Molecular studies on streptococcal surface proteins

Streptococcus agalactiae and Streptococcus pyogenes are two related human pathogens causing different diseases. This thesis focuses on a number of surface proteins expressed by S. agalactiae and S. pyogenes and, more specifically, the molecular and biological characterization of these proteins. The first paper describes the novel Blr protein of S. agalactiae, which together with the previously described Slr protein of S. pyogenes identifies a family of streptococcal proteins with leucine-rich repeats (LRRs). Characterization of Blr and Slr revealed that the two proteins are efficiently camouflaged by other surface components. In S. agalactiae, exposure of Blr was increased ~100-fold in a mutant lacking the polysaccharide capsule, whereas S. pyogenes mutants lacking M protein and/or protein F displayed ~20-fold increased surface exposure of Slr. It seems possible that the camouflaging structures are downregulated during some parts of the infection process, thus exposing the Blr and Slr proteins on the streptococcal surface. In the second paper, we analyzed a key problem in the vaccine field, the identification of antigens that elicit protective immunity. Our studies were focused on the immune response to the S. agalactiae proteins Rib and alpha. These proteins contain a unique N-terminal region and long repetitive C-terminal sequences. The immune response against pure Rib or alpha was almost exclusively directed against the repeat region, i.e. very few antibodies were directed against the N-terminal regions. Thus, the N-terminal region is nonimmunodominant in both Rib and alpha. Nevertheless, a fusion protein comprising the N-terminal regions of Rib and alpha elicited antibodies that were protective. Importantly, studies of the S. pyogenes M22 protein showed that the N-terminal region, which is targeted by opsonic antibodies, was also nonimmunodominant. Together, these results indicate that nonimmunodominant regions are of general interest for vaccine development. The third paper addresses the role of fibrinogen (Fg) in bacterial virulence. We characterized the Fg-binding B repeat region of the S. pyogenes M5 protein. In a mouse model, a mutant lacking the Fg-binding domain was severely attenuated, indicating that binding of Fg plays a key role in virulence. Similarly, a bacterial mutant lacking the most N-terminal part of the M5 protein was avirulent. Together, these data indicate that two separate regions of M5 are important for virulence. The function of bacteria-bound Fg is not known, but our data support the notion that bound Fg protects the bacteria against complement deposition and thereby against phagocytosis

Functional Dissection of Streptococcus pyogenes M5 Protein: the Hypervariable Region is Essential for Virulence

The surface-localized M protein of Streptococcus pyogenes is a major virulence factor that inhibits phagocytosis, as determined ex vivo. Because little is known about the role of M protein in vivo we analyzed the contribution of different M protein regions to virulence, using the fibrinogen (Fg)-binding M5 protein and a mouse model of acute invasive infection. This model was suitable, because M5 is required for mouse virulence and binds mouse and human Fg equally well, as shown here. Mixed infection experiments with wild type bacteria demonstrated that mutants lacking the N-terminal hypervariable region (HVR) or the Fg-binding B-repeat region were strongly attenuated, while a mutant lacking the conserved C-repeats was only slightly attenuated. Because the HVR of M5 is not required for phagocytosis resistance, our data imply that this HVR plays a major but unknown role during acute infection. The B-repeat region is required for phagocytosis resistance and specifically binds Fg, suggesting that it promotes virulence by binding Fg. However, B-repeat mutants were attenuated even in Fg-deficient mice, implying that the B-repeats may have a second function, in addition to Fg-binding. These data demonstrate that two distinct M5 regions, including the HVR, are essential to virulence during the early stages of an infection. In particular, our data provide the first in vivo evidence that the HVR of an M protein plays a major role in virulence, focusing interest on the molecular role of this region

