| 1. |
- Aung, Kyaw Min, et al.
(författare)
-
Naturally Occurring IgG Antibodies Provide Innate Protection against Vibrio cholerae Bacteremia by Recognition of the Outer Membrane Protein U
- 2016
-
Ingår i: Journal of Innate Immunity. - : S. Karger AG. - 1662-811X .- 1662-8128. ; 8:3, s. 269-283
-
Tidskriftsartikel (refereegranskat)abstract
- Cholera epidemics are caused by Vibrio cholerae serogroups O1 and O139, whereas strains collectively known as non-O1/non-O139 V. cholerae are found in cases of extraintestinal infections and bacteremia. The mechanisms and factors influencing the occurrence of bacteremia and survival of V. cholerae in normal human serum have remained unclear. We found that naturally occurring IgG recognizing V. cholerae outer membrane protein U (OmpU) mediates a serum-killing effect in a complement C1q-dependent manner. Moreover, outer membrane vesicles (OMVs) containing OmpU caused enhanced survival of highly serum-sensitive classical V. cholerae in a dose-dependent manner. OMVs from wild-type and ompU mutant V. cholerae thereby provided a novel means to verify by extracellular transcomplementation the involvement of OmpU. Our data conclusively indicate that loss, or reduced expression, of OmpU imparts resistance to V. cholerae towards serum killing. We propose that the difference in OmpU protein levels is a plausible reason for differences in serum resistance and the ability to cause bacteremia observed among V. cholerae biotypes. Our findings provide a new perspective on how naturally occurring antibodies, perhaps induced by members of the microbiome, may play a role in the recognition of pathogens and the provocation of innate immune defense against bacteremia.
|
|
| 2. |
- Berggren, Kristina, 1971, et al.
(författare)
-
3-Aminopiperidine Based Peptide Analogues as the First Selective Noncovalent Inhibitors of the Bacterial Cysteine Protease IdeS
- 2012
-
Ingår i: Journal of Medicinal Chemistry. - : American Chemical Society (ACS). - 0022-2623 .- 1520-4804. ; 55:6, s. 2549-2560
-
Tidskriftsartikel (refereegranskat)abstract
- A series of eight peptides corresponding to the amino acid sequence of the hinge region of IgG and 17 newly synthesized peptide analogues containing a piperidine moiety as a replacement of a glycine residue were tested as potential inhibitors of the bacterial IgG degrading enzyme of Streptococcus pyogenes, IdeS. None of the peptides showed any inhibitory activity of IdeS, but several piperidine-based analogues were identified as inhibitors. Two different analysis methods were used: an SDS-PAGE based assay to detect IgG cleavage products and a surface plasmon resonance spectroscopy based assay to quantify the degree of inhibition. To investigate the selectivity of the inhibitors for IdeS, all compounds were screened against two other related cysteine proteases (SpeB and papain). The selectivity results show that larger analogues that are active inhibitors of IdeS are even more potent as inhibitors of papain, whereas smaller analogues that are active inhibitors of IdeS inhibit neither SpeB nor papain. Two compounds were identified that exhibit high selectivity against IdeS and will be used for further studies.
|
|
| 3. |
- Bober, Marta, et al.
(författare)
-
The membrane bound LRR lipoprotein Slr, and the cell wall-anchored M1 protein from Streptococcus pyogenes both interact with type I collagen.
- 2011
-
Ingår i: PloS one. - : Public Library of Science. - 1932-6203. ; 6:5, s. e20345-
-
Tidskriftsartikel (refereegranskat)abstract
- Streptococcus pyogenes is an important human pathogen and surface structures allow it to adhere to, colonize and invade the human host. Proteins containing leucine rich repeats (LRR) have been identified in mammals, viruses, archaea and several bacterial species. The LRRs are often involved in protein-protein interaction, are typically 20-30 amino acids long and the defining feature of the LRR motif is an 11-residue sequence LxxLxLxxNxL (x being any amino acid). The streptococcal leucine rich (Slr) protein is a hypothetical lipoprotein that has been shown to be involved in virulence, but at present no ligands for Slr have been identified. We could establish that Slr is a membrane attached horseshoe shaped lipoprotein by homology modeling, signal peptidase II inhibition, electron microscopy (of bacteria and purified protein) and immunoblotting. Based on our previous knowledge of LRR proteins we hypothesized that Slr could mediate binding to collagen. We could show by surface plasmon resonance that recombinant Slr and purified M1 protein bind with high affinity to collagen I. Isogenic slr mutant strain (MB1) and emm1 mutant strain (MC25) had reduced binding to collagen type I as shown by slot blot and surface plasmon resonance. Electron microscopy using gold labeled Slr showed multiple binding sites to collagen I, both to the monomeric and the fibrillar structure, and most binding occurred in the overlap region of the collagen I fibril. In conclusion, we show that Slr is an abundant membrane bound lipoprotein that is co-expressed on the surface with M1, and that both these proteins are involved in recruiting collagen type I to the bacterial surface. This underlines the importance of S. pyogenes interaction with extracellular matrix molecules, especially since both Slr and M1 have been shown to be virulence factors.
|
|
| 4. |
- Brännström, Kristoffer, et al.
(författare)
-
Characterization of SPINK9, a KLK5-specific inhibitor expressed in palmo-plantar epidermis
- 2012
-
Ingår i: Biological chemistry (Print). - : Walter de Gruyter. - 1431-6730 .- 1437-4315. ; 393:5, s. 369-377
-
Tidskriftsartikel (refereegranskat)abstract
- SPINK9, a Kazal-type serine protease inhibitor, is almost exclusively expressed in the palmo-plantar epidermis. SPINK9 selectively inhibits kallikrein-related peptidase 5 (KLK5), no other target enzyme is known at present. In this study, we defined the reactive loop to residues 48 and 49 of SPINK9 and characterized the inhibition and binding of different SPINK9 variants towards KLK5, KLK7, KLK8 and KLK14. Substitutions of single amino acids in the reactive loop had a large impact on both inhibitory efficiency and specificity. Binding studies showed that it is mainly the dissociation rate that is affected by the amino acid substitutions. The inhibitory effect of wild-type SPINK9 was clearly pH-dependent with an improved effect at a pH similar to that of the outer layers of the skin. Modeling of the enzyme-inhibitor complexes showed that the reactive loop of SPINK9 fits very well into the deep negatively charged binding pocket of KLK5. A decrease in pH protonates His48 of the wild-type protein resulting in a positively charged residue, thereby explaining the observed decreased dissociation rate. Interestingly, substitution with a positively charged amino acid at position 48 resulted in a more efficient inhibitor at higher pH.
|
|
| 5. |
- Johansson, Björn, et al.
