Inhibition of Streptococcus pyogenes adherence to HaCaT cells by a peptide corresponding to the streptococcal fibronectin-binding protein, SfbI, is strain dependent

Streptococcus pyogenes (group A streptococcus, GAS) is a human-specific pathogen, which employs a large number of adhesins for colonization. Fibronectin-binding proteins (FBPs) play a major role in GAS adhesion to host cells. SfbI, a major streptococcal FBP, has been well studied. A peptide (peptide-MSG) based on this adhesin inhibits fibronectin (Fn)-binding by the pathogen. To test whether this peptide also inhibits adherence of GAS to host cells, adhesion assays were performed with strains possessing different combinations of genes for three distinct FBPs. Peptide-MSG inhibited GAS adherence to human keratinocytes (HaCaT) in a strain dependent manner. There is no consistent pattern between the effect and the ability to express one or more of the FBPs. A single peptide may be insufficient to prevent GAS adherence to host cells.     

Paxillin phosphorylation: bifurcation point downstream of integrin-linked kinase (ILK) in streptococcal invasion

Efficient group A streptococcus (GAS) invasion of mammalian cells requires fibronectin (Fn) binding proteins, such as M1 and PrtF1/SfbI, that bridge bacteria to integrins and activate cellular signalling for ingestion. Previous studies of GAS invasion, mediated by both proteins, suggest a common signalling pathway. However, distinct cellular morphological changes at the port of bacterial entry suggest that different signals are also induced. Here we report that paxillin is phosphorylated in response to Fn-bound GAS that express either M1 or PrtF1/SfbI protein, but is not phosphorylated in response to a mutant deficient in both proteins. Inhibition of paxillin phosphorylation by a tyrosine kinase inhibitor, PP2, or by expression of a dominant negative form of paxillin significantly reduced invasion by M1+ but did not affect ingestion of PrtF1/SfbI+ strains. In contrast, another tyrosine inhibitor, genistein, did not significantly prevent paxillin phosphorylation and had no effect on ingestion of the M1+ strain, but reduced PrtF1/SfbI-mediated entry. This suggests that paxillin phosphorylation is induced by both proteins but only required for M1-mediated invasion. A bifurcation point, downstream of integrin-linked kinase (ILK) and phosphoinositide 3-kinase, likely accounts for the distinct morphological changes. Furthermore, ILK activity is indispensable for M1-induced paxillin recruitment and phosphorylation.     

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.     

Protein F2, a novel fibronectin-binding protein from Streptococcus pyogenes, possesses two binding domains

Binding of the group A streptococcus (GAS) to respiratory epithelium is mediated by the fibronectin (Fn)-binding adhesin, protein F1. Previous studies have suggested that certain GAS strains express Fn-binding proteins that are different from protein F1. In this study, we have cloned, sequenced, and characterized a gene ( prtF2 ) from GAS strain 100076 encoding a novel Fn-binding protein, termed protein F2. Insertional inactivation of prtF2 in strain 100076 abolishes its high-affinity Fn binding. prtF2 -related genes exist in most GAS strains that lack prtF1 (encoding protein F1) but bind Fn with high affinity. These observations suggest that protein F2 is a major Fn-binding protein in GAS. Protein F2 is highly homologous to Fn-binding proteins from Streptococcus dysgalactiae and Strep-tococcus equisimilis , particularly in its carboxy-terminal portion. Two domains are responsible for Fn binding by protein F2. One domain (FBRD) consists of three consecutive repeats, whereas the other domain (UFBD) resides on a non-repeated stretch of approximately 100 amino acids and is located 100 amino acids amino-terminal of FBRD. Each of these domains is capable of binding Fn when expressed as a separate protein. In strain 100076, protein F2 activity is regulated in response to alterations in the concentration of atmospheric oxygen.     

