M41型A群链球菌的Ⅰ型胶原样蛋白与人低密度脂蛋白的相互作用

【目的】检测M41型A群链球菌(GAS)ATCC12373中Ⅰ型胶原样蛋白(Scl1)与人低密度脂蛋白(LDL)的相互作用。【方法】克隆了M41型GAS ATCC12373的Ⅰ型胶原样蛋白(Scl1)及其V区(Scl1-V)基因,并表达、纯化重组蛋白rScl1(C176)和rScl1-V(C176V)。通过重组蛋白与人血浆的亲和色谱层析、Western blot及酶联免疫吸附试验(ELISA)检测C176、C176V与LDL的相互作用;通过GAS与LDL的ELISA试验和人血浆与GAS的共孵育试验,检测GAS与LDL的相互作用。【结果】结果证明C176和C176V可以与LDL特异性结合;表达Scl1的M41型GAS可以与LDL相结合。【结论】M41型GAS的Scl1可以与LDL特异性结合。     

The Scl1 protein of M6-type group A Streptococcus binds the human complement regulatory protein, factor H, and inhibits the alternative pathway of complement

Non-specific activation of the complement system is regulated by the plasma glycoprotein factor H (FH). Bacteria can avoid complement-mediated opsonization and phagocytosis through acquiring FH to the cell surface. Here, we characterize an interaction between the streptococcal collagen-like protein Scl1.6 of M6-type group A Streptococcus (GAS) and FH. Using affinity chromatography with immobilized recombinant Scl1.6 protein, we co-eluted human plasma proteins with molecular weight of 155 kDa, 43 kDa and 38 kDa. Mass spectrometry identified the 155 kDa band as FH and two other bands as isoforms of the FH-related protein-1. The identities of all three bands were confirmed by Western immunoblotting with specific antibodies. Structure-function relation studies determined that the globular domain of the Scl1.6 variant specifically binds FH while fused to collagenous tails of various lengths. This binding is not restricted to Scl1.6 as the phylogenetically linked Scl1.55 variant also binds FH. Functional analyses demonstrated the cofactor activity of the rScl1.6-bound FH for factor I-mediated cleavage of C3b. Finally, purified FH bound to the Scl1.6 protein present in the cell wall material obtained from M6-type GAS. In conclusion, we have identified a functional interaction between Scl1 and plasma FH, which may contribute to GAS evasion of complement-mediated opsonization and phagocytosis.     

Binding of the low-density lipoprotein by streptococcal collagen-like protein Scl1 of Streptococcus pyogenes

Several bacterial genera express proteins that contain collagen-like regions, which are associated with variable (V) non-collagenous regions. The streptococcal collagen-like proteins, Scl1 and Scl2, of group A Streptococcus (GAS) are members of this 'prokaryotic collagen' family, and they too contain an amino-terminal non-collagenous V region of unknown function. Here, we use recombinant rScl constructs, derived from several Scl1 and Scl2 variants, and affinity chromatography to identify Scl ligands present in human plasma. First, we show that Scl1, but not Scl2, proteins from different GAS serotypes bind the same ligand identified as apolipoprotein B (ApoB100), which is a major component of the low-density lipoprotein (LDL). Scl1 binding to purified ApoB100 and LDL is specific and concentration-dependent. Furthermore, the non-collagenous V region of the Scl1 protein is responsible for LDL/ApoB100 binding because only those rScls, constructed by domain swapping, which contain the V region from Scl1 proteins, were able to bind to ApoB100 and LDL ligands, and this binding was inhibited by antibodies directed against the Scl1-V region. Electron microscopy images of Scl1-LDL complexes showed that the globular V domain of Scl1 interacted with spherical particles of LDL. Importantly, live M28-type GAS cells absorbed plasma LDL on the cell surface and this binding depended on the surface expression of the Scl1.28, but not Scl2.28, protein. Phylogenetic analysis showed that the non-collagenous globular domains of Scl1 and Scl2 evolved independently to form separate lineages, which differ in amino acid sequence, and these differences may account for the variations in binding patterns of Scl1 and Scl2 proteins. Present studies provide insight into the structure-function relationship of the Scl proteins and also underline the importance of lipoprotein binding by GAS.     