(författare)
-
The protein expression of Streptococcus pyogenes is significantly influenced by human plasma
- 2005
-
Ingår i: Journal of Proteome Research. - Washington : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 4:6, s. 2302-2311
-
Tidskriftsartikel (refereegranskat)abstract
- During the courser of infection, the common human pathogen Streptococcus pyogenes encounters plasma. We show that plasma causes S. pyogenes to rapidly remodel its cellular metabolism and virulence pathways. We also identified a variant of the major virulence factor, M1 protein, lacking 13 amino acids at the NH2-terminus in bacteria grown with plasma. The pronounced effect of plasma on protein expression, suggests this is an important adaptive mechanism with implications for S. pyogenes pathogenicity.
|
|
| 6. |
- Johansson Söderberg, Jenny, 1983-
(författare)
-
The streptococcal IgG degrading enzyme IdeS : studies on host-pathogen interactions
- 2012
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The important human pathogen Streptococcus pyogenes causes both mild infections such as pharyngitis and impetigo but also severe life threatening invasive infections. Specific antibodies (IgG) recognize pathogens and are important mediators for pathogen clearance by the immune defence. S.ipyogenes expresses a highly effective and specific IgG endopeptidase called IdeS (immunoglobulin degrading enzyme of S.ipyogenes). IdeS rescues bacteria from opsonising IgG by cleavage of IgG generating two fragments F(ab´)2 and ½Fc. Moreover, IdeS block ROS production by neutrophils. In this thesis I have studied (i) allelic variants of IdeS and their biological potential, (ii) consequences of ½Fc production for host-pathogen interactions and (iii) IdeS processing by streptococcal and neutrophil proteases. When investigating the allelic variants of IdeS we could show that in respect to IgG degradation and inhibition of ROS production the allelic variants where indistinguishable, however the allelic variant of serotype M28 appears to be an unique exception as this protein was deficient in IgG cleavage but still inhibited ROS production. Further, the ½Fc fragments produced when IgG is cleaved by IdeS were shown to prime human neutrophils and under ex vivo experimental conditions this increased the bactericidal activity of the neutrophils. Finally, we made the interesting finding that IdeS is N-terminally processed by neutrophil proteases and by the streptococcal protease SpeB, but retain enzymatic activity and was less immunogenic compared to the full length protein.
|
|
| 7. |
- Keeney, Jill B, et al.
(författare)
-
Multiple molecular determinants for retrotransposition in a primer tRNA
- 1995
-
Ingår i: Molecular and Cellular Biology. - : American Society for Microbiology. - 0270-7306 .- 1098-5549. ; 15:1, s. 217-226
-
Tidskriftsartikel (refereegranskat)abstract
- Retroviruses and long terminal repeat-containing retroelements use host-encoded tRNAs as primers for the synthesis of minus strong-stop DNA, the first intermediate in reverse transcription of the retroelement RNA. Usually, one or more specific tRNAs, including the primer, are selected and packaged within the virion. The reverse transcriptase (RT) interacts with the primer tRNA and initiates DNA synthesis. The structural and sequence features of primer tRNAs important for these specific interactions are poorly understood. We have developed a genetic assay in which mutants of tRNA(iMet), the primer for the Ty1 retrotransposon of Saccharomyces cerevisiae, can be tested for the ability to serve as primers in the reverse transcription process. This system allows any tRNA mutant to be tested, regardless of its ability to function in the initiation of protein synthesis. We find that mutations in the T psi C loop and the acceptor stem regions of the tRNA(iMet) affect transposition most severely. Conversely, mutations in the anticodon region have only minimal effects on transposition. Further study of the acceptor stem and other mutants demonstrates that complementarity to the element primer binding site is a necessary but not sufficient requirement for effective tRNA priming. Finally, we have used interspecies hybrid initiator tRNA molecules to implicate nucleotides in the D arm as additional recognition determinants. Ty3 and Ty1, two very distantly related retrotransposons, require similar molecular determinants in this primer tRNA for transposition.
|
|
| 8. |
- Nordlund, Monica E, et al.
(författare)
-
Identification of the TRM2 gene encoding the tRNA(m5U54)methyltransferase of Saccharomyces cerevisiae.
- 2000
-
Ingår i: RNA. - 1355-8382 .- 1469-9001. ; 6:6, s. 844-60
-
Tidskriftsartikel (refereegranskat)abstract
- The presence of 5-methyluridine (m5U) at position 54 is a ubiquitous feature of most bacterial and eukaryotic elongator tRNAs. In this study, we have identified and characterized the TRM2 gene that encodes the tRNA(m5U54)methyltransferase, responsible for the formation of this modified nucleoside in Saccharomyces cerevisiae. Transfer RNA isolated from TRM2-disrupted yeast strains does not contain the m5U54 nucleoside. Moreover, a glutathione S-transferase (GST) tagged recombinant, Trm2p, expressed in Escherichia coli displayed tRNA(m5U54)methyltransferase activity using as substrate tRNA isolated from a trm2 mutant strain, but not tRNA isolated from a TRM2 wild-type strain. In contrast to what is found for the tRNA(m5U54)methyltransferase encoding gene trmA+ in E. coli, the TRM2 gene is not essential for cell viability and a deletion strain shows no obvious phenotype. Surprisingly, we found that the TRM2 gene was previously identified as the RNC1/NUD1 gene, believed to encode the yNucR endo-exonuclease. The expression and activity of the yNucR endo-exonuclease is dependent on the RAD52 gene, and does not respond to increased gene dosage of the RNC1/NUD1 gene. In contrast, we find that the expression of a trm2-LacZ fusion and the activity of the tRNA(m5U54)methyltransferase is not regulated by the RAD52 gene and does respond on increased gene dosage of the TRM2 (RNC1/NUD1) gene. Furthermore, there was no nuclease activity associated with a GST-Trm2 recombinant protein. The purified yNucR endo-exonuclease has been reported to have an NH2-D-E-K-N-L motif, which is not found in the Trm2p. Therefore, we suggest that the yNucR endo-exonuclease is encoded by a gene other than TRM2.
|
|
| 9. |
- Nyberg, Patrik, et al.
(författare)
-
SpeB modulates fibronectin-dependent internalization of Streptococcus pyogenes by efficient proteolysis of cell-wall-anchored protein F1.
- 2004
-
Ingår i: Microbiology. - : Microbiology Society. - 1465-2080 .- 1350-0872. ; 150:Pt 5, s. 1559-1569
-
Tidskriftsartikel (refereegranskat)abstract
- SpeB is a cysteine proteinase and virulence determinant secreted by the important human pathogen Streptococcus pyogenes. Recent investigations have suggested a role for SpeB in streptococcal entry into human cells. However, conflicting data concerning the contribution of SpeB to internalization have been presented. Protein F1 is a cell-wall-attached fibronectin (Fn)-binding protein that is present in a majority of streptococcal isolates and is important for internalization. This study shows that protein F1 is efficiently degraded by SpeB, and that removal of protein F1 from the bacterial surface leads to reduced internalization. Whereas M1 protein and protein H, two additional surface proteins of S. pyogenes that bind human plasma proteins, are protected from proteolytic degradation by their ligands, protein F1 is readily cleaved by SpeB also when in complex with Fn. This finding, and the connection between the presence of Fn at the bacterial surface and entry into human cells, suggest that SpeB plays a role in the regulation of the internalization process.
|
|
| 10. |
- Okumura, Cheryl, et al.
