Cellular aspects of the distinct M protein and SfbI anchoring pathways in Streptococcus pyogenes.

Wall-anchored surface proteins are critical for the in vivo survival of Streptococcus pyogenes. Cues in the signal sequence direct the membrane translocation of surface proteins: M protein to the septum, and SfbI to the poles. Both proteins are subsequently anchored to the wall by the membrane bound enzyme sortase A. However, the cellular features of these pathways are not fully understood. Here we show that M protein and SfbI are anchored simultaneously throughout the cell cycle. M protein is rapidly anchored at the septum, and in part of the cell cycle, is anchored simultaneously at the mother and daughter septa. Conversely, SfbI accumulates gradually on peripheral peptidoglycan, resulting in a polar distribution. Sortase is not required for translocation of M protein or SfbI at their respective locations. Methicillin-induced unbalanced peptidoglycan synthesis diminishes surface M protein but not SfbI. Furthermore, overexpression of the division regulator DivIVA also diminishes surface M protein but increases SfbI. These results demonstrate a close connection between the regulation of cell division and protein anchoring. Better understanding of the spatial regulation of surface anchoring may lead to the identification of novel targets for the development of anti-infective agents, given the importance of surface molecules for pathogenesis.

Streptococcus pneumoniae induces exocytosis of Weibel-Palade bodies in pulmonary endothelial cells.

Invasive pneumococcal infections due to Streptococcus pneumoniae lead to inflammatory infiltration of leucocytes into lung alveolus, meninges and to septic dissemination within the vascular system. The lung microvasculature is covered by pulmonary endothelial cells containing Weibel-Palade bodies (WPB) releasing procoagulant von Willebrand factor (vWF) and other proteins in response to inflammatory stimuli. The influence of pathogenic pneumococci on secretion of WPB proteins is unknown. Here, we report that adherence of S. pneumoniae to primary human pulmonary microvascular endothelial cells (HPMEC) stimulates the WPB exocytosis and the secretion of vWF and interleukin 8 (IL-8). Moreover, infection analyses performed with pneumococcal mutants deficient in the expression of cytotoxic pneumolysin demonstrated that, in addition to direct bacterial adherence, sublytic concentrations of pneumolysin stimulated vWF secretion. The release of vWF was induced after infection with pneumococci from both the apical and the basal cell surfaces, which implies a stimulation of WPB exocytosis in both directions: from inside the vasculature and also following invasive pneumococcal transmigration from pulmonary tissue into the bloodstream. In conclusion, this study demonstrates that the most relevant pulmonary pathogen S. pneumoniae induces release of proinflammatory and procoagulative components directly contributing to pathophysiological processes leading to fatal tissue injury during course of infection.

Cooperative binding and activation of fibronectin by a bacterial surface protein.

Integrin-dependent cell invasion of some pathogenic bacteria is mediated by surface proteins targeting the extracellular matrix protein fibronectin (FN). Although the structural basis for bacterial FN recognition is well understood, it has been unclear why proteins such as streptococcal SfbI contain several FN-binding sites. We used microcalorimetry to reveal cooperative binding of FN fragments to arrays of binding sites in SfbI. In combination with thermodynamic analyses, functional cell-based assays show that SfbI induces conformational changes in the N-terminal 100-kDa region of FN (FN100kDa), most likely by competition with intramolecular interactions defining an inactive state of FN100kDa. This study provides insights into how long range conformational changes resulting in FN activation may be triggered by bacterial pathogens.

The FbaB-type fibronectin-binding protein of Streptococcus pyogenes promotes specific invasion into endothelial cells.

Invasive serotype M3 Streptococcus pyogenes are among the most frequently isolated organisms from patients suffering from invasive streptococcal disease and have the potential to invade primary human endothelial cells (EC) via a rapid and efficient mechanism. FbaB protein, the fibronectin-binding protein expressed by M3 S. pyogenes, was herein identified as a potent invasin for EC. By combining heterologous gene expression with allelic replacement, we demonstrate that FbaB is essential and sufficient to trigger EC invasion via a Rac1-dependent phagocytosis-like uptake. FbaB-mediated uptake follows the classical endocytic pathway with lysosomal destination. FbaB is demonstrated to be a streptococcal invasin exhibiting EC tropism. FbaB thus initiates a process that may contribute to the deep tissue tropism and spread of invasive S. pyogenes isolates into the vascular EC lining.