Scl1-dependent internalization of group A Streptococcus via direct interactions with the alpha2beta(1) integrin enhances pathogen survival and re-emergence

The molecular pathogenesis of infections caused by group A Streptococcus (GAS) is not fully understood. We recently reported that a recombinant protein derived from the collagen-like surface protein, Scl1, bound to the human collagen receptor, integrin α₂β₁. Here, we investigate whether the same Scl1 variant expressed by GAS cells interacts with the integrin α₂β₁ and affects the biological outcome of host–pathogen interactions. We demonstrate that GAS adherence and internalization involve direct interactions between surface expressed Scl1 and the α₂β₁ integrin, because (i) both adherence and internalization of the scl1- inactivated mutant were significantly decreased, and were restored by in-trans complementation of Scl1 expression, (ii) GAS internalization was reduced by pre-treatment of HEp-2 cells with anti-α₂ integrin-subunit antibody and type I collagen, (iii) recombinant α₂-I domain bound the wild-type GAS cells and (iv) internalization of wild-type cells was significantly increased in C2C12 cells expressing the α₂β₁ integrin as the only collagen-binding integrin. Next, we determined that internalized GAS re-emerges from epithelial cells into the extracellular environment. Taken together, our data describe a new molecular mechanism used by GAS involving the direct interaction between Scl1 and integrins, which increases the overall capability of the pathogen to survive and re-emerge.     

Adaptation of the group A Streptococcus adhesin Scl1 to bind fibronectin type III repeats within wound‐associated extracellular matrix: implications for cancer therapy

The human‐adapted pathogen group A Streptococcus (GAS) utilizes wounds as portals of entry into host tissue, wherein surface adhesins interact with the extracellular matrix, enabling bacterial colonization. The streptococcal collagen‐like protein 1 (Scl1) is a major adhesin of GAS that selectively binds to two fibronectin type III (FnIII) repeats within cellular fibronectin, specifically the alternatively spliced extra domains A and B, and the FnIII repeats within tenascin‐C. Binding to FnIII repeats was mediated through conserved structural determinants present within the Scl1 globular domain and facilitated GAS adherence and biofilm formation. Isoforms of cellular fibronectin that contain extra domains A and B, as well as tenascin‐C, are present for several days in the wound extracellular matrix. Scl1‐FnIII binding is therefore an example of GAS adaptation to the host's wound environment. Similarly, cellular fibronectin isoforms and tenascin‐C are present in the tumor microenvironment. Consistent with this, FnIII repeats mediate GAS attachment to and enhancement of biofilm formation on matrices deposited by cancer‐associated fibroblasts and osteosarcoma cells. These data collectively support the premise for utilization of the Scl1‐FnIII interaction as a novel method of anti‐neoplastic targeting in the tumor microenvironment.     

The group A streptococcal collagen‐like protein‐1, Scl1, mediates biofilm formation by targeting the extra domain A‐containing variant of cellular fibronectin expressed in wounded tissue

Wounds are known to serve as portals of entry for group A Streptococcus (GAS). Subsequent tissue colonization is mediated by interactions between GAS surface proteins and host extracellular matrix components. We recently reported that the streptococcal collagen‐like protein‐1, Scl1, selectively binds the cellular form of fibronectin (cFn) and also contributes to GAS biofilm formation on abiotic surfaces. One structural feature of cFn, which is predominantly expressed in response to tissue injury, is the presence of a spliced variant containing extra domain A (EDA/EIIIA). We now report that GAS biofilm formation is mediated by the Scl1 interaction with EDA‐containing cFn. Recombinant Scl1 proteins that bound cFn also bound recombinant EDA within the C‐C’ loop region recognized by the α₉β₁ integrin. The extracellular 2‐D matrix derived from human dermal fibroblasts supports GAS adherence and biofilm formation. Altogether, this work identifies and characterizes a novel molecular mechanism by which GAS utilizes Scl1 to specifically target an extracellular matrix component that is predominantly expressed at the site of injury in order to secure host tissue colonization.     