Two distinct genotypes of prtF2, encoding a fibronectin binding protein, and evolution of the gene family in Streptococcus pyogenes

The group A Streptococcus (GAS) is an important pathogen that is responsible for a wide range of human diseases. Fibronectin binding proteins (FBPs) play an important role in promoting GAS adherence and invasion of host cells. The prtF2 gene encodes an FBP and is present in approximately 60% of GAS strains. In the present study we examined 51 prtF2-positive GAS strains isolated from the Northern Territory of Australia, and here we describe two genotypes of prtF2 which are mutually exclusive. The two genotypes have been identified previously as pfbp and fbaB. We show that these genotypes map to the same chromosomal location within the highly recombinatorial fibronectin-collagen-T antigen (FCT) locus, indicating that they arose from a common ancestor, and in this study these genotypes were designated the pfbp type and the fbaB type. Phylogenetic analysis of seven pfbp types, 14 fbaB types, and 11 prtF2-negative GAS strains by pulsed-field gel electrophoresis (PFGE) produced 32 distinct PFGE patterns. Interpretation of evolution based on the PFGE dendrogram by parsimony suggested that the pfbp type had a recent origin compared to the fbaB type. A comparison of multiple DNA sequences of the pfbp and fbaB types revealed a mosaic pattern for the amino-terminal region of the pfbp types. The fbaB type is generally conserved at the amino terminus but varies in the number of fibronectin binding repeats in the carboxy terminus. Our data also suggest that there is a possible association of the pfbp genotype with sof (84.2%), while the fbaB genotype was found in a majority of the GAS strains negative for sof (90.6%), indicating that these two prtF2 subtypes may be under different selective pressures.     

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.     

High affinity streptococcal binding to human fibronectin requires specific recognition of sequential F1 modules

Fibronectin (Fn) binding by the Streptococcus pyogenes protein SfbI has been shown to trigger integrin-dependent internalization of this pathogen by human epithelial and endothelial cells. Here, using nuclear magnetic resonance spectroscopy and isothermal titration calorimetry in a dissection approach, the basis for the specificity and high affinity of the interaction between the N-terminal domain of Fn and SfbI is revealed. Each of the five Fn type 1 modules is directly involved in the interaction and is recognized by short consecutive motifs within the repeat region of SfbI. Crucially, these motifs must be combined in the correct order to form a high affinity ligand for the N-terminal domain of Fn.     

Presence of fibronectin-binding protein gene prtF2 in invasive group A streptococci in tropical Australia is associated with increased internalisation efficiency

The fibronectin-binding proteins (FnBPs) PrtF1 and PrtF2 are considered to be major group A streptococcal virulence factors, mediating adherence to and internalisation of host cells. The present study investigated an association between the presence of prtF1 and prtF2 genes and internalisation efficiency in group A streptococci (GAS) isolated from patients with invasive disease. Of the 80 isolates tested, 58 (73%) had prtF1 and 71 (89%) possessed prtF2. Three isolates (4%) had neither gene, seven (9%) had prtF1 only, 19 (24%) had prtF2 only and 51 isolates (64%) had both prtF1 and prtF2. prtF2-positive isolates internalised up to three times more efficiently than isolates that had prtF1 alone (P<0.001), and 1.5-fold better than isolates that had neither gene. No significant association was found between internalisation efficiency and presence of the prtF1 gene. Analysis of the fibronectin-binding repeat domain (FBRD) of prtF2 revealed that this gene can contain 2, 3, 4 or 5 repeat regions and that five repeat regions conferred very high internalisation efficiency in invasive GAS isolates.     

