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Aung, Kyaw Min
Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
Sjöström, Annika E
von Pawel-Rammingen, Ulrich
Riesbeck, Kristian
Uhlin, Bernt Eric
Wai, Sun Nyunt
Naturally Occurring IgG Antibodies Provide Innate Protection against Vibrio cholerae Bacteremia by Recognition of the Outer Membrane Protein U2016In: Journal of Innate Immunity, ISSN 1662-811X, E-ISSN 1662-8128, Vol. 8, no 3, p. 269-283Article in journal (Refereed)
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
Vindebro, Reine
Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
Bergström, Claes
Spoerry, Christian
Persson, Helena
Fex, Tomas
Kihlberg, Jan
Luthman, Kristina
3-aminopiperidine-based peptide analogues as the first selective noncovalent inhibitors of the bacterial cysteine protease IdeS2012In: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 55, no 6, p. 2549-2560Article in journal (Refereed)
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
Mörgelin, Matthias
Olin, Anders I
Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
Collin, Mattias
The membrane bound LRR lipoprotein Slr, and the cell wall-anchored M1 protein from Streptococcus pyogenes both interact with type I collagen.2011In: PloS one, ISSN 1932-6203, Vol. 6, no 5, p. e20345-Article in journal (Refereed)
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.
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
Öhman, Anders
Umeå University, Faculty of Science and Technology, Department of Chemistry.
Brattsand, Maria
Umeå University, Faculty of Medicine, Department of Public Health and Clinical Medicine, Dermatology and Venerology.
Characterization of SPINK9, a KLK5-specific inhibitor expressed in palmo-plantar epidermis2012In: Biological chemistry (Print), ISSN 1431-6730, E-ISSN 1437-4315, Vol. 393, no 5, p. 369-377Article in journal (Refereed)
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 P
Levander, Fredrik
Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
Berggård, Tord
Björck, Lars
James, Peter
The protein expression of Streptococcus pyogenes is significantly influenced by human plasma2005In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 4, no 6, p. 2302-2311Article in journal (Refereed)
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. Keeney, Jill B
Chapman, Karen B
Lauermann, Vit
Voytas, Daniel F
Åström, Stefan U
Byström, Anders
Boeke, Jeff D
Multiple molecular determinants for retrotransposition in a primer tRNA1995In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 15, no 1, p. 217-226Article in journal (Refereed)
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.
Okumura, Cheryl
University of California.
Anderson, Ericka L
Döhrmann, Simon
Tran, Dan N
Olson, Joshua
Nizet, Victor
IgG protease Mac/IdeS is not essential for phagocyte resistance or mouse virulence of M1T1 group A Streptococcus2013In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 4, no 4, p. e00499-e00513 Article in journal (Refereed)
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.
Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
Johansson Söderberg, Jenny
Johansson, Björn P
Proteolytic processing of the streptococcal IgG endopeptidase IdeS modulates the functional properties of the enzyme and results in reduced immunorecognition2015In: Molecular Immunology, ISSN 0161-5890, E-ISSN 1872-9142, Vol. 68, no 2, p. 176-184Article in journal (Refereed)
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.
Proteolytic processing of the streptococcal IgG cleaving enzyme IdeS reduces immunorecognition without affecting the biological activity of the enzymeManuscript (preprint) (Other academic)
The streptococcal cysteine protease SpeB is not a natural immunoglobulin-cleaving enzyme2013In: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 81, no 6, p. 2236-2241Article in journal (Refereed)
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.
The Streptococcal Cysteine Protease SpeB Is Not a Natural Immunoglobulin-Cleaving Enzyme
11. Rungelrath, Viktoria
Wohlsein, Jan Christian
Siebert, Ursula
Stott, Jeffrey
Prenger-Berninghoff, Ellen
Valentin-Weigand, Peter
Baums, Christoph G.