(författare)
-
IgG protease Mac/IdeS is not essential for phagocyte resistance or mouse virulence of M1T1 group A Streptococcus
- 2013
-
Ingår i: mBio. - 2161-2129 .- 2150-7511. ; 4:4, s. e00499-e00513
-
Tidskriftsartikel (refereegranskat)abstract
- The Mac/IdeS protein of group A Streptococcus (GAS) is a secreted cysteine protease with cleavage specificity for IgG and is highly expressed in the GAS serotype M1T1 clone, which is the serotype most frequently isolated from patients with life-threatening invasive infections. While studies of Mac/IdeS with recombinant protein have shown that the protein can potentially prevent opsonophagocytosis of GAS by neutrophils, the role of the protein in immune evasion as physiologically produced by the living organism has not been studied. Here we examined the contribution of Mac/IdeS to invasive GAS disease by generating a mutant lacking Mac/IdeS in the hyperinvasive M1T1 background. While Mac/IdeS was highly expressed and proteolytically active in the hyperinvasive strain, elimination of the bacterial protease did not significantly influence GAS phagocytic uptake, oxidative-burst induction, cathelicidin sensitivity, resistance to neutrophil or macrophage killing, or pathogenicity in pre- or postimmune mouse infectious challenges. We conclude that in the highly virulent M1T1 background, Mac/IdeS is not essential for either phagocyte resistance or virulence. Given the conservation of Mac/IdeS and homologues across GAS strains, it is possible that Mac/IdeS serves another important function in GAS ecology or contributes to virulence in other strain backgrounds.IMPORTANCE Group A Streptococcus (GAS) causes human infections ranging from strep throat to life-threatening conditions such as flesh-eating disease and toxic shock syndrome. Common disease-associated clones of GAS can cause both mild and severe infections because of a characteristic mutation and subsequent change in the expression of several genes that develops under host immune selection. One of these genes encodes Mac/IdeS, a protease that has been shown to cleave antibodies important to the immune defense system. In this study, we found that while Mac/IdeS is highly expressed in hypervirulent GAS, it does not significantly contribute to the ability of the bacteria to survive white blood cell killing or produce invasive infection in the mouse. These data underscore the importance of correlating studies on virulence factor function with physiologic expression levels and the complexity of streptococcal pathogenesis and contribute to our overall understanding of how GAS causes disease.
|
|
| 11. |
|
|
| 12. |
- Persson, Helena, et al.
(författare)
-
Proteolytic processing of the streptococcal IgG endopeptidase IdeS modulates the functional properties of the enzyme and results in reduced immunorecognition
- 2015
-
Ingår i: Molecular Immunology. - : Elsevier. - 0161-5890 .- 1872-9142. ; 68:2, s. 176-184
-
Tidskriftsartikel (refereegranskat)abstract
- The important human gram positive bacterial pathogen Streptococcus pyogenes employs various virulence factors to promote inflammation and to facilitate invasive disease progression. In this study we explored the relation of the secreted streptococcal cysteine proteases IdeS and SpeB, and neutrophil (PMN) proteases. We found that SpeB is resistant to proteolytic attack in an inflammatory environment, emphasizing the importance of SpeB for streptococcal pathogenicity, while PMN enzymes and SpeB itself process the IgG degrading endopeptidase IdeS. Processing occurs as NH2-terminal cleavage of IdeS resulting in reduced immunorecognition of the protease by specific antibodies. While the endopeptidase retains IgG cleaving activity, its ability to suppress the generation of reactive oxygen species is abolished. We suggest that the cleavage of NH2-terminal peptides by SpeB and/or neutrophil proteases is a mechanism evolved to prevent early inactivation of this important streptococcal virulence factor, albeit at the cost of impaired functionality.
|
|
| 13. |
- Persson, Helena, et al.
(författare)
-
The streptococcal cysteine protease SpeB is not a natural immunoglobulin-cleaving enzyme
- 2013
-
Ingår i: Infection and Immunity. - 0019-9567 .- 1098-5522. ; 81:6, s. 2236-2241
-
Tidskriftsartikel (refereegranskat)abstract
- The human bacterial pathogen Streptococcus pyogenes has developed a broad variety of virulence mechanisms to evade the actions of the host immune defense. One of the best-characterized factors is the streptococcal cysteine protease SpeB, an important multifunctional protease that contributes to group A streptococcal pathogenesis in vivo. Among many suggested activities, SpeB has been described to degrade various human plasma proteins, including immunoglobulins (Igs). In this study, we show that SpeB has no Ig-cleaving activity under physiological conditions and that only Igs in a reduced state, i.e., semimonomeric molecules, are cleaved and degraded by SpeB. Since reducing conditions outside eukaryotic cells have to be considered nonphysiological and IgG in a reduced state lacks biological effector functions, we conclude that SpeB does not contribute to S. pyogenes virulence through the proteolytic degradation of Igs.
|
|
| 14. |
- Rungelrath, Viktoria, et al.
(författare)
-
Identification of a novel host-specific IgG protease in Streptococcus phocae subsp phocae
- 2017
-
Ingår i: Veterinary Microbiology. - : Elsevier. - 0378-1135 .- 1873-2542. ; 201, s. 42-48
-
Tidskriftsartikel (refereegranskat)abstract
- Streptococcus (S.) phocae subsp. phocae causes bronchopneumonia and septicemia in a variety of marine mammals. Especially in harbor seals infected with phocine distemper virus it plays an important role as an opportunistic pathogen. This study was initiated by the detection of IgG cleavage products in Western blot analysis after incubation of bacterial supernatant with harbor seal serum. Hence, the objectives of this study were the identification and characterization of a secreted IgG cleaving protease in S. phocae subsp. phocae isolated from marine mammals. To further identify the responsible factor of IgG cleavage a protease inhibitor profile was generated. Inhibition of the IgG cleaving activity by iodoacetamide and Z-LVG-CHN2 indicated that a cysteine protease is involved. Moreover, an anti-IdeS antibody directed against the IgG endopeptidase IdeS of S. pyogenes showed cross reactivity with the putative IgG protease of S. phocae subsp. phocae. The IgG cleaving factor of S. phocae subsp. phocae was identified through an inverse PCR approach and designated IdeP (Immunoglobulin G degrading enzyme of S. phocae subsp. phocae) in analogy to the cysteine protease IdeS. Notably, recombinant (r) IdeP is a host and substrate specific protease as it cleaves IgG from grey and harbor seals but not IgG from harbor porpoises or non-marine mammals. The identification of IdeP represents the first description of a protein in S. phocae subsp. phocae involved in immune evasion. Furthermore, the fact that IdeP cleaves solely IgG of certain marine mammals reflects functional adaption of S. phocae subsp. phocae to grey and harbor seals as its main hosts.
|
|
| 15. |
- Seele, J., et al.
(författare)
-
Identification of a novel host-specific IgM protease in Streptococcus suis
- 2013
-
Ingår i: International Journal of Medical Microbiology. - : Elsevier. - 1438-4221 .- 1618-0607. ; 303:Suppl. 1, MPP35, s. 58-58
-
Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Streptococcus (S.) suis is an important invasive, extracellular pathogen in pigs, which causes meningitis, arthritis, serositis and other diseases. Furthermore, it is also an emerging zoonotic agent. This study was initiated by the finding that IgM degradation products are released after opsonization of S. suis with porcine serum. The objective of this work was to identify and characterize the factor responsible for IgM cleavage. The results showed that a protein of S. suis with high homology to the well characterized IgG endopeptidase of S. pyogenes IdeS (or Mac1) [1, 2], designated IdeSsuis, degrades immunoglobulins (Ig) of the isotype M, but not IgG, IgA or other proteins present in porcine cerebrospinal fluid, joint fluid or serum. Western Blot analysis revealed that IdeSsuis is host-specific as it exclusively cleaves porcine IgM but not IgM from six other species. Flow cytometry and immunofluorescence microscopy demonstrated that this protein modulates binding of IgM to the bacterial surface. Furthermore the isogenic ideSsuis deletion mutant is significantly attenuated in survival in porcine blood [3]. IdeSsuis is the first prokaryotic IgM-specific protease described indicating a novel host-pathogen interaction at an early stage of the host immune response. Furthermore cleavage of porcine IgM by IdeSsuis is the first identified phenotype reflecting functional adaptation of S. suis to pigs as the main host.
|
|
| 16. |
- Seele, Jana, et al.