Game Design : Social Learning Games for Kids

This is the report for the final thesis in Game Design at Luleå University of Technology. The work was done in Skellefteå University which is part of LTU. This document contains the background and goal of the project, how it was done and the results together with the final conclusions of the work. The work was done for a project called Meet, Play and Learn, a collaboration made up by several institutes and universities around Scandinavia. Their goal was to research and develop social learning games for children with diabetes and the goal of this thesis was to create a few ideas and game concepts for that project. The work of this thesis include the process of researching type 1 diabetes, children and their gaming habits, games and their potential as a learning tool and how all of it comes together in the design process. The result are five game concepts, each with their own unique way of approaching the challenge of designing social learning games for kids.Validerat; 20120511 (anonymous

The Streptococcal Blr and Slr Proteins Define a Family of Surface Proteins with Leucine-Rich Repeats: Camouflaging by Other Surface Structures

Regions with tandemly arranged leucine-rich repeats (LRRs) have been found in many prokaryotic and eukaryotic proteins, in which they provide a remarkably versatile framework for the formation of ligand-binding sites. Bacterial LRR proteins include the recently described Slr protein of Streptococcus pyogenes, which is related to internalin A of Listeria monocytogenes. Here, we show that strains of the human pathogen Streptococcus agalactiae express a protein, designated Blr, which together with Slr defines a family of internalin A-related streptococcal LRR proteins. Analysis with specific antibodies demonstrated that Blr is largely inaccessible on S. agalactiae grown in vitro, but surface exposure was increased ∼100-fold on mutants lacking polysaccharide capsule. In S. pyogenes, surface exposure of Slr was not affected in a mutant lacking hyaluronic acid capsule but was increased >20-fold in mutants lacking M protein or protein F. Thus, both Blr and Slr are efficiently camouflaged by other surface structures on bacteria grown in vitro. When Blr and Slr exposed on the bacterial surface were compared, they exhibited only little immunological cross-reactivity, in spite of extensive residue identity, suggesting that their surface-exposed parts have been under evolutionary pressure to diverge functionally and/or antigenically. These data identify a family of immunologically diverse streptococcal LRR proteins that show unexpected complexity in their interactions with other bacterial surface components

The Hypervariable Region of Streptococcus pyogenes M Protein Escapes Antibody Attack by Antigenic Variation and Weak Immunogenicity.

Sequence variation of antigenic proteins allows pathogens to evade antibody attack. The variable protein commonly includes a hypervariable region (HVR), which represents a key target for antibodies and is therefore predicted to be immunodominant. To understand the mechanism(s) of antibody evasion, we analyzed the clinically important HVR-containing M proteins of the human pathogen Streptococcus pyogenes. Antibodies elicited by M proteins were directed almost exclusively against the C-terminal part and not against the N-terminal HVR. Similar results were obtained for mice and humans with invasive S. pyogenes infection. Nevertheless, only anti-HVR antibodies protected efficiently against infection, as shown by passive immunizations. The HVR fused to an unrelated protein elicited no antibodies, implying that it is inherently weakly immunogenic. These data indicate that the M protein HVR evades antibody attack not only through antigenic variation but also by weak immunogenicity, a paradoxical observation that may apply to other HVR-containing proteins

Nonimmunodominant regions are effective as building blocks in a streptococcal fusion protein vaccine.

Identification of antigens that elicit protective immunity is essential for effective vaccine development. We investigated the related surface proteins of group B Streptococcus, Rib and alpha, as potential vaccine candidates. Paradoxically, nonimmunodominant regions proved to be of particular interest as vaccine components. Mouse antibodies elicited by Rib and alpha were directed almost exclusively against the C-terminal repeats and not against the N-terminal regions. However, a fusion protein derived from the nonimmunodominant N-terminal regions of Rib and alpha was much more immunogenic than one derived from the repeats and was immunogenic even without adjuvant. Moreover, antibodies to the N-terminal fusion protein protected against infection and inhibited bacterial invasion of epithelial cells. Similarly, the N-terminal region of Streptococcus pyogenes M22 protein, which is targeted by opsonic antibodies, is nonimmunodominant. These data indicate that nonimmunodominant regions of bacterial antigens could be valuable for vaccine development