Differences in the aromatic domain of homologous streptococcal fibronectin-binding proteins trigger different cell invasion mechanisms and survival rates.

Group A streptococci (GAS, Streptococcus pyogenes) and Group G streptococci (GGS, Streptococcus dysgalactiae ssp. equisimilis) adhere to and invade host cells by binding to fibronectin. The fibronectin-binding protein SfbI from GAS acts as an invasin by using a caveolae-mediated mechanism. In the present study we have identified a fibronectin-binding protein, GfbA, from GGS, which functions as an adhesin and invasin. Although there is a high degree of similarity in the C-terminal sequence of SfbI and GfbA, the invasion mechanisms are different. Unlike caveolae-mediated invasion by SfbI-expressing GAS, the GfbA-expressing GGS isolate trigger cytoskeleton rearrangements. Heterologous expression of GfbA on the surface of a commensal Streptococcus gordonii and purified recombinant protein also triggered actin rearrangements. Expression of a truncated GfbA (lacking the aromatic domain) and chimeric GfbA/SfbI protein (replacing the aromatic domain of SfbI with the GfbA aromatic domain) on S. gordonii or recombinant proteins alone showed that the aromatic domain of GfbA is responsible for different invasion mechanisms. This is the first evidence for a biological function of the aromatic domain of fibronectin-binding proteins. Furthermore, we show that streptococci invading via cytoskeleton rearrangements and intracellular trafficking along the classical endocytic pathway are less persistence than streptococci entering via caveolae.

The CXC chemokine-degrading protease SpyCep of Streptococcus pyogenes promotes its uptake into endothelial cells.

Streptococcus pyogenes expresses the LPXTG motif-containing cell envelope serine protease SpyCep (also called ScpC, PrtS) that degrades and inactivates the major chemoattractant interleukin 8 (IL-8), thereby impairing host neutrophil recruitment. In this study, we identified a novel function of SpyCep: the ability to mediate uptake into primary human endothelial cells. SpyCep triggered its uptake into endothelial cells but not into human epithelial cells originating from pharynx or lung, indicating an endothelial cell-specific uptake mechanism. SpyCep mediated cellular invasion by an endosomal/lysosomal pathway distinct from the caveolae-mediated invasion pathway of S. pyogenes. Recombinant expression and purification of proteolytically active SpyCep and a series of subfragments allowed functional dissection of the domains responsible for endothelial cell invasion and IL-8 degradation. The N-terminal PR domain was sufficient to mediate endothelial cell invasion, whereas for IL-8-degrading activity, the protease domain and the flanking A domain were required. A polyclonal rabbit serum raised against the recombinant protease efficiently blocked the invasion-mediating activity of SpyCep but not its proteolytic function, further indicating that SpyCep-mediated internalization is independent from its enzymatic activity. SpyCep may thus specifically mediate its own uptake as secreted protein into human endothelial cells.

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.

The M1 protein of Streptococcus pyogenes triggers an innate uptake mechanism into polarized human endothelial cells.

Serotype M1 Streptococcus pyogenes is a major human pathogen associated with severe invasive diseases causing high morbidity and mortality. In a substantial number of cases, invasive disease develops in previously healthy individuals with no obvious port of entry. This has led to the hypothesis that the source of streptococci in these cases is a transient bacteraemia. This study focuses on the analysis of interaction of tissue-invasive serotype M1 S. pyogenes with human endothelial cells (EC) of the vascular system. We identify the M1 surface protein of S. pyogenes as the EC invasin which is recognised by polarized human blood EC, thereby triggering rapid, phagocytosis-like uptake of streptococci into polarized EC layers. Upon internalization, the M1 S. pyogenes serotype is incorporated into phagosomes which traffic via the endosomal/lysosomal pathway. However, some of the streptococci successfully evade this innate killing process and hereby mediate their escape into the cytoplasm of the host cell. The results of this study demonstrate that blood EC possess an efficient uptake mechanism for serotype M1 S. pyogenes. Despite efficient phagocytosis, streptococcal survival within EC constitutes one potential mechanism which favours intracellular persistence and thus facilitates continuous infection and dissemination from the primary side of infection into deep tissue.