(författare)
-
Identification of a Novel Host-Specific IgM Protease in Streptococcus suis
- 2013
-
Ingår i: Journal of Bacteriology. - Washington : American society of microbiology. - 0021-9193 .- 1098-5530. ; 195:5, s. 930-940
-
Tidskriftsartikel (refereegranskat)abstract
- Streptococcus suis serotype 2 is a highly invasive, extracellular pathogen in pigs with the capacity to cause severe infections in humans. This study was initiated by the finding that IgM degradation products are released after opsonization of S. suis. The objective of this work was to identify the bacterial factor responsible for IgM degradation. The results of this study showed that a member of the IdeS family, designated Ide(Ssuis) (Immunoglobulin M-degrading enzyme of S. suis), is responsible and sufficient for IgM cleavage. Recombinant Ide(Ssuis) was found to degrade only IgM but neither IgG nor IgA. Interestingly, Western blot analysis revealed that Ide(Ssuis) is host specific, as it exclusively cleaves porcine IgM but not IgM from six other species, including a closely related member of the Suidae family. As demonstrated by flow cytometry and immunofluorescence microscopy, Ide(Ssuis) modulates binding of IgM to the bacterial surface. Ide(Ssuis) is the first prokaryotic IgM-specific protease described, indicating that this enzyme is involved in a so-far-unknown mechanism of host-pathogen interaction at an early stage of the host immune response. Furthermore, cleavage of porcine IgM by Ide(Ssuis) is the first identified phenotype reflecting functional adaptation of S. suis to pigs as the main host.
|
|
| 17. |
- Seele, Jana, et al.
(författare)
-
The immunoglobulin M-degrading enzyme of Streptococcus suis, Ide(Ssuis), is a highly protective antigen against serotype 2
- 2015
-
Ingår i: Vaccine. - : Elsevier BV. - 0264-410X .- 1873-2518. ; 33:19, s. 2207-2212
-
Tidskriftsartikel (refereegranskat)abstract
- Streptococcus suis (S. suis) is a major porcine pathogen causing meningitis, arthritis and several other pathologies. Recently, we identified a highly specific immunoglobulin M degrading enzyme of S. suis, designated IdeSsuis, which is expressed by various serotypes. The objective of this work was to access the immunogenicity and protective efficacy of a recombinant vaccine including IdeSsuis. Vaccination with rIdeSsuis elicited antibodies efficiently neutralizing the IgM protease activity. Importantly, 18 piglets vaccinated with rIdeSsuis alone or in combination with bacterin priming were completely protected against mortality and severe morbidity after S. suis serotype 2 challenge. In contrast, 12 of the 17 piglets either treated with the placebo or primed with the bacterin only, succumbed to S. suis disease. Immunity against Idessuis was associated with increased killing of S. suis wt in porcine blood ex vivo leading to a tenfold difference in the bacterial survival factor in blood of placebo-treated and rIdeSsuis-vaccinated piglets. In conclusion, the results of this study indicate that rIdeSsuis is a highly protective antigen in pigs.
|
|
| 18. |
- Seele, Jana, et al.
(författare)
-
The immunoglobulin M-degrading enzyme of Streptococcus suis, Ide(Ssuis), is involved in complement evasion
- 2015
-
Ingår i: Veterinary research (Print). - : Springer Science and Business Media LLC. - 0928-4249 .- 1297-9716. ; 46
-
Tidskriftsartikel (refereegranskat)abstract
- Streptococcus (S.) suis is one of the most important pathogens in pigs causing meningitis, arthritis, endocarditis and serositis. Furthermore, it is also an emerging zoonotic agent. In our previous work we identified a highly specific IgM protease in S. suis, designated IdeSsuis. The objective of this study was to characterize the function of IdeSsuis in the host-pathogen interaction. Edman-sequencing revealed that Ide(Ssuis) cleaves the heavy chain of the IgM molecule between constant domain 2 and 3. As the C1q binding motif is located in the C3 domain, we hypothesized that IdeSsuis is involved in complement evasion. Complement-mediated hemolysis induced by porcine hyperimmune sera containing erythrocyte-specific IgM was abrogated by treatment of these sera with recombinant IdeSsuis. Furthermore, expression of IdeSsuis reduced IgM-triggered complement deposition on the bacterial surface. An infection experiment of prime-vaccinated growing piglets suggested attenuation in the virulence of the mutant 10 Delta IdeSsuis. Bactericidal assays confirmed a positive effect of IdeSsuis expression on bacterial survival in porcine blood in the presence of high titers of specific IgM. In conclusion, this study demonstrates that IdeSsuis is a novel complement evasion factor, which is important for bacterial survival in porcine blood during the early adaptive (IgM-dominated) immune response.
|
|
| 19. |
- Spoerry, Christian, et al.
(författare)
-
Identification and Characterization of IgdE, a Novel IgG-degrading Protease of Streptococcus suis with Unique Specificity for Porcine IgG.
- 2016
-
Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 291:15, s. 7915-7925
-
Tidskriftsartikel (refereegranskat)abstract
- Streptococcus suis is a major endemic pathogen of pigs causing meningitis, arthritis, and other diseases. Zoonotic S. suis infections are emerging in humans causing similar pathologies as well as severe conditions such as toxic shock-like syndrome. Recently, we discovered an IdeS family protease of S. suis that exclusively cleaves porcine IgM and represents the first virulence factor described, linking S. suis to pigs as their natural host. Here we report the identification and characterization of a novel, unrelated protease of S. suis that exclusively targets porcine IgG. This enzyme, designated IgdE for immunoglobulin G-degrading enzyme of S. suis, is a cysteine protease distinct from previous characterized streptococcal immunoglobulin degrading proteases of the IdeS family and mediates efficient cleavage of the hinge region of porcine IgG with a high degree of specificity. The findings that all S. suis strains investigated possess the IgG proteolytic activity and that piglet serum samples contain specific antibodies against IgdE strongly indicate that the protease is expressed in vivo during infection and represents a novel and putative important bacterial virulence/colonization determinant, and a thus potential therapeutic target.
|
|
| 20. |
- Spoerry, Christian, et al.
(författare)
-
Novel IgG-Degrading Enzymes of the IgdE Protease Family Link Substrate Specificity to Host Tropism of Streptococcus Species.