Seele, Jana
Identification of a novel host-specific IgG protease in Streptococcus phocae subsp phocae2017In: Veterinary Microbiology, ISSN 0378-1135, E-ISSN 1873-2542, Vol. 201, p. 42-48Article in journal (Refereed)
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.
Seele, J.
University of Veterinary Medicine Hannover, Inst Microbiol, Hannover, Germany.
Singpiel, A.
Valentin-Weigand, P.
Baums, C. G.
Identification of a novel host-specific IgM protease in Streptococcus suis2013In: International Journal of Medical Microbiology, ISSN 1438-4221, E-ISSN 1618-0607, Vol. 303, no Suppl. 1, MPP35, p. 58-58Article in journal (Other academic)
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.
13. Seele, Jana
Beineke, Andreas
Hillermann, Lena-Maria
Jaschok-Kentner, Beate
Baums, Christoph Georg
The immunoglobulin M-degrading enzyme of Streptococcus suis, Ide(Ssuis), is involved in complement evasion2015In: Veterinary research (Print), ISSN 0928-4249, E-ISSN 1297-9716, Vol. 46, article id 45 Article in journal (Refereed)
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.
Seitz, Maren
The immunoglobulin M-degrading enzyme of Streptococcus suis, Ide(Ssuis), is a highly protective antigen against serotype 22015In: Vaccine, ISSN 0264-410X, E-ISSN 1873-2518, Vol. 33, no 19, p. 2207-2212Article in journal (Refereed)
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.
Singpiel, Alena
Identification of a Novel Host-Specific IgM Protease in Streptococcus suis2013In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 195, no 5, p. 930-940Article in journal (Refereed)
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.
Hessle, Pontus
Lewis, Melanie J
Paton, Lois
Woof, Jenny M
Novel IgG-Degrading Enzymes of the IgdE Protease Family Link Substrate Specificity to Host Tropism of Streptococcus Species.2016In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 11, no 10 Article in journal (Refereed)
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.
Identification and Characterization of IgdE, a Novel IgG-degrading Protease of Streptococcus suis with Unique Specificity for Porcine IgG.2016In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 291, no 15, p. 7915-7925Article in journal (Refereed)
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.
Söderberg, Jenny Johansson
Engström, Patrik
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 strains2008In: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 76, no 5, p. 2183-2188Article in journal (Refereed)
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.
The streptococcal protease IdeS modulates bacterial IgGFc binding and generates 1/2Fc fragments with the ability to prime polymorphonuclear leucocytes2008In: Molecular Immunology, ISSN 0161-5890, E-ISSN 1872-9142, Vol. 45, no 12, p. 3347-3353Article in journal (Refereed)
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.
20. Vincents, Bjarne
Guentsch, Arndt
Kostolowska, Dominika
Eick, Sigrun
Potempa, Jan
Abrahamson, Magnus
Cleavage of IgG(1) and IgG(3) by gingipain K from Porphyromonas gingivalis may compromise host defense in progressive periodontitis2011In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 25, no 10, p. 3741-3750Article in journal (Refereed)
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
The human protease inhibitor cystatin C is an activating cofactor for the streptococcal cysteine protease IdeS2008In: Chemistry and Biology, ISSN 1074-5521, E-ISSN 1879-1301, Vol. 15, no 9, p. 960-968Article in journal (Refereed)
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.
Enzymatic characterization of the streptococcal endopeptidase, IdeS, reveals that it is a cysteine protease with strict specificity for IgG cleavage due to exosite binding.2004In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 43, no 49, p. 15540-15549Article in journal (Refereed)
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.
Rapid IgG heavy chain cleavage by the streptococcal IgG endopeptidase IdeS is mediated by IdeS monomers and is not due to enzyme dimerization2013In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 587, no 12, p. 1818-1822Article in journal (Refereed)
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.