Molecular mechanisms of Streptococcus dysgalactiae subsp equisimilis enabling intravascular persistence.

Streptococcus dysgalactiae subsp. equisimilis (SDSE) can cause recurrent bacteremic infection. We have characterized novel virulence properties of an SDSE isolate of type stG485.0 that caused severe sepsis three times in a patient despite that he had opsonizing antibodies to the isolate. An infected aortic aneurysm was suspected to be the focus for the persisting bacteria. For the first time we show that this SDSE isolate, as well as other invasive SDSE isolates, aggregate human platelets and efficiently internalize into human endothelial cells. These properties may aid SDSE to persist and could explain the tendency of SDSE to cause recurrent bacteremia.

Lack of the delta subunit of RNA polymerase increases virulence related traits of Streptococcus mutans.

The delta subunit of the RNA polymerase, RpoE, maintains the transcriptional specificity in gram-positive bacteria. Lack of RpoE results in massive changes in the transcriptome of the human dental caries pathogen Streptococcus mutans. In this study, we analyzed traits of the ΔrpoE mutant which are important for biofilm formation and interaction with oral microorganisms and human cells and performed a global phenotypic analysis of its physiological functions. The ΔrpoE mutant showed higher self-aggregation compared to the wild type and coaggregated with other oral bacteria and Candida albicans. It formed a biofilm with a different matrix structure and an altered surface attachment. The amount of the cell surface antigens I/II SpaP and the glucosyltransferase GtfB was reduced. The ΔrpoE mutant displayed significantly stronger adhesion to human extracellular matrix components, especially to fibronectin, than the wild type. Its adhesion to human epithelial cells HEp-2 was reduced, probably due to the highly aggregated cell mass. The analysis of 1248 physiological traits using phenotype microarrays showed that the ΔrpoE mutant metabolized a wider spectrum of carbon sources than the wild type and had acquired resistance to antibiotics and inhibitory compounds with various modes of action. The reduced antigenicity, increased aggregation, adherence to fibronection, broader substrate spectrum and increased resistance to antibiotics of the ΔrpoE mutant reveal the physiological potential of S. mutans and show that some of its virulence related traits are increased.

Invasion of endothelial cells by tissue-invasive M3 type group A streptococci requires Src kinase and activation of Rac1 by a phosphatidylinositol 3-kinase-independent mechanism.

Streptococcus pyogenes can cause invasive diseases in humans, such as sepsis or necrotizing fasciitis. Among the various M serotypes of group A streptococci (GAS), M3 GAS lacks the major epithelial invasins SfbI/PrtF1 and M1 protein but has a high potential to cause invasive disease. We examined the uptake of M3 GAS into human endothelial cells and identified host signaling factors required to initiate streptococcal uptake. Bacterial uptake is accompanied by local F-actin accumulation and formation of membrane protrusions at the entry site. We found that Src kinases and Rac1 but not phosphatidylinositol 3-kinases (PI3Ks) are essential to mediate S. pyogenes internalization. Pharmacological inhibition of Src activity reduced bacterial uptake and abolished the formation of membrane protrusions and actin accumulation in the vicinity of adherent streptococci. We found that Src kinases are activated in a time-dependent manner in response to M3 GAS. We also demonstrated that PI3K is dispensable for internalization of M3 streptococci and the formation of F-actin accumulations at the entry site. Furthermore, Rac1 was activated in infected cells and accumulated with F-actin in a PI3K-independent manner at bacterial entry sites. Genetic interference with Rac1 function inhibited streptococcal internalization, demonstrating an essential role of Rac1 for the uptake process of streptococci into endothelial cells. In addition, we demonstrated for the first time accumulation of the actin nucleation complex Arp2/3 at the entry port of invading M3 streptococci.

