Studies on streptococcal M proteins. Interactions with IgA and human complement regulators

The human pathogenic bacterium Streptococcus pyogenes (group A Streptococcus) expresses several different virulence factors. Of these, the M protein is regarded as one of the most important, because it confers resistance to phagocytosis, allowing the bacterium to multiply in blood. A remarkable property of M proteins is their ability to bind different human plasma proteins, in particular components of the immune system. This property is believed to be of importance for the biological functions of M protein. This thesis describes studies of two such interactions: binding of IgA and binding of regulators of complement activation. Many M proteins bind to the Fc part of IgA, the major immunoglobulin on mucosal surfaces. The biological role of this binding is not yet known, but the IgA-binding proteins are valuable model systems for studies of IgA. Alignment of the amino acid residue sequences of five different IgA-binding M proteins identified a putative IgA-binding region. Further analysis of one M protein demonstrated that a 29-residue region was necessary and sufficient for IgA-binding. Subsequent work showed that the IgA-binding region can be studied in the form of a synthetic peptide. Indeed, a 50-residue peptide including the IgA-binding region binds IgA with high specificity, and the properties of this peptide indicate that it corresponds to an isolated IgA-binding domain. An important property of M proteins is the presence of an N-terminal hypervariable region, which probably plays a major role in pathogenesis. However, the function of the hypervariable region has remained unknown. We have found that the hypervariable region of many M proteins specifically binds a ligand, the human complement regulator C4BP. Alignment of the amino acid residue sequences of several C4BP-binding regions did not reveal any identities sufficient to explain the binding of C4BP. Thus, hypervariable regions with highly divergent primary sequences all bind the same ligand, C4BP. This ability to bind C4BP may explain the capacity of these M proteins to confer phagocytosis resistance, since bound C4BP may protect the bacterium against complement attack. However, some M proteins do not bind C4BP. Analysis of two such M proteins demonstrated that their hypervariable regions bind another human complement regulator, FHL-1. Thus, the studies with C4BP and FHL-1 made it possible to attribute a specific function to the hypervariable region of many M proteins: the binding of a human complement regulator

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

Isolated hypervariable regions derived from streptococcal M proteins specifically bind human C4b-binding protein: Implications for antigenic variation

Antigenic variation in microbial surface proteins represents an apparent paradox, because the variable region must retain an important function, while exhibiting extensive immunological variability. We studied this problem for a group of streptococcal M proteins in which the similar to 50-residue hypervariable regions (HVRs) show essentially no residue identity but nevertheless bind the same ligand, the human complement regulator C4b-binding protein (C4BP). Synthetic peptides derived from different HVRs were found to retain the ability to bind C4BP, implying that the HVR corresponds to a distinct ligand-binding domain that can be studied in isolated form. This finding allowed direct characterization of the ligand-binding properties of isolated HVRs and permitted comparisons between different HV Rs in the absence of conserved parts of the M proteins. Affinity chromatography of human serum on immobilized peptides showed that they bound C4BP with high specificity and inhibition experiments indicated that different peptides bound to the same site in C4BP. Different C4BP-binding peptides did not exhibit any immunological cross-reactivity, but structural analysis suggested that they have similar folds. These data show that the HVR of streptococcal M protein can exhibit extreme variability in sequence and immunological properties while retaining a highly specific ligand-binding function

Excretion of Urinary Metabolites of the Phthalate Esters DEP and DEHP in 16 Volunteers after Inhalation and Dermal Exposure

Phthalate esters are suspected endocrine disruptors that are found in a wide range of applications. The aim of this study was to determine the excretion of urinary metabolites in 16 individuals after inhalation and/or dermal exposure to 100⁻300 µg/m³ of deuterium-labelled diethyl phthalate (D₄-DEP) and bis(2-ethylhexyl) phthalate (D₄-DEHP). Dermal exposure in this study represents a case with clean clothing acting as a barrier. After inhalation, D₄-DEP and D₄-DEHP metabolites were excreted rapidly, though inter-individual variation was high. D₄-DEP excretion peaked 3.3 h (T½ of 2.1 h) after combined inhalation and dermal exposure, with total excreted metabolite levels ranging from 0.055 to 2.351 nmol/nmol/m³ (nmol of urinary metabolites per phthalates air concentration in (nmol/m³)). After dermal exposure to D₄-DEP, metabolite excretion peaked 4.6 h (T½ of 2.7 h) after exposure, with excreted metabolite levels in between 0.017 and 0.223 nmol/nmol/m³. After combined inhalation and dermal exposure to D₄-DEHP, the excretion of all five analysed metabolites peaked after 4.7 h on average (T½ of 4.8 h), and metabolite levels ranged from 0.072 to 1.105 nmol/nmol/m³ between participants. No dermal uptake of particle phase D₄-DEHP was observed. In conclusion, the average excreted levels of metabolites after combined inhalation and dermal exposure to D₄-DEP was three times higher than after combined exposure to D₄-DEHP; and nine times higher than after dermal exposure of D₄-DEP. This study was made possible due to the use of novel approaches, i.e., the use of labelled phthalate esters to avoid the background concentration, and innovative technique of phthalate generation, both in the particle and the gas phase

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

Selective Nonsteroidal Glucocorticoid Receptor Modulators for the Inhaled Treatment of Pulmonary Diseases

A class of potent, nonsteroidal, selective indazole ether-based glucocorticoid receptor modulators (SGRMs) was developed for the inhaled treatment of respiratory diseases. Starting from an orally available compound with demonstrated anti-inflammatory activity in rat, a soft-drug strategy was implemented to ensure rapid elimination of drug candidates to minimize systemic GR activation. The first clinical candidate <b>1b</b> (AZD5423) displayed a potent inhibition of lung edema in a rat model of allergic airway inflammation following dry powder inhalation combined with a moderate systemic GR-effect, assessed as thymic involution. Further optimization of inhaled drug properties provided a second, equally potent, candidate, <b>15m</b> (AZD7594), that demonstrated an improved therapeutic ratio over the benchmark inhaled corticosteroid <b>3</b> (fluticasone propionate) and prolonged the inhibition of lung edema, indicating potential for once-daily treatment