Susanne R. Talay

SPSA, A NOVEL PNEUMOCOCCAL SURFACE PROTEIN WITH SPECIFIC BINDING TO SECRETORY IMMUNOGLOBULIN A AND SECRETORY COMPONENT

The interaction of pathogenic bacteria with host serum and matrix proteins is a common strategy to enhance their virulence. Streptococcus pneumoniae colonizes the human upper respiratory tract in healthy individuals and is also able to cause invasive diseases. Here, we describe a novel pneumococcal surface protein, SpsA, capable of binding specifically to human secretory immunoglobulin A (SIgA). The dissociation constant of SIgA binding to SpsA was 9.3 × 10−9 M. Free secretory component (SC) also binds to S. pneumoniae, whereas serum IgA does not, suggesting that pneumococcal binding to SIgA is mediated by the SC. To our knowledge, this is the first defined interaction of SC with a prokaryotic protein. The spsA gene encodes a polypeptide of 523 amino acids with a predicted molecular mass of 59 151 Da. The SIgA‐ or SC‐binding domain is located in the N‐terminal part of SpsA and exhibits no significant homology to any other proteins. The purified SIgA‐binding domain of SpsA could completely inhibit the binding of SIgA to pneumococci. SpsA was expressed by 73% of the tested S. pneumoniae isolates and was substantially conserved between different serotypes. The interaction between S. pneumoniae and SC via SpsA represents a novel biological interaction that might increase virulence by the impairment of bacterial clearance.

Characterization of a novel fibronectin‐binding surface protein in group A streptococci

Streptococcus pyogenes interacts with host fibronectin via distinct surface components. One of these components is the Sfbl protein (streptococcal fibronectin‐binding protein, now specified as class I), an adhesin that represents a protein family with characteristic features. Here we present the complete structure of a novel fibronectin‐binding protein of S. pyogenes, designated SfbII, which is distinct from the previously described Sfbl proteins. The sfbII gene originated from a λ EMBL3 library of chromosomal DNA from group A streptococcal strain A75 and coded for a 113kDa protein exhibiting features of membrane‐anchored surface proteins of Gram‐positive cocci. The expression of biologically active fusion proteins allowed the determination of the location of the fibronectin‐binding domain within the C‐terminal part of the protein. It consisted of two and a half repeats which share common motifs with fibronectin‐binding repeats of other streptococcal and staphylococcal proteins. Purified recombinant fusion protein containing this domain competitively inhibited the binding of fibronectin to the parental S. pyogenes strain. Furthermore, polyclonal antibodies against the binding domain specifically blocked the Sfbll receptor site on the streptococcal surface. No cross‐reactivity could be detected between anti‐Sfbll antibodies and the sfbl gene product, and vice versa, indicating that the two proteins do not share common immunogenic epitopes. Southern hybridization experiments performed with specific sfbll gene probes revealed the presence of the sfbll gene in more than 55% of 93 streptococcal isolates tested. The majority of the strains also harboured the sfbl gene, and 86% carried at least one of the two sfb genes.

Protective Immune Response against Streptococcus pyogenes in Mice after Intranasal Vaccination with the Fibronectin-Binding Protein SfbI

Despite the significant impact on human health of Streptococcus pyogenes, an efficacious vaccine has not yet been developed. Here, the potential as a vaccine candidate of a major streptococcal adhesin, the fibronectin-binding protein SfbI, was evaluated. Intranasal immunization of mice with either SfbI alone or coupled to cholera toxin B subunit (CTB) triggered efficient SfbI-specific humoral (mainly IgG) and lung mucosal (14% of total IgA) responses. CTB-immunized control mice were not protected against challenge with S. pyogenes (90%-100% lethality), whereas SfbI-vaccinated animals showed 80% and 90% protection against homologous and heterologous challenge, respectively. Multiple areas of consolidation with diffused cellular infiltrates (macrophages and neutrophils) were observed in lungs from control mice; the histologic structure was preserved in SfbI-vaccinated animals, which occasionally presented focal infiltrates confined to the perivascular, peribronchial, and subpleural areas. These results suggest that SfbI is a promising candidate for inclusion in acellular vaccines against S. pyogenes.