- 2016
-
Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 11:10
-
Tidskriftsartikel (refereegranskat)abstract
- Recently we have discovered an IgG degrading enzyme of the endemic pig pathogen S. suis designated IgdE that is highly specific for porcine IgG. This protease is the founding member of a novel cysteine protease family assigned C113 in the MEROPS peptidase database. Bioinformatical analyses revealed putative members of the IgdE protease family in eight other Streptococcus species. The genes of the putative IgdE family proteases of S. agalactiae, S. porcinus, S. pseudoporcinus and S. equi subsp. zooepidemicus were cloned for production of recombinant protein into expression vectors. Recombinant proteins of all four IgdE family proteases were proteolytically active against IgG of the respective Streptococcus species hosts, but not against IgG from other tested species or other classes of immunoglobulins, thereby linking the substrate specificity to the known host tropism. The novel IgdE family proteases of S. agalactiae, S. pseudoporcinus and S. equi showed IgG subtype specificity, i.e. IgdE from S. agalactiae and S. pseudoporcinus cleaved human IgG1, while IgdE from S. equi was subtype specific for equine IgG7. Porcine IgG subtype specificities of the IgdE family proteases of S. porcinus and S. pseudoporcinus remain to be determined. Cleavage of porcine IgG by IgdE of S. pseudoporcinus is suggested to be an evolutionary remaining activity reflecting ancestry of the human pathogen to the porcine pathogen S. porcinus. The IgG subtype specificity of bacterial proteases indicates the special importance of these IgG subtypes in counteracting infection or colonization and opportunistic streptococci neutralize such antibodies through expression of IgdE family proteases as putative immune evasion factors. We suggest that IgdE family proteases might be valid vaccine targets against streptococci of both human and veterinary medical concerns and could also be of therapeutic as well as biotechnological use.
|
|
| 21. |
- Spoerry, Christian, 1985-
(författare)
-
Streptococcal immunoglobulin degrading enzymes of the IdeS and IgdE family
- 2017
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Bacteria of the genus Streptococcus are common asymptomatic colonisers of humans and animals. As opportunistic pathogens they can however, depending on their host’s immune status and other circumstances, cause mild to very severe infections. Streptococci are highly intertwined with specific host species, but can also cause zoonosis or anthroponosis in more uncommon hosts. Prolonged and reoccurring infections require immune evasion strategies to circumvent detection and eradication by the host’s immune defence. A substantial part of the immune defence against bacterial pathogens is mediated by immunoglobulins. This thesis is based on work to identify and characterise immunoglobulin degrading enzymes secreted by different Streptococcus species as a means to sabotage and evade antibody-mediated immune responses.Stoichiometric and kinetic analysis of the IgG degrading enzyme IdeS from the important human pathogen S. pyogenes revealed that IdeS cleaves IgG, opposed to previous publications, as a monomer following classical Michaelis-Menten kinetics.The IdeS homologue of S. suis, IdeSsuis, did however not cleave IgG, but was highly specific fo rporcine IgM. S. suis was found to possess yet another protease, IgdE, capable of cleaving porcine IgG. Both of these proteases were shown to promote increased bacterial survival in porcine blood during certain conditions.IgdE is the founding member of a novel cysteine protease family (C113). Novel streptococcal members of this protease family were shown to specifically degrade certain IgG subtypes of the respective Streptococcus species’ main host. The observed substrate specificity of IgdE family proteases reflects the host tropism of these Streptococcus species, thereby giving insights into host-pathogen co-evolution.The abundance of immunoglobulin degrading enzymes among Streptococcus species indicates the importance of evasion from the antibody mediated immune responses for streptococci. These novel identified immunoglobulin degrading enzymes of the IdeS and IgdE protease families are potential valid vaccine targets and could also be of biotechnological use.
|
|
| 22. |
- Söderberg, Jenny Johansson, et al.
(författare)
-
The intrinsic immunoglobulin g endopeptidase activity of streptococcal Mac-2 proteins implies a unique role for the enzymatically impaired Mac-2 protein of M28 serotype strains
- 2008
-
Ingår i: Infection and Immunity. - 0019-9567 .- 1098-5522. ; 76:5, s. 2183-2188
-
Tidskriftsartikel (refereegranskat)abstract
- IdeS, a secreted cysteine protease of the important human pathogen Streptococcus pyogenes, interferes with phagocytic killing by specifically cleaving the heavy chain of immunoglobulin G (IgG). Two allelic variants of the enzyme have been described, the IgG-specific endopeptidase, IdeS (or Mac-1) and Mac-2, a protein with only weak IgG endopeptidase activity, which has been suggested to interfere with opsonophagocytosis by blocking Fcgamma receptors of phagocytic cells. However, despite the fact that Mac-2 proteins interact with Fcgamma receptors, no inhibition of reactive oxygen species (ROS) production, opsonophagocytosis, or streptococcal killing by Mac-2 has been reported. In the present study, Mac-2 proteins are shown to contain IgG endopeptidase activity indistinguishable from the enzymatic activity exhibited by IdeS/Mac-1 proteins. The earlier reported weak IgG endopeptidase activity appears to be unique to Mac-2 of M28 serotype strains (Mac-2(M28)) and is most likely due to the formation of a disulfide bond between the catalytic site cysteine and a cysteine residue in position 257 of Mac-2(M28). Furthermore, Mac-2 proteins are shown to inhibit ROS production ex vivo, independently of the IgG endopeptidase activity of the proteins. Inhibition of ROS generation per se, however, was not sufficient to mediate streptococcal survival in bactericidal assays. Thus, in contrast to earlier studies, implicating separate functions for IdeS and Mac-2 protein variants, the current study suggests that Mac-2 and IdeS are bifunctional proteins, combining Fcgamma receptor binding and IgG endopeptidase activity. This finding implies a unique role for Mac-2 proteins of the M28 serotype, since this serotype has evolved and retained a Mac-2 protein lacking IgG endopeptidase activity.
|
|
| 23. |
- Söderberg, Jenny Johansson, et al.
(författare)
-
The streptococcal protease IdeS modulates bacterial IgGFc binding and generates 1/2Fc fragments with the ability to prime polymorphonuclear leucocytes
- 2008
-
Ingår i: Molecular Immunology. - : Elsevier BV. - 0161-5890 .- 1872-9142. ; 45:12, s. 3347-3353
-
Tidskriftsartikel (refereegranskat)abstract
- The important human bacterial pathogen Streptococcus pyogenes has evolved a variety of mechanisms to evade the actions of the human immune system. M protein and M-like proteins are major virulence factors that bind with high affinity to the Fc-part of IgG. However, the contribution of non-immune binding of IgG to bacterial virulence is not fully established. Importantly, the capacity of S. pyogenes to bind IgG is limited and due to the presence of large amounts of IgG present in vivo, the majority of IgGFc binding sites at the streptococcal surface are likely to be occupied by non-specific IgG. S. pyogenes also secretes a highly effective IgG-endopeptidase, IdeS that inhibits phagocytic killing by cleavage of specific IgG creating F(ab')2 and 1/2Fc fragments. In the present work, IgG and 1/2Fc binding to the streptococcal surface was studied and correlated to IdeS activity. Binding of IgG to the streptococcal surface is shown to be equilibrium and thus not designed to mediate a lasting protection against specific antibodies. However, non-immune binding of IgG to the bacterial surface is followed by the proteolytic cleavage of the antibody by the IgG-endopeptidase IdeS. IdeS generated 1/2Fc fragments do not compete efficiently with intact IgG in binding to the bacterial surface and rapid dissociation of 1/2Fc allows binding of new IgG. Thus, a correlated binding and proteolytic cleavage of IgG also increases the probability that the bacteria can resist specific IgG, despite the presence of a large excess of non-specific IgG in the circulation. As a consequence of IdeS activity, circulating 1/2Fc fragments are generated. These 1/2Fc fragments were shown to be biological active by acting as priming agents for polymorphonuclear leucocytes, suggesting a new mechanism of immune evasion employed by S. pyogenes.
|
|
| 24. |
- Vincents, Bjarne, et al.