Streptococcal Scl1 and Scl2 proteins form collagen-like triple helices

The collagens are a family of animal proteins containing segments of repeated Gly-Xaa-Yaa (GXY) motifs that form a characteristic triple-helical structure. Genes encoding proteins with repeated GXY motifs have also been reported in bacteria and phages; however, it is unclear whether these prokaryotic proteins can form a collagen-like triple-helical structure. Here we used two recently identified streptococcal proteins, Scl1 and Scl2, containing extended GXY sequence repeats as model proteins. First we observed that prior to heat denaturation recombinant Scl proteins migrated as homotrimers in gel electrophoresis with and without SDS. We next showed that the collagen-like domain of Scl is resistant to proteolysis by trypsin. We further showed that circular dichroism spectra of the Scl proteins contained features characteristic of collagen triple helices, including a positive maximum of ellipticity at 220 nm. Furthermore the triple helices of Scl1 and Scl2 showed a temperature-dependent unfolding with melting temperatures of 36.4 and 37.6 degrees C, respectively, which resembles those seen for collagens. We finally demonstrated by electron microscopy that the Scl proteins are organized into "lollipop-like" structures, similar to those seen in human proteins with collagenous domains. This implies that the repeated GXY tripeptide motif is a structural indicator of collagen-like triple helices in proteins from such phylogenetically distant sources as bacteria and humans.     

Post-proteomic identification of a novel phage-encoded streptodornase, Sda1, in invasive M1T1 Streptococcus pyogenes

The M1T1 strain remains the most frequently isolated strain from group A streptococcal (GAS) infection cases worldwide. We previously reported that M1T1 differs from the fully sequenced M1 SF370 strain. To better understand the reason for the persistence and increased virulence of M1T1, we analysed its secreted proteome and identified two virulence proteins that are not present in the sequenced M1 SF370 strain: streptococcal pyrogenic exotoxin A (SpeA) and a streptodornase D (SdaD) homologue. In the present study, we determined the nucleotide sequence of the M1T1 streptodornase and found that its deduced amino acid sequence is highly similar to other streptococcal streptodornases, and is most closely related to the SdaD of GAS strain M49. M1T1 Sda shares two highly conserved domains with several DNases and putative DNases in streptococci; however, it possesses a unique C-terminal amino acid sequence. Thus, we named the protein Sda1, and we detected the presence of the sda1 gene in 16 M1T1 clinical isolates. The cloned and expressed Sda1 degrades both streptococcal and mammalian DNA at physiological pH. Amino acid similarity analyses of known GAS deoxyribonucleases suggest that Sda1 may be a chimeric protein created through recombination events. Moreover, a natural mutation that resulted in longer Sda1 and SdaD as compared to other GAS DNases was found to confer increased activity on the protein. Analysis of the sequences flanking sda1 determined that it is carried by a prophage or a prophage-like element inserted in the tRNA-Ser gene of M1T1 GAS. Ongoing studies in our laboratory aim to determine the contribution of Sda1 to the virulence of this globally disseminated M1T1 strain.     

Identification of the first prokaryotic collagen sequence motif that mediates binding to human collagen receptors, integrins alpha2beta1 and alpha11beta1