Insights into substrate recognition and biochemical properties of the streptococcal IgG endopeptidase IdeSManuscript (preprint) (Other academic)
Streptococcal IdeS and Its Impact on Immune Response and Inflammation2012In: JOURNAL OF INNATE IMMUNITY, ISSN 1662-811X, Vol. 4, no 2, p. 132-140Article in journal (Refereed)
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.
Telepnev, Maxim V
Schmidt, Gudula
Aktories, Klaus
Wolf-Watz, Hans
Rosqvist, Roland
GAP activity of the Yersinia YopE cytotoxin specifically targets the Rho pathway: a mechanism for disruption of actin microfilament structure.2000In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 36, no 3, p. 737-748Article in journal (Refereed)
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.
Åström, Stefan
Byström, Anders S
Mutational analysis of conserved positions potentially important for initiator tRNA function in Saccharomyces cerevisiae1992In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 12, no 4, p. 1432-1442Article in journal (Refereed)
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.
28. Wenig, Katja
Chatwell, Lorenz
Huber, Robert
Sondermann, Peter
Structure of the streptococcal endopeptidase IdeS, a cysteine proteinase with strict specificity for IgG.2004In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 101, no 50, p. 17371-17376Article in journal (Refereed)
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-A 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.
29. Åkesson, Per
Moritz, Linnea
Truedsson, Mikael
Christensson, Bertil
IdeS, a highly specific immunoglobulin G (IgG)-cleaving enzyme from Streptococcus pyogenes, is inhibited by specific IgG antibodies generated during infection2006In: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 74, no 1, p. 497-503Article in journal (Refereed)
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.
Åström, Stefan U.
Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
The yeast initiator tRNAMet can act as an elongator tRNA(Met) in vivo1993In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 233, no 1, p. 43-58Article in journal (Refereed)
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.


text:
Joining the Air Force Reserve
A C-17 Globemaster III assigned to the 911th Airlift Wing sits on the flight line while waiting to receive a basic postflight inspection at the Pittsburgh International Airport Air Reserve Station, Pennsylvania, Feb. 3, 2020. (Joshua J. Seybert/U.S. Air Force)
The Air Force Reserve supports the regular Air Force in fulfilling its national security objectives by providing professionals in mission critical roles. In addition, it is a major force in your community by contributing manpower and expertise in the wake of national disaster or civil unrest to protect life and property, and to preserve peace, order and public safety.
Although the Air Force Reserve is a separate command of the U.S. Air Force, its aviators and personnel have played significant roles in all of America's wars and most of its major contingencies since the beginning of the 20th century.
If you are interested in enlisting in the Air Force Reserve, you must:
-- Join before you turn 40 with some exceptions (medical professionals, clergy, attorneys). If you have served previously, you must join before you are 40, plus time served. So if you served in the military for six years, you could join in an enlisted position until about age 46.
-- Have a high school diploma, although in some instances GED certificates can be accepted.
-- Pass a physical.
-- Complete an aptitude test (the ASVAB), which will determine career paths available for consideration. For tips on doing well on the ASVAB, see the Ace the ASVAB section.
-- For more on general Reserve benefits and pay, see the Reserve Help Pages.
Education Benefits and Skills Training
Community College of the Air Force -- The CCAF enables students to earn an associate in applied science degree. The college is only open to enlisted active-duty Air Force, Air National Guard and Air Force Reserve members. CCAF offers 71 degree programs and has more than 270,000 airmen enrolled, making it the largest multi-campus community college in the world.
To earn their two-year degrees, students combine Air Force technical training, professional military education and general education credits from accredited colleges and universities. Most technical training is completed at schools on four bases: Keesler Air Force Base, Mississippi; or Lackland, Sheppard and Goodfellow Air Force Bases in Texas. All technical courses are completed at CCAF-affiliated Air Force schools. For liberal arts prerequisites, students can complete these at civilian colleges and universities.
Visit the Community College of the Air Force's website for more information.