(författare)
-
Cleavage of IgG(1) and IgG(3) by gingipain K from Porphyromonas gingivalis may compromise host defense in progressive periodontitis
- 2011
-
Ingår i: The FASEB Journal. - Bethesda, Md. : Federation of American Societies for Experimental Biology. - 0892-6638 .- 1530-6860. ; 25:10, s. 3741-3750
-
Tidskriftsartikel (refereegranskat)abstract
- Degradation of immunoglobulins is an effective strategy of bacteria to evade the immune system. We have tested whether human IgG is a substrate for gingipain K of Porphyromonas gingivalis and found that the enzyme can hydrolyze subclass 1 and 3 of human IgG. The heavy chain of IgG(1) was cleaved at a single site within the hinge region, generating Fab and Fc fragments. IgG(3) was also cleaved within the heavy chain, but at several sites around the CH(2) region. Investigation of the enzyme kinetics of IgG proteolysis by gingipain K, using FPLC- and isothermal titration calorimetry-based assays followed by Hill plots, revealed non-Michaelis-Menten kinetics involving a mechanism of positive cooperativity. In ex vivo studies, it was shown that gingipain K retained its IgG hydrolyzing activity in human plasma despite the high content of natural protease inhibitors; that IgG(1) cleavage products were detected in gingival crevicular fluid samples from patients with severe periodontitis; and that gingipain K treatment of serum samples from patients with high antibody titers against P. gingivalis significantly hindered opsonin-dependent phagocytosis of clinical isolates of P. gingivalis by neutrophils. Altogether, these findings underline a biological function of gingipain K as an IgG protease of pathophysiological importance.-Vincents, B., Guentsch, A., Kostolowska, D., von Pawel-Rammingen, U., Eick, S., Potempa, J., Abrahamson, M. Cleavage of IgG(1) and IgG(3) by gingipain K from Porphyromonas gingivalis may compromise host defense in progressive periodontitis. FASEB J. 25, 3741-3750 (2011). www.fasebj.org
|
|
| 25. |
- Vincents, Bjarne, et al.
(författare)
-
Enzymatic characterization of the streptococcal endopeptidase, IdeS, reveals that it is a cysteine protease with strict specificity for IgG cleavage due to exosite binding.
- 2004
-
Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 43:49, s. 15540-15549
-
Tidskriftsartikel (refereegranskat)abstract
- Streptococcus pyogenes, an important pathogen in humans, secretes an IgG specific endopeptidase named IdeS. To elucidate the mechanism that is responsible for this specificity, we have here characterized the activity of IdeS in detail. Both gamma chains of human IgG or its Fc fragment were cleaved in the hinge region after Gly236 by IdeS, but other proteins or synthetic peptides containing sequences such as the P(4)-P(1) segment in the IgG cleavage site, or long peptides resembling the IgG hinge, were not hydrolyzed at all. This is likely due to a second binding site interacting with the Fc part of IgG. The lack of IdeS activity on peptide substrates necessitated the development of an assay with IgG as the substrate for kinetic studies. IdeS showed a sigmoidal velocity curve at physiological IgG concentrations, and a declining enzyme rate at higher IgG concentrations. This atypical velocity curve suggests product inhibition and/or allosteric control, which again indicates the presence of an exosite involved in substrate binding. The pseudoequilibrium constant for IdeS hydrolysis of IgG was 90 microM. The enzyme exhibited activity in the pH range of 5.1-7.6, with an optimum at pH 6.6. IdeS was stable above pH 10 but not at acidic pH. It exhibited an activity maximum around 37 degrees C and a decreased thermal stability at 42 degrees C. Iodoacetate and iodoacetamide inhibited IdeS, as expected for a cysteine protease, and biochemical evidence verified this classification. E-64 and chicken cystatin, specific inhibitors of family C1 and C13 cysteine proteases, were without effect on enzyme activity, as were class specific serine, aspartic, and metallo protease inhibitors. No significant similarities were found in protein sequence comparisons with known enzyme families, suggesting that IdeS represents a novel family of cysteine proteases.
|
|
| 26. |
- Vincents, Bjarne, et al.
(författare)
-
The human protease inhibitor cystatin C is an activating cofactor for the streptococcal cysteine protease IdeS
- 2008
-
Ingår i: Chemistry and Biology. - : Elsevier BV. - 1074-5521 .- 1879-1301. ; 15:9, s. 960-968
-
Tidskriftsartikel (refereegranskat)abstract
- Human cystatin C is considered the physiologically most important inhibitor of endogenous papain-like cysteine proteases. We present here an unexpected function of cystatin C. Instead of acting as an inhibitor, cystatin C acts as a facultative, endogenous cofactor for the papain-like IgG-cleaving enzyme IdeS of the human pathogen Streptococcus pyogenes. IdeS activity is not dependent on cystatin C, but is significantly enhanced in the presence of cystatin C. We report a protease inhibitor that accelerates the activity of its putative target protease and a unique example of how a host protease inhibitor is "hijacked" by a bacterial protease to increase its activity. This finding has important implications for the view on protease-inhibitor interactions, and is relevant to consider in the therapeutic use of protease inhibitors.
|
|
| 27. |
|
|
| 28. |
- Vindebro, Reine, et al.
(författare)
-
Rapid IgG heavy chain cleavage by the streptococcal IgG endopeptidase IdeS is mediated by IdeS monomers and is not due to enzyme dimerization
- 2013
-
Ingår i: FEBS Letters. - : Elsevier. - 0014-5793 .- 1873-3468. ; 587:12, s. 1818-1822
-
Tidskriftsartikel (refereegranskat)abstract
- Streptococcus pyogenes employs an IgG specific endopeptidase, IdeS, to counteract the effector functions of specific IgG. The physiological significant step in disarming specific IgG is the cleavage of one IgG heavy chain. So far, characterizations of IdeS enzymatic activity have employed techniques that failed to differentiate between the first and the second cleavage step. The present data demonstrate that IdeS is active as a monomer and that IdeS activity follows classical Michaelis-Menten kinetics arguing against the previously proposed formation of a functional IdeS dimer. Our results show that IdeS inactivates IgG 100-fold faster than previously reported. (C) 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
|
|
| 29. |
- Vindebro, Reine, 1983-
(författare)
-
Studies on secreted cysteine proteases of Streptococcus pyogenes : IdeS and SpeB
- 2014
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The pathogen Streptococcus pyogenes is a significant cause of human morbidity and mortality. Most of the work in this thesis is focused on streptococcal virulence factor IdeS, but the thesis also features work on SpeB, another streptococcal virulence factor. Both IdeS and SpeB are secreted cysteine proteases and both have previously been shown to degrade human IgG. IgG is the only known substrate for IdeS while SpeB is a more promiscuous protease with a larger number of identified substrates. A significant part of the data presented in this thesis is the result of designing and optimizing methods to detect and accurately measure the proteolytic degradation of IgG. Methods aimed at measuring the binding interactions between enzyme and substrate have also been frequently utilized. I show that IdeS is a monomeric protease, as opposed to previously published data that suggested it to be dimeric. IdeS cleaves the two heavy chains of IgG in a two-step reaction and I demonstrate that the first cleavage is magnitudes faster than the second one. This means that IdeS is a more efficient enzyme than previously thought. The difference in rate cannot completely be explained by a loss of affinity between IdeS and IgG after the cleavage of the first heavy chain. The velocity of IdeS is further increased by the presence of human Cystatin C, via an unknown mechanism. Cystatin C is normally a protease inhibitor and it having an opposite effect is puzzling.The synthesis and evaluation of novel inhibitors are also described. Peptide analogues mimicking the sequence surrounding the scissile bond on IgG - with an amino acid replaced with a more rigid motif - act as specific, but low-affinity, inhibitors of IdeS. The peptide analogues’ inhibitory capacity for SpeB and papain was also assayed.When it comes to SpeB, I show that it does not have IgG as a substrate under physiological conditions, in contrast to what was previously thought. This thesis does not only present findings on the IgG degrading capacity of IdeS and SpeB but also include data on fundamental enzymatic properties for these proteases.