Domain structure and conserved epitopes of Sfb protein, the fibronectin‐binding adhesin of Streptococcus pyogenes

Streptococcus pyogenes expresses a fibronectin‐binding surface protein (Sfb protein) which mediates adherence to human epithelial cells. The nucleotide sequence of the sfb gene was determined and the primary sequence of the Sfb protein was analysed. The protein consists of 638 amino acids and comprises five structurally distinct domains. The protein starts with an N‐terminal signal peptide followed by an aromatic domain. The central part of the protein is formed by four proline‐rich repeats which are flanked by non‐repetitive spacer sequences. A second repeat region, consisting of four repeats that are distinct from the proline repeats and have been shown to form the fibronectin‐binding domain, is located in the Cterminal part of the protein. The protein ends with a typical cell wall and membrane anchor region. Comparative sequence analysis of the N‐terminal aromatic domain revealed similarities with carbohydrate‐binding sites of other proteins. The proline repeat region of the Sfb protein shares characteristic features with proline‐rich repeats of functionally distinct surface proteins from pathogenic Gram‐positive cocci. Immunoelectron microscopy revealed an even distribution of the fibronectin‐binding domain of Sfb protein on the surface of streptococcal cells. Analyses of 38 sfb genes originating from different S. pyogenes isolates revealed primary sequence variability in regions coding for the N‐termini of mature Sfb proteins, whereas sequences coding for the central and C‐terminal repeats were highly conserved. The repeat sequences are postulated to act as target sites for intragenic recombination events that result in variable numbers of repeats within the different sfb genes. A model of the Sfb protein is presented.

Co-operative binding of human fibronectin to SfbI protein triggers streptococcal invasion into respiratory epithelial cells

Streptococcal fibronectin binding protein I (SfbI) mediates adherence to and invasion of Streptococcus pyogenes into human epithelial cells. In this study, we analysed the binding activity of distinct domains of SfbI protein towards its ligand, the extracellular matrix component fibronectin, as well as the biological implication of the binding events during the infection process. By using purified recombinant SfbI derivatives as well as in vivo expressed SfbI domains on the surface of heterologous organism Streptococcus gordonii, we were able to dissociate the two major streptococcal target domains on the human fibronectin molecule. The SfbI repeat region exclusively bound to the 30 kDa N‐terminal fragment of fibronectin, whereas the SfbI spacer region exclusively bound to the 45 kDa collagen‐binding fragment of fibronectin. In the case of native surface‐expressed SfbI protein, an induced fit mode of bacteria–fibronectin interaction was identified. We demonstrate that binding of the 30 kDa fibronectin fragment to the repeat region of SfbI protein co‐operatively activates the adjacent SfbI spacer domain to bind the 45 kDa fibronectin fragment. The biological consequence arising from this novel mode of fibronectin targeting was analysed in eukaryotic cell invasion assays. The repeat region of SfbI protein is mediating adherence and constitutes a prerequisite for subsequent invasion, whereas the SfbI spacer domain efficiently triggers the invasion process of streptococci into the eukaryotic cell. Thus, we were able to dissect bacterial adhesion from invasion by manipulating one protein. SfbI protein therefore represents a highly evolved prokaryotic molecule that exploits the host factor fibronectin not only for extracellular targeting but also for its subsequent activation that leads to efficient cellular invasion.

Pharyngeal carriage of group C and group G streptococci and acute rheumatic fever in an Aboriginal population

Acute rheumatic fever (ARF) and rheumatic heart disease (RHD) represent an autoimmune response to group A streptococcal pharyngitis. In the Aboriginal population of the Northern Territory of Australia, some of the highest rates of ARF in the world have been reported, although throat carriage rates of group A streptococcus in this population are extremely low and symptomatic group A streptococcal pharyngitis is uncommon; by contrast, carriage rates of group C and G streptococci are high. Therefore, we questioned the involvement of these groups in ARF and showed that they have the potential to elicit an autoimmune response that may trigger ARF.

Rheumatic fever–associated Streptococcus pyogenes isolates aggregate collagen

Acute rheumatic fever is a serious autoimmune sequel of Streptococcus pyogenes infection. This study shows that serotype M3 and M18 S. pyogenes isolated during outbreaks of rheumatic fever have the unique capability to bind and aggregate human basement membrane collagen type IV. M3 protein is identified as collagen-binding factor of M3 streptococci, whereas M18 isolates bind collagen through a hyaluronic acid capsule, revealing a novel function for M3 protein and capsule. Following in vivo mouse passage, conversion of a nonencapsulated and collagen-binding negative M1 S. pyogenes into an encapsulated, collagen-binding strain further supports the crucial role of capsule in mediating collagen binding. Collagen binding represents a novel colonization mechanism, as it is demonstrated that S. pyogenes bind to collagen matrix in vitro and in vivo. Moreover, immunization of mice with purified recombinant M3 protein led to the generation of anti-collagen type IV antibodies. Finally, sera from acute rheumatic fever patients had significantly increased titers of anti-collagen type IV antibodies as compared with healthy controls. These findings may suggest a link between the potential of rheumatogenic S. pyogenes isolates to bind collagen, and the presence of collagen-reactive autoantibodies in the serum of rheumatic fever patients, which may form a basis for post-streptococcal rheumatic disease. These anti-collagen antibodies may form a basis for poststreptococcal rheumatic disease.