Roles of integrins and fibronectin in the entry of Streptococcus pyogenes into cells via protein F1

Entry of group A streptococcus (GAS) into cells has been suggested as an important trait in GAS pathogenicity. Protein F1, a fibronectin (Fn) binding protein, mediates GAS adherence to cells and the extracellular matrix, and efficient cell internalization. We demonstrate that the cellular receptors responsible for protein F1-mediated internalization of GAS are integrins capable of Fn binding. In HeLa cells, bacterial entry is blocked by anti-β1 integrin monoclonal antibody. In the mouse cell line GD25, a β1 null mutant, the αvβ3 integrin promotes GAS entry. Internalization of these cells by GAS is blocked by a peptide that specifically binds to αvβ3 integrin. In both cell lines, entry of GAS requires the occupancy of protein F1 by Fn. Neither the 29 kDa nor the 70 kDa N-terminal fragments or the 120 kDa cell-binding fragment of Fn promote bacterial entry. Fn-coated beads are taken up efficiently by HeLa cells. Both the entry of GAS via protein F1 and the uptake of Fn-coated beads are blocked by anti-β1 antibody but are unaffected by a large excess of soluble Fn. Internalization of HeLa cells by bacteria bearing increasing amounts of prebound Fn to protein F1 reveals a sigmoidal ultrasensitive curve. These suggest that the ability of particles to interact via Fn with multiple integrin sites plays a central role in their ability to enter cells.     

Integrin-linked kinase is an essential link between integrins and uptake of bacterial pathogens by epithelial cells

Entry of Streptococcus pyogenes or group A streptococcus (GAS) into host cells is mediated by fibronectin bound to surface proteins, M1 or PrtF1, forming a bridge to alpha5beta1 integrins. This interaction leads to cytoskeletal rearrangement and uptake of streptococci. We postulated that integrin-linked kinase (ILK), which directly associates with integrins, is the universal link between integrins and several bacterial pathogens. We showed that inhibition of ILK expression by siRNA silencing, or ILK kinase activity by chemical inhibitors or expression of a dominant negative form of ILK reduced M1-mediated invasion of epithelial cells up to 80%. To evaluate the ILK requirement for PrtF1-mediated GAS invasion, a M1-PrtF1+ recombinant strain within the M1 background was constructed. Inhibition of ILK kinase activity also significantly reduced invasion of epithelial cells by this recombinant and wild-type strain JRS4 that expresses PrtF1. In addition, impaired ILK kinase activity results in significant reduction of integrin-dependent invasion mediated by invasins of two other important pathogens, Staphylococcus aureus and Yersinia spp. This study suggests that bacterial pathogens evolved different molecules and strategies to exploit the host integrin signalling pathway for their survival.     

Co-operative binding of human fibronectin to Sfbl 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.     

Novel mycobacteria antigen 85 complex binding motif on fibronectin

The members of the antigen 85 protein family (Ag85), consisting of members Ag85A, Ag85B, and Ag85C, are the predominantly secreted proteins of mycobacteria and possess the ability to specifically interact with fibronectin (Fn). Because Fn-binding proteins are likely to be important virulence factors of Mycobacterium spp., Ag85 may contribute to the adherence, invasion, and dissemination of organisms in host tissue. In this study, we reported the Fn binding affinity of Ag85A, Ag85B, and Ag85C from Mycobacterium avium subsp. paratuberculosis (MAP) (K(D) values were determined from 33.6 to 68.4 nm) and mapped the Ag85-binding motifs of Fn. Fn14, a type III module located on the heparin-binding domain II (Hep-2) of Fn, was discovered to interact with Ag85 from MAP. The peptide inhibition assay subsequently demonstrated that a peptide consisting of residues 17-26 from Fn14 ((17)SLLVSWQPPR(26), termed P17-26) could interfere with Ag85B binding to Fn (73.3% reduction). In addition, single alanine substitutions along the sequence of P17-26 revealed that the key residues involved in Ag85-Fn binding likely contribute through hydrophobic and charge interactions. Moreover, binding of Ag85 on Fn siRNA-transfected Caco2 cells was dramatically reduced (44.6%), implying the physiological significance of the Ag85-Fn interaction between mycobacteria and host cells during infection. Our results indicate that Ag85 binds to Fn at a novel motif and plays a critical role in mycobacteria adherence to host cells by initiating infection. Ag85 might serve as an important colonization factor potentially contributing to mycobacterial virulence.     