|
|
| 30. |
- Von Pawel-Rammingen, Ulrich, et al.
(författare)
-
GAP activity of the Yersinia YopE cytotoxin specifically targets the Rho pathway : a mechanism for disruption of actin microfilament structure.
- 2000
-
Ingår i: Molecular Microbiology. - : Wiley. - 0950-382X .- 1365-2958. ; 36:3, s. 737-748
-
Tidskriftsartikel (refereegranskat)abstract
- The YopE cytotoxin of Yersinia pseudotuberculosis is an essential virulence determinant that is injected into the eukaryotic target cell via a plasmid-encoded type III secretion system. Injection of YopE into eukaryotic cells induces depolymerization of actin stress fibres. Here, we show that YopE exhibits a GTPase-activating protein (GAP) activity and that the presence of YopE stimulates downregulation of Rho, Rac and Cdc42 activity. YopE has an arginine finger motif showing homology with those found in other GAP proteins. Exchange of arginine 144 with alanine, located in this arginine finger motif, results in an inactive form of YopE that can no longer stimulate GTP hydrolysis by the GTPase. Furthermore, a yopE(R144A) mutant is unable to induce cytotoxicity on cultured HeLa cells in contrast to the corresponding wild-type strain. Expression of wild-type YopE in cells of Saccharomyces cerevisiae inhibits growth, while in contrast, expression of the inactive form of YopE, YopE(R144A), does not affect the yeast cells. Co-expression of proteins belonging to the Rho1 pathway of yeast, Rho1, Rom2p, Bck1 and Ste20, suppressed the growth phenotype of YopE in yeast cells. These results provide evidence that YopE exhibits a GAP activity to inactivate RhoGTPases, leading to depolymerization of the actin stress fibres in eukaryotic cells and growth inhibition in yeast.
|
|
| 31. |
- von Pawel-Rammingen, Ulrich, et al.
(författare)
-
IdeS, a novel streptococcal cysteine proteinase with unique specificity for immunoglobulin G.
- 2002
-
Ingår i: EMBO Journal. - : Wiley. - 1460-2075. ; 21:7, s. 1607-1615
-
Tidskriftsartikel (refereegranskat)abstract
- Recent work from several laboratories has demonstrated that proteolytic mechanisms significantly contribute to the molecular interplay between Streptococcus pyogenes, an important human pathogen, and its host. Here we describe the identification, purification and characterization of a novel extracellular cysteine proteinase produced by S.pyogenes. This enzyme, designated IdeS for Immunoglobulin G-degrading enzyme of S.pyogenes, is distinct from the well-characterized streptococcal cysteine proteinase, SpeB, and cleaves human IgG in the hinge region with a high degree of specificity. Thus, other human proteins, including immunoglobulins M, A, D and E, are not degraded by IdeS. The enzyme efficiently cleaves IgG antibodies bound to streptococcal surface structures, thereby inhibiting the killing of S.pyogenes by phagocytic cells. This and additional observations on the distribution and expression of the ideS gene indicate that IdeS represents a novel and significant bacterial virulence determinant, and a potential therapeutic target.
|
|
| 32. |
- von Pawel-Rammingen, Ulrich, et al.
(författare)
-
IdeS and SpeB: immunoglobulin-degrading cysteine proteinases of Streptococcus pyogenes.
- 2003
-
Ingår i: Current Opinion in Microbiology. - 1879-0364. ; 6:1, s. 50-55
-
Forskningsöversikt (refereegranskat)abstract
- The Gram-positive bacterium Streptococcus pyogenes is a major human pathogen causing substantial morbidity and mortality in society. S. pyogenes has evolved numerous molecular mechanisms to avoid the various actions of the human immune system and has established means to modulate both adaptive and innate immune responses. S. pyogenes produces and secretes proteolytic enzymes, which have an important impact on the ability of the bacteria to survive in the human host. Prominent among these are two immunoglobulin-degrading enzymes: the newly discovered streptococcal cysteine proteinase, IdeS, and the classical cysteine proteinase of S. pyogenes, SpeB.
|
|
| 33. |
- von Pawel-Rammingen, Ulrich
(författare)
-
IdeS Peptidase
- 2013
-
Ingår i: Handbook of proteolytic enzymes. - : Academic Press. - 9780123822192 ; , s. 2175-2178
-
Bokkapitel (refereegranskat)
|
|
| 34. |
- von Pawel-Rammingen, Ulrich, et al.
(författare)
-
Mutational analysis of conserved positions potentially important for initiator tRNA function in Saccharomyces cerevisiae
- 1992
-
Ingår i: Molecular and Cellular Biology. - : American Society for Microbiology. - 0270-7306 .- 1098-5549. ; 12:4, s. 1432-1442
-
Tidskriftsartikel (refereegranskat)abstract
- The conserved positions of the eukaryotic cytoplasmic initiator tRNA have been suggested to be important for the initiation of protein synthesis. However, the role of these positions is not known. We describe in this report a functional analysis of the yeast initiator methionine tRNA (tRNA(iMet)), using a novel in vivo assay system which is not dependent on suppressor tRNAs. Strains of Saccharomyces cerevisiae with null alleles of the four initiator methionine tRNA (IMT) genes were constructed. Consequently, growth of these strains was dependent on tRNA(iMet) encoded from a plasmid-derived gene. We used these strains to investigate the significance of the conserved nucleosides of yeast tRNA(iMet) in vivo. Nucleotide substitutions corresponding to the nucleosides of the yeast elongator methionine tRNA (tRNA(MMet)) have been made at all conserved positions to identify the positions that are important for tRNA(iMet) to function in the initiation process. Surprisingly, nucleoside changes in base pairs 3-70, 12-23, 31-39, and 29-41, as well as expanding loop I by inserting an A at position 17 (A17) had no effect on the tester strain. Nucleotide substitutions in positions 54 and 60 to cytidines and guanosines (C54, G54, C60, and G60) did not prevent cell growth. In contrast, the double mutation U/rT54C60 blocked cell growth, and changing the A-U base pair 1-72 to a G-C base pair was deleterious to the cell, although these tRNAs were synthesized and accepted methionine in vitro. From our data, we suggest that an A-U base pair in position 1-72 is important for tRNA(iMet) function, that the hypothetical requirement for adenosines at positions 54 and 60 is invalid, and that a U/rT at position 54 is an antideterminant distinguishing an elongator from an initiator tRNA in the initiation of translation.