If your hope is to become an Air Force officer, then Air Force ROTC is one of three ways to achieve that goal. With detachments at more than 143 universities and colleges nationwide, and more than 850 schools offering the program with host schools in their locations, AFROTC is an excellent way to work toward a commission while obtaining your degree. The program annually commissions more than 2,000 new second lieutenants and is the oldest and largest source of commissioned officers for the Air Force.
Air Force ROTC offers 3.5-, 3-, and 2.5-year scholarships in all majors. There are two categories of scholarships:
Type I -- This scholarship offers full tuition and fees with a textbook allowance per year.
Type II -- An annual payment for tuition and fees, and an additional annual book allowance.
All scholarship cadets receive a monthly nontaxable stipend throughout the school year. To be eligible you must:
-- Have a GPA of 2.5 or higher.
-- Prior to applying for the scholarship, you must complete at least one term of full-time, college-level work (does not include college-level work during high school) and have at least 24 semester hours of college-level credits.
-- Meet weight, fitness, medical, age, citizenship, Air Force Officer Qualifying Test score and other eligibility requirements.
Selection goes to the most qualified students. Those selected must participate in the AFROTC program and successfully complete a field-training encampment after selection.
After completing all AFROTC program and degree requirements, students will be commissioned as a second lieutenant in the Air Force and serve a minimum of four years on active duty.
If you are interested in becoming a pilot, all pilot slots are selected during the junior year on a competitive basis. Good college grades, physical fitness, medical qualification and military performance are the keys to selection.
Visit the Air Force ROTC's website for more information.
Officer Training School
Officer Training School is an intensive 12-week program that prepares officer candidates for the technical, physical and professional requirements of commissioned service. Approximately 1,000 OTS graduates enter skill training or supervisory positions each year in all major career areas. In years past, OTS has surged to meet wartime officer requirements in its "flexible partner role," producing as many as 7,000 new second lieutenants in one year.
Montgomery GI Bill for Selected Reserve
The MGIB-SR allows you to attend school full time while serving in the Reserve or National Guard and get more than $10,000 for school in addition to your paycheck and any other educational benefits you may be eligible to receive. For more on the GI Bill for Selected Reservists, go to the Reserve GI Bill section.
The Air Force Reserve offers you tuition assistance. You can be reimbursed up to 100% tuition assistance up to $250 per semester hour, or $166 per quarter hour, not to exceed $4,500 annually per service member. For more on this program, see the Tuition Assistance Overview.
College Credits By Exam
Members of the Reserve also can take advantage of free College Level Examination Program tests (CLEP). For every test you pass on a particular subject, you earn up to six transferable college credits.
Voluntary Education
As a member of the National Guard, you also can pursue college and university courses through the Community College of the Air Force. The Community College of the Air Force provides two-year educational programs to enlisted members of the active-duty Air Force, Air National Guard and Air Force Reserves. These programs combine Air Force technical training with general education coursework from civilian accredited colleges.
Promotions and Career Path
In the Air Force Reserve, you can expect steady advancement in rank and pay. Promotions are made with what the Air Force considers the "whole person" approach:
-- Job performance
-- Time in rank
-- Career specialty
-- Leadership
-- Job responsibility
The Air Force Reserve also looks favorably on your education. Being part of one of the most advanced branches of the military, the USAFR constantly looks for qualified people to advance to more technical positions.
Prior Service
If you have prior military service, then the Air Force Reserve is an excellent way to continue accruing time and money for retirement while continuing to serve your country and passing on your experience and knowledge. When you join the Air Force Reserve, you'll receive benefits for your prior military and reserve experience.
There are opportunities if you currently are transitioning from active duty or if you've been out of the service for a while.
Even your experience in another service could be put to good use in the Air Force Reserve, and this could be coupled with the financial and educational opportunities in locations close to where you live, work or study.