Role of fibronectin in the adhesion of Acinetobacter baumannii to host cells

Adhesion to host cells is an initial and important step in Acinetobacter baumannii pathogenesis. However, there is relatively little information on the mechanisms by which A. baumannii binds to and interacts with host cells. Adherence to extracellular matrix proteins, such as fibronectin, affords pathogens with a mechanism to invade epithelial cells. Here, we found that A. baumannii adheres more avidly to immobilized fibronectin than to control protein. Free fibronectin used as a competitor resulted in dose-dependent decreased binding of A. baumannii to fibronectin. Three outer membrane preparations (OMPs) were identified as fibronectin binding proteins (FBPs): OMPA, TonB-dependent copper receptor, and 34 kDa OMP. Moreover, we demonstrated that fibronectin inhibition and neutralization by specific antibody prevented significantly the adhesion of A. baumannii to human lung epithelial cells (A549 cells). Similarly, A. baumannii OMPA neutralization by specific antibody decreased significantly the adhesion of A. baumannii to A549 cells. These data indicate that FBPs are key adhesins that mediate binding of A. baumannii to human lung epithelial cells through interaction with fibronectin on the surface of these host cells.     

Fc-mediated nonspecific binding between fibronectin-binding protein I of Streptococcus pyogenes and human immunoglobulins.

Fibronectin-binding protein I (SfbI) from Streptococcus pyogenes plays a key role in bacterial adhesion to, and invasion of, eukaryotic cells. In addition, SfbI exhibits a considerable potential as mucosal adjuvant and can trigger polyclonal activation of B cells. Here, we report that SfbI is also capable of binding human IgG in a nonimmune fashion. SfbI was reactive with IgG1, IgG2, IgG3, and IgG4 isotypes (type IIo IgG-binding profile). The affinity constant (Kd) of the SfbI-IgG interaction was in the range of 1-2 x 10(-5) M. Further studies demonstrated that the SfbI binding was mediated by the Fc component of the IgG molecule. Experiments performed using purified recombinant proteins spanning different domains of SfbI showed that the IgG-binding activity was restricted to the fibronectin-binding domains, and in particular to the fibronectin-binding repeats. Finally, the presence of recombinant SfbI resulted in an impairment of both phagocytosis of IgG-coated RBCs and Ab-dependent cell cytotoxicity by macrophages. These results demonstrated for the first time that, in addition to its major role during the colonization process, SfbI may also favor bacterial immune evasion after the onset of the infection by interfering with host clearance mechanisms.     

Lactobacillus rhamnosus GG inhibits invasion of cultured human respiratory cells by prtF1-positive macrolide-resistant group A streptococci

Aims:  This study was designed to determine whether the probiotic strain Lactobacillus GG, which is extensively used in the treatment and prevention of intestinal disorders, is able to inhibit invasion of cultured human respiratory cells by macrolide-resistant group A streptococci (GAS) carrying the prtF1 gene, which encodes the fibronectin (Fn)-binding invasin F1.
Methods and Results:  Eight prtF1 -positive erythromycin-resistant GAS strains were used to infect A549 monolayers in competition and displacement assays with Lactobacillus GG. Live (L-LGG) and heat-killed (HK-LGG) lactobacilli and their spent culture supernatant (SCS) significantly reduced ( P  <   0·001) GAS invasion efficiency in both assays. No antibacterial activity of Lactobacillus GG against GAS was detected. Both L-LGG and HK-LGG and all prtF1 -positive GAS induced a strong agglutination reaction using Fn-coated particles.
Conclusions:  Lactobacillus GG exerts an antagonistic action against GAS by inhibiting cell invasion. Competitive binding of Lactobacillus GG and GAS to Fn might be involved in the inhibition process.
Significance and Impact of the Study:  The finding that Lactobacillus GG can prevent in vitro invasion of respiratory cells by GAS suggests new applications for this probiotic strain and warrants further studies of its capacity to prevent GAS throat infections.