|
|
| 35. |
- von Pawel-Rammingen, Ulrich
(författare)
-
Streptococcal IdeS and Its Impact on Immune Response and Inflammation
- 2012
-
Ingår i: JOURNAL OF INNATE IMMUNITY. - : S. Karger AG. - 1662-811X .- 1662-8128. ; 4:2, s. 132-140
-
Tidskriftsartikel (refereegranskat)abstract
- Survival of the important bacterial pathogen Streptococcus pyogenes relies on its ability to circumvent the antimicrobial actions of innate and specific immune responses and to modulate the inflammatory responses induced during the course of an infection. Inflammatory processes play key roles during streptococcal pathogenesis and streptococcal infections are accompanied by an intense inflammatory state. As an exclusively human pathogen, S. pyogenes has adapted to the various countermeasures employed by its host to fight bacterial infections, in particular to interfere with the effector functions of immunoglobulin G (IgG). For this purpose, S. pyogenes has evolved an IgG-specific endopeptidase, IdeS, which is highly specific for the lower hinge region of IgG. This review summarizes the current knowledge about this intriguing enzyme as well as its role in inflammation and in the attenuation of human immune responses towards streptococcal infection.
|
|
| 36. |
- von Pawel-Rammingen, Ulrich, et al.
(författare)
-
Streptococcus pyogenes and phagocytic killing.
- 2002
-
Ingår i: Nature Medicine. - : Springer Science and Business Media LLC. - 1546-170X .- 1078-8956. ; 8:10, s. 1043-1044
-
Tidskriftsartikel (refereegranskat)
|
|
| 37. |
- Wenig, K, et al.
(författare)
-
Structure of the streptococcal endopeptidase IdeS, a cysteine proteinase with strict specificity for IgG
- 2004
-
Ingår i: Proceedings of the National Academy of Sciences. - : Proceedings of the National Academy of Sciences. - 1091-6490 .- 0027-8424. ; 101:50, s. 17371-17376
-
Tidskriftsartikel (refereegranskat)abstract
- Pathogenic bacteria have developed complex and diverse virulence mechanisms that weaken or disable the host immune defense system. IdeS (IgG-degrading enzyme of Streptococcus pyogenes) is a secreted cysteine endopeptidase from the human pathogen S. pyogenes with an extraordinarily high degree of substrate specificity, catalyzing a single proteolytic cleavage at the lower hinge of human IgG. This proteolytic degradation promotes inhibition of opsonophagocytosis and interferes with the killing of group A Streptococcus. We have determined the crystal structure of the catalytically inactive mutant Ides-C94S by x-ray crystallography at 1.9-Angstrom resolution. Despite negligible sequence homology to known proteinases, the core of the structure resembles the canonical papain fold although with major insertions and a distinct substrate-binding site. Therefore IdeS belongs to a unique family within the CA clan of cysteine proteinases. Based on analogy with inhibitor complexes of papain-like proteinases, we propose a model for substrate binding by IdeS.
|
|
| 38. |
- Åkesson, Per, et al.
(författare)
-
IdeS, a highly specific immunoglobulin G (IgG)-cleaving enzyme from Streptococcus pyogenes, is inhibited by specific IgG antibodies generated during infection
- 2006
-
Ingår i: Infection and Immunity. - : American Society for Microbiology. - 0019-9567 .- 1098-5522. ; 74:1, s. 497-503
-
Tidskriftsartikel (refereegranskat)abstract
- IdeS, a recently discovered cysteine proteinase secreted by the important human pathogen Streptococcus pyogenes, interferes with phagocytic killing by specifically cleaving the heavy chain of immunoglobulin G. The fact that the enzyme targets one of the key molecules of the adapted immune response raised the question of whether an antibody response against IdeS could inhibit, i.e., neutralize, enzyme activity. Paired acute- and convalescent-phase serum samples from patients with pharyngotonsillitis (n = 10), bacteremia (n = 7), and erysipelas (n = 4) were analyzed. Antibodies with the ability to neutralize IdeS enzymatic activity were already found in two-thirds of acute-phase sera. However, patients who seroconverted to IdeS, in particular patients with pharyngotonsillitis and erysipelas, developed specific antibodies during convalescence with an increased capability to efficiently neutralize the enzymatic activity of IdeS. Also, the presence of neutralizing antibodies decreased the ability of IdeS to mediate bacterial survival in human immune blood. In patients with bacteremia, several acute-phase sera contained neutralizing antibodies, but no correlation was found to severity or outcome of invasive infections. Still, the fact that the human immune response targets the enzymatic activity of IdeS supports the view that the enzyme plays an important role during streptococcal infection.
|
|
| 39. |
|
|
| 40. |
- Åström, Stefan U., et al.
(författare)
-
The yeast initiator tRNAMet can act as an elongator tRNA(Met) in vivo
- 1993
-
Ingår i: Journal of Molecular Biology. - : Academic Press. - 0022-2836 .- 1089-8638. ; 233:1, s. 43-58
-
Tidskriftsartikel (refereegranskat)abstract
- Saccharomyces cerevisiae uses two different methionine accepting tRNAs during protein synthesis. One, tRNA(iMet), is used exclusively during the initiation of translation whereas the other, tRNA(mMet), is used during the elongation of translation. To study the unique features of each methionine tRNA species, we constructed yeast strains with null alleles of the five elongator methionine tRNA (EMT) genes and strains with null alleles of the four initiator methionine tRNA (IMT) genes, respectively. Consequently, growth of these strains was dependent either on a tRNA(mMet) or a tRNA(iMet), respectively, encoded from a plasmid-derived gene. For both null mutants, the plasmid carrying the wild-type gene can be selected against and exchanged for another plasmid derived EMT or IMT gene (wild-type or mutant). A high gene dosage of the wild-type IMT gene could restore growth to the elongator-depleted strain. However, wild-type EMT genes in a high gene dosage never restored growth of the initiator depleted strain. Thus, the elongator tRNA(Met) is much more restricted to participate in the initiation of translation than the initiator tRNA(Met) is restricted to participate in the elongation process. Using the two null mutants, we have identified tRNA(mMet) mutants, which show reduced elongator activity, and tRNA(iMet) mutants, with improved elongator activity in the elongator depleted strain. Also, tRNA(mMet) mutants that function as an initiator tRNA in the initiator depleted strain were identified. From this mutant analysis, we showed that the conserved U/rT at position 54 of the elongator tRNA(Met) is an important determinant for an elongator tRNA. The most important determinant for an initiator was shown to be the acceptor stem and especially the conserved A1.U72 base-pair. Mutant tRNAs, with reduced activity in either process, were investigated for enhanced activity during overproduction of the alpha and beta-subunits of the eukaryotic initiation factor 2 (eIF-2) or the eukaryotic elongation factor 1 alpha (eEF-1 alpha). The data suggest that the U/rT of the elongator at position 54 is important for eEF-1 alpha recognition and that the acceptor stem of the initiator is important for eIF-2 recognition.
|
|