Many pathogenic bacteria interact with human integrins to enter host cells and to augment host colonization. Group A Streptococcus (GAS) employs molecular mimicry by direct interactions between the cell surface streptococcal collagen-like protein-1 (Scl1) and the human collagen receptor, integrin alpha2beta1. The collagen-like (CL) region of the Scl1 protein mediates integrin-binding, although, the integrin binding motif was not defined. Here, we used molecular cloning and site-directed mutagenesis to identify the GLPGER sequence as the alpha2beta1 and the alpha11beta1 binding motif. Electron microscopy experiments mapped binding sites of the recombinant alpha2-integrin-inserted domain to the GLPGER motif of the recombinant Scl (rScl) protein. rScl proteins and a synthetic peptide harboring the GLPGER motif mediated the attachment of C2C12-alpha2+myoblasts expressing the alpha2beta1 integrin as the sole collagen receptor. The C2C12-alpha11+myoblasts expressing the alpha11beta1 integrin also attached to GLPGER-harboring rScl proteins. Furthermore, the C2C12-alpha11+cells attached to rScl1 more efficiently than C2C12-alpha2+cells, suggesting that the alpha11beta1 integrin may have a higher binding affinity for the GLPGER sequence. Human endothelial cells and dermal fibroblasts adhered to rScl proteins, indicating that multiple cell types may recognize and bind the Scl proteins via their collagen receptors. This work is a stepping stone toward defining the utilization of collagen receptors by microbial collagen-like proteins that are expressed by pathogenic bacteria.     

Development and Evaluation of Capture Enzyme-Linked Immunosorbent Assays for Detection of Immunoglobulin G and M Antibodies to Group A Streptococcal Antigens

Capture enzyme-linked immunosorbent assays (ELISAs) were developed to detect immunoglobulin G and M antibodies to group A streptococcal (GAS) antigens, streptolysin O, streptokinase, and group A carbohydrate. The sensitivities and the specificities of the IgM capture ELISAs to each GAS antigen were high enough to distinguish the patients with GAS infections (diagnosed as GAS pharyngitis or scarlet fever) from the control groups (healthy people and patients with pharyngitis from whom GAS could not be isolated). On the other hand, the specificities of the IgG capture ELISAs were not very effective in diagnosis of GAS infections. When the capture ELISA and an indirect ELISA detecting IgM antibodies to group A carbohydrate were compared, false-positive reactions due to rheumatoid factor occurred in the indirect ELISA, but did not occur in the capture ELISA. These results indicate that the capture ELISA works better than the indirect ELISA in detecting the IgM antibody, and that the IgM capture ELISA to GAS antigen provides a rapid and highly reliable serodiagnosis for GAS infections employing only a single serum.     

HLA transgenic mice provide evidence for a direct and dominant role of HLA class II variation in modulating the severity of streptococcal sepsis

Our epidemiologic studies on invasive Group A Streptococci (GAS) infections identified specific HLA class II haplotypes/alleles conferring high-risk or protection from streptococcal toxic shock syndrome with a strong protection conferred by the DRB1*15/DQB1*06 haplotype. We used HLA-transgenic mice to provide an in vitro and in vivo validation for the direct role of HLA class II allelic variation in streptococcal toxic shock syndrome. When splenocytes from mice expressing the protective HLA-DQB1*06 (DQ6) allele were stimulated with a mixture of streptococcal superantigens (SAgs), secreted by the prevalent M1T1 strain, both proliferative and cytokine responses were significantly lower than those of splenocytes from mice expressing the neutral DRB1*0402/DQB1*0302 (DR4/DQ8) alleles (p < 0.001). In crisscross experiments, the presentation of SAgs to pure T cells from either the DQ6 or the DR4/DQ8 mice resulted in significantly different levels of response depending on the HLA type expressed on the APCs. Presentation by HLA-DQ6 APCs elicited significantly lower responses than the presentation by HLA-DR4/DQ8 APCs. Our in vitro data were supported by in vivo findings, as the DQ6 mice showed significantly longer survival post-i.v. infection with live M1T1 GAS (p < 0.001) and lower inflammatory cytokine responses as compared with the DR4/DQ8 mice (p < 0.01). The data presented here provide evidence for a direct role of HLA class II molecules in modulating responses to GAS SAgs and underscore the dominant role of HLA class II allelic variation in potentiating the severity of GAS systemic infections.     