The Air Force Reserve Experience
The Air Force Reserve offers you the ability to go to school full time or pursue a civilian career while serving your country. As a member of the Air Force Reserve, your commitment -- after going through basic training and your initial technical schools -- will be for one drill a month and one period of annual training per year. People with no prior military experience who join the Reserve will incur an eight-year obligation. These eight years are broken down into two segments. The first six years, you will be an active participating member. The last two years, if you choose, you will be in the inactive ready reserve. Although you are not active within the Reserve, you will be subject to being recalled in the event of a national emergency.
Drills -- A drill consists of two days of training per month. Your pay will be adjusted automatically for cost-of-living increases. You'll get pay increases for every two years of accumulated service and may earn special duty pay. To calculate your drill pay, visit the Drill Calculator.
Annual training -- Annual training in the Air Force Reserve is held for two weeks per year. Depending on the unit and your specialty, you could be stationed at a shore location, with an aviation squadron or aboard ship.
Travel -- As a member of the Air Force Reserve, you will have the opportunity to travel on duty and off. You also qualify for military space available travel within and between the continental United States, Alaska, Hawaii, Guam and Puerto Rico. For more on military travel options and benefits, see the Travel Center.
Life insurance, medical care -- The Air Force Reserve provides comprehensive medical care while on duty. You also qualify for low-cost life insurance. For more on these and other benefits, visit the Reserve Help Pages.
Home loan program -- Members of the Air Force Reserve with at least six years of service are eligible to apply for the Department of Veterans Affairs Home-Loan Guarantee program. For more on this program, go to the Home section.
Base privileges -- As a member, you can access all recreational facilities on military bases, such as gyms, tennis courts and libraries. You and your family also can enjoy unlimited access shopping at any Military Exchange nationwide. Air Force Reserve members and their families are entitled to use base commissaries for up to 24 days annually, plus any days spent on active duty. For more on recreational and family benefits, see the Reserve Family and Individual Help page.
Need more information? Fill out this form, and recruiters will be in touch with you about military career opportunities. Great information with no obligation!
Interested in Joining the Military?
We can put you in touch with recruiters from the different military branches. Learn about the benefits of serving your country, paying for school, military career paths, and more: sign up now and hear from a recruiter near you.
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redpajama_set_name: RedPajamaCommonCrawl


Lemonade Day is coming to Fort Atkinson May 4th, 2019!
Lemonade Day is a free, fun, experiential learning program that teaches kids how to start, own and operate their very own business – a lemonade stand. Lemonade Day has spread to over 77 cities in 23 states and 3 countries. We are proud to announce that this year Fort Atkinson kids will get to join in on the fun.
Each child that registers receives a FREE backpack with an Entrepreneur Workbook or access to an online interactive program that teaches them the valuable lessons of Lemonade Day - including how to set a goal, make a plan, work the plan and achieve their dreams. Once a youth pays their investor back, they keep all of the money they earn and are encouraged to spend some, save some, and share some of their profit.
Anyone and everyone can be involved in Lemonade Day! Young entrepreneurs with lemonade stands need mentors, investors, business partners, great locations, and customers! Sponsors and volunteers are also needed to make Lemonade Day a success.
Your workbooks and backpacks are in and ready for pick-up! If your students are affiliated with BASE (Badgerland After School Enrichment), their supplies will be distributed at the location your child attends, beginning Monday, March 11th.
The Fort Atkinson Area Chamber of Commerce is the pick-up site for all other registrants. The Chamber is open Monday-Friday, 8:00am-5:00pm, and you are welcome to stop in and pick up your supplies between those hours. If this time is inconvenient, please notify the Chamber by calling 920-563-3210, and we can arrange for your supplies to be picked up on a Saturday at Fort Community Credit Union.
Please be prepared to provide your children's t-shirt sizes when you pick up your materials.
Good luck, and have fun! The Lemon Council will be awarding prizes for the Best Tasting Lemonade, the Best Stand, and the Entrepreneur of the Year, and one lucky child will win a new bike, just for participating!
