Evasion of phagocytosis through cooperation between two ligand-binding regions in Streptococcus pyogenes M protein.

The M protein of Streptococcus pyogenes is a major bacterial virulence factor that confers resistance to phagocytosis. To analyze how M protein allows evasion of phagocytosis, we used the M22 protein, which has features typical of many M proteins and has two well-characterized regions binding human plasma proteins: the hypervariable NH2-terminal region binds C4b-binding protein (C4BP), which inhibits the classical pathway of complement activation; and an adjacent semivariable region binds IgA-Fc. Characterization of chromosomal S. pyogenes mutants demonstrated that each of the ligand-binding regions contributed to phagocytosis resistance, which could be fully explained as cooperation between the two regions. Deposition of complement on S. pyogenes occurred almost exclusively via the classical pathway, even under nonimmune conditions, but was down-regulated by bacteria-bound C4BP, providing an explanation for the ability of bound C4BP to inhibit phagocytosis. Different opsonizing antisera shared the ability to block binding of both C4BP and IgA, suggesting that the two regions in M22 play important roles also under immune conditions, as targets for protective antibodies. These data indicate that M22 and similar M proteins confer resistance to phagocytosis through ability to bind two components of the human immune system

A Proline-Rich Region with a Highly Periodic Sequence in Streptococcal beta Protein Adopts the Polyproline II Structure and Is Exposed on the Bacterial Surface.

Proline-rich regions have been identified in many surface proteins of pathogenic streptococci and staphylococci. These regions have been suggested to be located in cell wall-spanning domains and/or to be required for surface expression of the protein. Because little is known about these regions, which are found in extensively studied and biologically important surface proteins, we characterized the proline-rich region in one such protein, the beta protein of group B streptococci. The proline-rich region in beta, designated the XPZ region, has a proline at every third position, and the sequence is highly periodic in other respects. Immunochemical analysis showed that the XPZ region was not associated with the cell wall but was exposed on the bacterial surface. Moreover, characterization of a beta mutant lacking the XPZ region demonstrated that this region was not required for surface expression of the beta protein. Comparison of the XPZ region in different beta proteins showed that it varied in size but always retained the typical sequence periodicity. Circular dichroism spectroscopy indicated that the XPZ region had the structure of a polyproline II helix, an extended and solvent-exposed structure with exactly three residues per turn. Because of the three-residue sequence periodicity in the XPZ region, it is expected to be amphipathic and to have distinct nonpolar and polar surfaces. This study identified a proline-rich structure with unique properties that is exposed on the surface of an important human pathogen

Human CEACAM1 is targeted by a Streptococcus pyogenes adhesin implicated in puerperal sepsis pathogenesis

Life-threatening bacterial infections in women after childbirth, known as puerperal sepsis, resulted in classical epidemics and remain a global health problem. While outbreaks of puerperal sepsis have been ascribed to Streptococcus pyogenes, little is known about disease mechanisms. Here, we show that the bacterial R28 protein, which is epidemiologically associated with outbreaks of puerperal sepsis, specifically targets the human receptor CEACAM1. This interaction triggers events that would favor development of puerperal sepsis, including adhesion to cervical cells, suppression of epithelial wound repair and subversion of innate immune responses. High-resolution structural analysis showed that an R28 domain with IgI3-like fold binds to the N-terminal domain of CEACAM1. Together, these findings demonstrate that a single adhesin-receptor interaction can drive the pathogenesis of bacterial sepsis and provide molecular insights into the pathogenesis of one of the most important infectious diseases in medical history

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

Protective Activity of Streptococcus pneumoniae Spr1875 Protein Fragments Identified Using a Phage Displayed Genomic Library

There is considerable interest in pneumococcal protein antigens capable of inducing serotype-independent immunoprotection and of improving, thereby, existing vaccines. We report here on the immunogenic properties of a novel surface antigen encoded by ORF spr1875 in the R6 strain genome. An antigenic fragment encoded by spr1875, designated R4, was identified using a Streptococcus pneumoniae phage displayed genomic library after selection with a human convalescent serum. Immunofluorescence analysis with anti-R4 antisera showed that Spr1875 was expressed on the surface of strains belonging to different serotypes. Moreover, the gene was present with little sequence variability in 27 different pneumococcal strains isolated worldwide. A mutant lacking Spr1875 was considerably less virulent than the wild type D39 strain in an intravenous mouse model of infection. Moreover, immunization with the R4 recombinant fragment, but not with the whole Spr1875 protein, induced significant protection against sepsis in mice. Lack of protection after immunization with the whole protein was related to the presence of immunodominant, non-protective epitopes located outside of the R4 fragment. In conclusion, our data indicate that Spr1875 has a role in pneumococcal virulence and is immunogenic. As the R4 fragment conferred immunoprotection from experimental sepsis, selected antigenic fragments of Spr1875 may be useful for the development of a pneumococcal protein-based vaccine

