSGL-1.The majority of previous adsorption studies has dealt with blood plasma proteins. However, the concentration of released cytosolic proteins may locally reach high levels upon a tissue injury. Actin is one of the most abundant proteins in the eukaryotic cytoplasm, and may tentatively accumulate at interfaces. Actin was immobilized to gold and aminated silicon surfaces and polymerized into F-actin by adjusting the osmotic conditions. Upon incubation in human serum, the actin surfaces adsorbed serum proteins, amongst them C3 and C1q. However, the complement deposition was apparendy not a result of true or prolonged complement activation, and immobilized actin evoked only a low ROS-generation, aggregation, spreading and adhesion of neutrophils and platelets (similar to low-activating albumin-surfaces). Yet, F-actin on gold recruited platelets in a C1q-dependent manner, indicating an immunoregulatory capacity of surface-bound actin.The results in the present thesis are relevant for a better understanding of the basic mechanisms that determine the fate of artificial devices in contact with human body fluids.
|
|
| 26. |
- Wistedt, AC, et al.
(author)
-
Identification of a plasminogen-binding motif in PAM, a bacterial surface protein.
- 1995
-
In: Molecular Microbiology. - 1365-2958. ; 18:3, s. 569-578
-
Journal article (peer-reviewed)abstract
- Surface-associated plasmin(ogen) may contribute to the invasive properties of various cells. Analysis of plasmin(ogen)-binding surface proteins is therefore of interest. The N-terminal variable regions of M-like (ML) proteins from five different group A streptococcal serotypes (33, 41, 52, 53 and 56) exhibiting the plasminogen-binding phenotype were cloned and expressed in Escherichia coli. The recombinant proteins all bound plasminogen with high affinity. The binding involved the kringle domains of plasminogen and was blocked by a lysine analogue, 6-aminohexanoic acid, indicating that lysine residues in the M-like proteins participate in the interaction. Sequence analysis revealed that the proteins contain common 13-16-amino-acid tandem repeats, each with a single central lysine residue. Experiments with fusion proteins and a 30-amino-acid synthetic peptide demonstrated that these repeats harbour the major plasminogen-binding site in the ML53 protein, as well as a binding site for the tissue-type plasminogen activator. Replacement of the lysine in the first repeat with alanine reduced the plasminogen-binding capacity of the ML53 protein by 80%. The results precisely localize the binding domain in a plasminogen surface receptor, thereby providing a unique ligand for the analysis of interactions between kringles and proteins with internal kringle-binding determinants.
|
|
| 27. |
- Wistedt, AC, et al.
(author)
-
Kringle 2 mediates high affinity binding of plasminogen to an internal sequence in streptococcal surface protein PAM.
- 1998
-
In: Journal of Biological Chemistry. - 1083-351X. ; 273:38, s. 24420-24424
-
Journal article (peer-reviewed)abstract
- Many cells express receptors for plasminogen (Pg), although the responsible molecules in most cases are poorly defined. In contrast, the group A streptococcal surface protein PAM contains a domain with two 13-amino acid residue long repeated sequences (a1 and a2) responsible for Pg binding. Here we identify the region in Pg that interacts with PAM. A radiolabeled proteolytic plasminogen fragment containing the first three kringles (K1-K3) interacted with streptococci expressing PAM or a chimeric surface protein harboring the a1a2 sequence. In contrast, plasminogen fragments containing kringle 4 or kringle 5 and the activable serine proteinase domain failed to bind to PAM-expressing group A streptococci. A synthetic and a recombinant polypeptide containing the a1a2 sequence both bound to immobilized recombinant K2 (rK2) but not to rK1 or rK3. The interaction between the a repeat region and rK2 was reversible, and rK2 completely blocked the binding of Pg to the a1a2 region. The binding of the a repeat containing polypeptide to K2 occurred with an equilibrium association constant of 4.5 x 10(7) M-1, as determined by surface plasmon resonance, a value close to that (1.6 x 10(7) M-1) calculated for the a1a2-Pg interaction. Inhibition experiments suggested involvement of the lysine-binding site of K2 in the interaction. These data demonstrate that K2 contains the major Pg-binding site for PAM, providing the first well defined example of an interaction between an internal Pg-binding region in a protein and a single kringle domain.
|
|