To find out more information or any questions about registration please call the Chamber at 920-563-3210.


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ITC Publishing House > PrintWeek > About Magazine
PrintWeek is a business magazine on graphic arts that is almost 50 years on the market. The first issue came out in the UK in 1958, and today it is also published in Russia, Ukraine, Germany and the United Arab Emirates.
A clear and comprehensible concept made PrintWeek a leader among European brunch periodical titles. PrintWeek provides quick and objective information for managers and owners in graphic arts industry. Therefore it is an effective marketing tool for the players of printing market.
A thorough look at the portrait of PrintWeek Russia#s reader allows to conclude that companies interested in communicating important information (for example, ads, PR etc.) to the directors of printing houses, advertising agencies or publishers can stake on this magazine. Naturally, advertising in PrintWeek not only stimulates direct sales — it is an element of marketing and is first of all to extend the firm#s market share and to form its image in their clients# opinion.
Russian printing market is one of the most promising in Europe. Its volume is estimated at $3,5—4 billion annually, and the number of printing houses working in the country exceeds 8000.
At the same time, as the year-on-year growth of Russian GDP is forecasted at the level of 8—9%, and with the population of more than 130 million, one can expect serious development of the graphic arts industry, closely bound with the economy of the country.
We offer effective partnership to all the production and trade companies engaged in Russian graphic arts market. You can rely on us!
ITC Publishing House > What Hi-Fi? > About Magazine
ITC Publishing House > Packaging News > About Magazine


OCEM's American affiliate company, Multi Electric, is kicking off its next century of quality and innovation in airfield ground lighting with a major rebranding effort.
The new company identity, Multi Electric-OCEM Airfield, reflects a closer relationship with Multi's parent company, the OCEM Airfield Technology Group, and its renewed investments in research and development.
Founded in Chicago, Illinois, in February of 1917, Multi started out in wiring components and street lighting before becoming one of the first American producers of airfield ground lighting when commercial aviation took off in the 1960s. Meanwhile, in Italy, OCEM was pioneering European AGL solutions thanks to its acquisition of Buini and Grandi, founded in 1921.
OCEM acquired Multi in 1999, and together they were among the first companies in the world to incorporate long-lasting, energy efficient LED technology into their raised and inset lighting signals.
Now, Multi is enjoying a renewed commitment from OCEM — including investments in personnel, R&D and manufacturing — to usher in the next generation of smart, efficient lighting signals and control and monitoring systems.
The newly strengthened partnership will bring OCEM's cutting-edge R&D to a company that boasts strong partnerships with key transportation agencies in the U.S., including the FAA, Air Force and Navy, and New York-New Jersey Port Authority, and has long served as OCEM's entre into the North American market.
The OCEM group also includes Augier, a French company specializing in power transport that was founded in 1954. These acquisitions allow the OCEM Group to provide fully integrated AGL solutions: Lighting Signals, Telecontrol Systems and Constant Current Regulators.
To find out which OCEM solution is right for you, contact a sales representative today.


"Jam Packed street of food and fun"
Ludlow really starts off with a bang with Katz' Deli right on the corner. Katz is one of the two best deli's in the city and it's quite famous. It's the deli with the famous scene from When Harry Met Sally. The pastrami is pretty killer. I recommend it to any visitors that want a downtown deli. There are a ton of bars on this block as well. Max Fish is across the street and it's probably the most popular one. It has been around since the 80's and it's very Lower East Side. There's a grilled cheese spot a few doors down that's perfect after a few too many hours at the Max Fish if you catch my drift.
The block between Stanton and Rivington is where the LES really gets ahold of the street. Pianos is right on the corner. And, it's a great dive bar with live acts ranging from music to comedy. Cakeshop, the creepiest but most awesome coffee shop is right next door to that. And, down the street is 'inoteca which is a fantastic restaurant that has a jammin brunch. There are also a lot of funky shops on this block so you could actually never leave this block for an entire day and have things to do.