Streptococcal protein Rib and related proteins: a family of repetitive surface proteins that elicit protective immunity

ABSTRACT Streptococci constitute a heterogeneous group of Gram-positive bacteria, divided into different serological groups on the basis of antigenic differences in a cell wall-associated carbohydrate. This thesis describes related surface proteins expressed by bacteria in two of these groups, the group B streptococcus (GBS) and the group A streptococcus (GAS). GBS is an encapsulated bacterium that is the major cause of invasive bacterial infections in the neonatal period. We have identified and purified a novel cell surface protein, Rib, which is expressed by many GBS strains causing invasive infections. Protein Rib confers protective immunity in a mouse model, making it of interest for analysis of pathogenetic mechanisms and for vaccine development. Sequence analysis demonstrated that the sequence of Rib is related to that of a previously described GBS protein, the alpha protein. Both of these proteins have exceptionally long signal peptides and their sequences are extremely repetitive. Although the two proteins show extensive amino acid residue identity they do not cross-react immunologically. Rib and alpha share several properties i.e. size variation between strains and protease resistance, and they show a characteristic laddering pattern when analyzed in Western blots, due to hydrolysis of acid-labile Asp-Pro bonds. These data show that the Rib and alfa proteins are members of a novel family of streptococcal surface proteins with unusual repetitive structure. R28 is a surface protein expressed by some strains of group A streptococci (Streptococcus pyogenes). The R28 protein cross-reacts immunologically with the Rib protein of GBS, and sequence analysis showed that R28 is a member of the same family of repetitive proteins as Rib and alpha. R28 promotes adhesion to human epithelial cells, and has the important property to elicit protective immunity. Interestingly, the R28 and Rib proteins, which are expressed by different bacterial species, confer cross-protection, i.e. immunization with R28 protects against Rib-expressing strains, and vice versa

Protein rib: a novel group B streptococcal cell surface protein that confers protective immunity and is expressed by most strains causing invasive infections

The group B Streptococcus, an important cause of invasive infections in the neonate, is classified into four major serotypes (Ia, Ib, II, and III) based on the structure of the polysaccharide capsule. Since the capsule is a known virulence factor, it has been extensively studied, in particular in type III strains, which cause the majority of invasive infections. Two cell surface proteins, alpha and beta, have also been studied in detail since they confer protective immunity, but these proteins are usually not expressed by type III strains. We describe here a cell surface protein, designated protein Rib (resistance to proteases, immunity, group B), that confers protective immunity and is expressed by most strains of type III. Protein Rib was first identified as a distinct 95-kD protein in extracts of a type III strain, and was purified to homogeneity from that strain. Rabbit antiserum to protein Rib was used to demonstrate that it is expressed on the cell surface of 31 out of 33 type III strains, but only on 1 out of 25 strains representing the other three serotypes. Mouse protection tests showed that antiserum to protein Rib protects against lethal infection with three different strains expressing this antigen, including a strain representing a recently identified high virulence type III clone. Protein Rib is immunologically unrelated to the alpha and beta proteins, but shares several features with the alpha protein. Most importantly, the NH2-terminal amino acid sequences of the Rib and alpha proteins are identical at 6 out of 12 positions. In addition, both protein Rib and the alpha protein are relatively resistant to trypsin (and Rib is also resistant to pepsin) and both proteins vary greatly in size between different clinical isolates. Finally, both protein Rib and the alpha protein exhibit a regular ladderlike pattern in immunoblotting experiments, which may reflect a repetitive structure. Taken together, these data suggest that the Rib and alpha proteins are members of a family of proteins with related structure and function. Since protein Rib confers protective immunity, it may be valuable for the development of a protein vaccine against the group B Streptococcus, an encapsulated bacterium