Host cell caveolae act as an entry-port for Group A streptococci

This study identified caveolae as an entry port for group A streptococci into epithelial and endothelial cells. Scanning electron microscopy as well as ultrathin sections of infected cells demonstrated accumulation of small omega‐shaped cavities in the host cell membrane close to adherent streptococci. During invasion, invaginations were formed that subsequentely revealed intracellular compartments surrounding streptococci. Caveolin‐1 was shown to be present in the membrane of invaginations and the compartment membranes. These compartments were devoid of any classic endosomal/lysosomal marker proteins and can thus be described as caveosomes. Disruption of caveolae with methyl‐β‐cyclodextrin and filipin abolished host cell invasion. Importantly, streptococci inside caveosomes avoid fusion with lysosomes. Expressing of SfbI protein on the surface of the non‐invasive S. gordonii resulted in identical morphological alterations on the host cell as for S. pyogenes. Incubation of HUVEC cells with purified recombinant sole SfbI protein also triggered accumulation of cavity‐like structures and formation of membrane invaginations. Tagged to colloidal gold‐particles, SfbI protein was shown to cluster following membrane contact. Thus, our results demonstrate that host cell caveolae initiate the invasion process of group A streptococci and that the streptococcal invasin SfbI is the triggering factor that activates the caveolae‐mediated endocytic pathway.

Streptococcus pyogenes recruits collagen via surface‐bound fibronectin: a novel colonization and immune evasion mechanism

This study aimed to characterize matrix assembly mechanisms on the surface of the human pathogen Streptococcus pyogenes. Among 125 S. pyogenes isolates, 61% were able to recruit collagen type IV via surface‐bound fibronectin. Streptococcus gordonii expressing the fibronectin‐binding repeat domain of S. pyogenes SfbI protein was equally potent in recruiting collagen, indicating that this domain was sufficient to promote fibronectin‐mediated collagen recruitment. Electron microscopic analysis of streptococci revealed that fibronectin‐mediated collagen recruitment led to matrix deposition on and between streptococcal cells, which induced the formation of large bacterial aggregates. Furthermore, collagen‐recruiting streptococci were able to colonize collagen fibres and were protected from adhering to human polymorphonuclear cells in the presence of op‐sonizing antibodies. Fibronectin‐mediated collagen recruitment thus represents a novel aggregation, colonization and immune evasion mechanism of S. pyogenes.

The role played by the group A streptococcal negative regulator Nra on bacterial interactions with epithelial cells

Group A streptococci (GAS) specifically attach to and internalize into human epithelial host cells. In some GAS isolates, fibronectin‐binding proteins were identified as being responsible for these virulence traits. In the present study, the previously identified global negative regulator Nra was shown to control the binding of soluble fibronectin probably via regulation of protein F2 and/or SfbII expression in the serotype M49 strain 591. According to results from a conventional invasion assay based on the recovery of viable intracellular bacteria, the increased fibronectin binding did not affect bacterial adherence to HEp‐2 epithelial cells, but was associated with a reduction in the internalization rates. However, when examined by confocal and electron microscopy techniques, the nra‐mutant bacteria were shown to exhibit higher adherence and internalization rates than the corresponding wild type. The mutant bacteria escaped from the phagocytic vacuoles much faster, promoting consistent morphological changes which resulted in severe host cell damage. The apoptotic and lytic processes observed in nra‐mutant infected host cells were correlated with an increased expression of the genes encoding superantigen SpeA, the cysteine protease SpeB, and streptolysin S in the nra‐mutant bacteria. Adherence and internalization rates of a nra/speB‐double mutant at wild‐type levels indicated that the altered speB expression in the nra mutant contributed to the observed changes in both processes. The Nra‐dependent effects on bacterial virulence were confined to infections carried out with stationary growth phase bacteria. In conclusion, the obtained results demonstrated that the global GAS regulator Nra modulates virulence genes, which are involved in host cell damage. Thus, by helping to achieve a critical balance of virulence factor expression that avoids the injury of target cells, Nra may facilitate GAS persistence in a safe intracellular niche.