But, you don't have to stay on that block because the good times keep going after you cross Rivington. Spitzer's, on the corner, is another lovely little restaurant with a long beer list. There's also a crazy, Bulgarian bar called Mehanata that's pretty fun if you're into crazy. But, my personal favorite spot on this block is Motor City. It is one of the scariest of dive bars of all time and I love it. Some of my favorite memories in New York have occurred in that disgusting bar. The rest of the street is all schools and apartment buildings until you get down to Canal where you hit yet another fantastic restaurant: Les Enfants Terribles. The food is great, the restaurant is lovely and it always has a great energy.
Ludlow is one of the most Lower East Side streets of the Lower East Side. It's fun and lively and loud. It's too loud at times and sometimes dirty but there's so much going on, it's kind of hard to care. I really enjoy Ludlow. It's one of my favorite streets in the neighborhood.
Delancey Street is in the SOHO district of New York City. It is bustling with culture everywhere you look. The part of Delancey I lived on would be considered a little "rough," but you can also find some good friends among neighbors. Nobody sleeps on this street, so for those that enjoy peace and quiet, you probably won't get it. I loved it!
Uraniumfish Hey, Delancey's definitely not in SoHo, no way. Maybe the Lower East Side, and yeah, there are parts that look a little rough.
Whether you love music, history or art-deco architecture and settings, Delancey Street is going to offer something for everyone. This famous location houses the Bowery Ballroom which plays host to a range of gigs on its different levels. So whether you want to rock out, enjoy a drink in the lounge or just admire the stunning design and feel of a venue that has seen and done it all, Delancey Street is the place to be.
Delancey is one of my favorite streets – it can be pretty busy with street traffic as well as pedestrian traffic during the day, but at night it slows down a lot. I've seen a few shows at the Bowery Ballroom music venue, and can't really complain (it's one of those large-yet-intimate venues).
Delancey Street offers bargain shopping for everyone who wants to indulge. The area is lined with bargain and discount shops for everything from apparel to kitchen equipment. Hyperactive is a hot spot along the road and a few coffeehouses and a Starbucks will satisfy the caffeine urges of the masses. it's filled with young professionals and aspiring artists offering an interesting mix of people to chat with. Overall it's a friendly and casual area that is not for the faint of heart.
"Old New York is All About Delancey Street"
Delancey Street is one of my favorite streets in New York. I went here first because it's mentioned in a song by Regina Spektor (who I love), and I fell in love with this neighborhood!
Delancey cuts across the island all the way from the Williamsburgh Bridge to the Bowery, and taking a walk here puts you back in the Manhattan of the movies, from the Godfather to every film Woody Allen ever made. I've walked here with my boyfriend and it's the quintessenial New York Experience.
The shops of Orchard Street are just to the south, and the entire street is jam packed with discount and bargain shops (my favorite kind!). At night there are lots of great old dive bars here, too.
"Delancey St, A cool place to hang out"
Delancey Street is one of the main thoroughfares of Manhattan's Lower East Side, running east from the Bowery to connect to the Williamsburg Bridge to Brooklyn. This is a busy street with a lot of billboards hanging. Delancey Street is named after James De Lancey. Delancey Street is famous for its discount and bargain clothing stores. I visited the famous Bowery Ballroom.
"Delancey St, A nice place"
Whenever I visit New York I have to visit Delancey St. Trust Me guys it's a very busy street with a lot of billboards hanging here & there. Delancey Street is famous for its discount and bargain clothing stores. Delancey Street is named after James De Lancey. I always visit the famous Bowery Ballroom.
"A good place to hang out"
This street was named in honor of James De Lancey. It is an eight lane, Median divided street. A wide range of buisnesses are found in this area. Delancy Street is known for its discount clothing stores. There are also professional artist and singers earning by performing on this street. I took some snaps of the famous Bowery Ballroom.