Crucial role of the CB3-region of collagen IV in PARF-induced acute rheumatic fever.

Acute rheumatic fever (ARF) and rheumatic heart disease are serious autoimmune sequelae to infections with Streptococcus pyogenes. Streptococcal M-proteins have been implicated in ARF pathogenesis. Their interaction with collagen type IV (CIV) is a triggering step that induces generation of collagen-specific auto-antibodies. Electron microscopy of the protein complex between M-protein type 3 (M3-protein) and CIV identified two prominent binding sites of which one is situated in the CB3-region of CIV. In a radioactive binding assay, M3-protein expressing S. pyogenes and S. gordonii bound the CB3-fragment. Detailed analysis of the interactions by surface plasmon resonance measurements and site directed mutagenesis revealed high affinity interactions with dissociation constants in the nanomolar range that depend on the recently described collagen binding motif of streptococcal M-proteins. Because of its role in the induction of disease-related collagen autoimmunity the motif is referred to as "peptide associated with rheumatic fever" (PARF). Both, sera of mice immunized with M3-protein as well as sera from patients with ARF contained anti-CB3 auto-antibodies, indicating their contribution to ARF pathogenesis. The identification of the CB3-region as a binding partner for PARF directs the further approaches to understand the unusual autoimmune pathogenesis of PARF-dependent ARF and forms a molecular basis for a diagnostic test that detects rheumatogenic streptococci.

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 subsequently 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-beta-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 opsonizing antibodies. Fibronectin-mediated collagen recruitment thus represents a novel aggregation, colonization and immune evasion mechanism of S. pyogenes.

Molecular analysis of group B protective surface protein, a new cell surface protective antigen of group B streptococci.

Group B streptococci (GBS) express various surface antigens designated c, R, and X antigens. A new R-like surface protein from Streptococcus agalactiae strain Compton R has been identified by using a polyclonal antiserum raised against the R protein fraction of this strain to screen a lambda Zap library. DNA sequence analysis of positive clones allowed the prediction of the primary structure of a 105-kDa protein designated BPS protein (group B protective surface protein) that exhibited typical features of streptococcal surface proteins such as a signal sequence and a membrane anchor region but did not show significant similarity with other known sequences. Immunogold electron microscopy using a BPS-specific antiserum confirmed the surface location of BPS protein on S. agalactiae strain Compton R. Anti-BPS antibodies did not cross-react with R1 and R4 proteins expressed by two variant type III GBS strains but reacted with the parental streptococcal strain in Western blot and immunoprecipitation analyses. Separate R3 and BPS immunoprecipitation bands were observed when a cell extract of strain Compton R was tested with an antiserum against Compton R previously cross-absorbed to remove R4 antibodies. Immunization of mice with recombinant BPS protein by the subcutaneous route produced an efficient antigen-specific response, and immunized animals survived challenge with a lethal dose of a virulent strain. Therefore, BPS protein represents a new R-like protective antigen of GBS.

Evolution of sfbI encoding streptococcal fibronectin-binding protein I: horizontal genetic transfer and gene mosaic structure.

Streptococcal fibronectin-binding protein is an important virulence factor involved in colonization and invasion of epithelial cells and tissues by Streptococcus pyogenes. In order to investigate the mechanisms involved in the evolution of sfbI, the sfbI genes from 54 strains were sequenced. Thirty-four distinct alleles were identified. Three principal mechanisms appear to have been involved in the evolution of sfbI. The amino-terminal aromatic amino acid-rich domain is the most variable region and is apparently generated by intergenic recombination of horizontally acquired DNA cassettes, resulting in a genetic mosaic in this region. Two distinct and divergent sequence types that shared only 61 to 70% identity were identified in the central proline-rich region, while variation at the 3' end of the gene is due to deletion or duplication of defined repeat units. Potential antigenic and functional variabilities in SfbI imply significant selective pressure in vivo with direct implications for the microbial pathogenesis of S. pyogenes.