Miami (August 7, 2017) Can a better understanding of leadership across multiple teams help send the right people to Mars? Dr. Dorothy Carter believes it may. Her doctoral research into leadership across teams with different goals reveals how people influence each other and achieve related outcomes - and may even help NASA choose the most cohesive team of astronauts for their mission to Mars.
The work from Dr. Carter, an assistant professor of Industrial and Organizational Psychology at the University of Georgia, won the Center for Leadership at Florida International University's Alvah H. Chapman Jr. Outstanding Dissertation Award.
Dr. Carter's dissertation, "The Motivational Drivers of Leadership Emergence in Multiteam Systems," reveals that when a team is embedded in a larger system comprised of multiple teams, the goals emphasized within each team affect members' willingness to participate in leadership processes in relation to other teams. Ultimately, her findings help explain why patterns of leadership relationships arise within and across teams to affect the success or failure of complex organizational systems.
"Studying leadership as a network is necessary in order to understand the ways that people influence one another, both formally, as well as informally, toward different goals," Dr. Carter said.
Established in partnership with the Network of Leadership Scholars (NLS), the award honors an individual whose dissertation makes an outstanding contribution to the field of leadership. Dr. Carter will be honored at the Network of Leadership Scholars meeting at the Academy of Management Annual Conference on August 6, 2017, and she will present her findings at The Leadership Research Colloquium, presented by the Center in April 2018. The award includes a $3,000 cash prize.
Dr. Carter's research has been published in such outlets as Journal of Applied Psychology, The Leadership Quarterly, Journal of Management, Group and Organization Management, and The Oxford Handbook of Leadership. She is also the Primary Investigator on "Project FUSION: Facilitating Unified Systems of Interdependent Organizational Networks", a grant funded project by the National Aeronautics and Space Administration (NASA) to enhance the multiteam collaboration processes that will be necessary to send a team of humans to Mars. Her dissertation research was overseen by adviser Dr. Leslie DeChurch of Northwestern University and supported in part through DeChurch's National Science Foundation CAREER grant.


Jersey Boys is based on the real life rags to riches story of Frankie Valli and the Four Seasons who went from the street corners for New Jersey to massive musical success with songs such as Oh What A Night, Big Girls Don't Cry, Rag Doll, Sherry, and Can't Take My Eyes Off of You. Hit the jump for more on the deal, the musical and even a video.
Jersey Boys is one of the most successful shows still running on Broadway. It opened in 2005 and has sold over $1 billion in tickets not only in New York, but all over the world. The show's success is partially because of its relatable, inspiring story of working hard and fulfilling your dreams but more so because it's the rare musical where almost everyone who sees it knows all the songs before stepping in to the theater. Below is an example of what the show is like. Notice the awesome way it incorporates live performance and what it would have been like to experience this moment as an audience member on TV. I love this show.
The film version will be written by the same people who wrote the book (basically the script) for the musical, Marshall Brickman and Rick Elice. Musicals have become hot properties ever since Mamma Mia made $600 million worldwide with versions of Wicked, In the Heights and others are all in development. But Jersey Boys is easily the most commercial out of the bunch, at least according to the man who bought the rights. "Jersey Boys is one of the most electrifying stage shows any of us has ever seen and it has all the ingredients to become a big commercial hit movie," King said to Deadline, who broke the story. They also reported that even though the show opened in 2005, the film rights were off the market until six weeks ago.
I'd venture to guess that many /Film readers like yourselves have seen Jersey Boys over the years. Do you think it can be a successful film? And, if you were making it, would you keep it as a straight up musical – like Mamma Mia or The Producers – or make the music less the focus – like in Purple Rain?


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Welcome to David Shutkin's Website
About educational technology, the theory of the extended mind & the experience of being in school
Web Vita
Beyond Representation Bibliography
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Dr. David Shutkin
Hello, my name is David Shutkin. I am a Professor of Education at John Carroll University