High‐density lipoprotein acts as an opsonin to enhance phagocytosis of group A streptococcus by U937 cells

We have previously demonstrated that high‐density lipoprotein (HDL) can specifically bind to streptococcal collagen‐like protein 1 (Scl1) of M41‐type group A Streptococcus (GAS). However, the pathological or physiological significance of Scl1?HDL interaction is unknown. Here, the hypothesis that HDL acts as an opsonin to enhance phagocytosis of HDL‐bound GAS by monocytes given that some scavenger receptors can mediate the endocytosis of HDL was tested by using FITC‐labeled bacteria, human U937 monocytes and HDL for phagocytic assays. HDL (10 µg/mL) was found to significantly enhance internalization of M41‐type (ATCC 12373) GAS by U937 cells after 60 min incubation, compared with an HDL‐free group. The internalized GAS were dead after 60 min incubation with U937 cells regardless of presence and absence of HDL. Although very‐low‐density lipoprotein (VLDL) could specifically bind to ATCC 12373 strain, it did not promote phagocytosis of GAS. Additionally, LDL, HDL and VLDL did not enhance phagocytosis of CMCC 32198 strain because this strain did not bind to these lipoproteins. A physiological concentration of HDL (1000 µg/mL) had a similar effect. Anti‐CD36 antibody completely abolished opsonic phagocytosis whereas anti‐CD4 antibody did not, indicating that CD36 is the major scavenger receptor mediating the uptake of HDL‐opsonized GAS by U937 cells. Furthermore, because rScl1 competitively blocked the interaction of ATCC 12373 strain with HDL recombinant Scl1 (rScl1) derived from M41‐type GAS, it significantly decreased opsonophagocytosis of ATCC 12373 strain but not of CMCC 32198 strain. Therefore, our findings suggest that HDL may be an opsonin that enhances CD36‐dependent opsonophagocytosis of GAS by U937 cells.

     

The Fibrinogen-binding M1 Protein Reduces Pharyngeal Cell Adherence and Colonization Phenotypes of M1T1 Group A Streptococcus

Group A Streptococcus (GAS) is a leading human pathogen producing a diverse array of infections from simple pharyngitis (“strep throat”) to invasive conditions, including necrotizing fasciitis and toxic shock syndrome. The surface-anchored GAS M1 protein is a classical virulence factor that promotes phagocyte resistance and exaggerated inflammation by binding host fibrinogen (Fg) to form supramolecular networks. In this study, we used a virulent WT M1T1 GAS strain and its isogenic M1-deficient mutant to examine the role of M1-Fg binding in a proximal step in GAS infection-interaction with the pharyngeal epithelium. Expression of the M1 protein reduced GAS adherence to human pharyngeal keratinocytes by 2-fold, and this difference was increased to 4-fold in the presence of Fg. In stationary phase, surface M1 protein cleavage by the GAS cysteine protease SpeB eliminated Fg binding and relieved its inhibitory effect on GAS pharyngeal cell adherence. In a mouse model of GAS colonization of nasal-associated lymphoid tissue, M1 protein expression was associated with an average 6-fold decreased GAS recovery in isogenic strain competition assays. Thus, GAS M1 protein-Fg binding reduces GAS pharyngeal cell adherence and colonization in a fashion that is counterbalanced by SpeB. Inactivation of SpeB during the shift to invasive GAS disease allows M1-Fg binding, increasing pathogen phagocyte resistance and proinflammatory activities.     

Molecular insight into invasive group A streptococcal disease

Streptococcus pyogenes is also known as group A Streptococcus (GAS) and is an important human pathogen that causes considerable morbidity and mortality worldwide. The GAS serotype M1T1 clone is the most frequently isolated serotype from life-threatening invasive (at a sterile site) infections, such as streptococcal toxic shock-like syndrome and necrotizing fasciitis. Here, we describe the virulence factors and newly discovered molecular events that mediate the in vivo changes from non-invasive GAS serotype M1T1 to the invasive phenotype, and review the invasive-disease trigger for non-M1 GAS. Understanding the molecular basis and mechanism of initiation for streptococcal invasive disease may expedite the discovery of novel therapeutic targets for the treatment and control of severe invasive GAS diseases.     

Clinical evaluation of autoantibodies to a novel PM/Scl peptide antigen

Anti-PM/Scl antibodies represent a specific serological marker for a subset of patients with scleroderma (Scl) and polymyositis (PM), and especially with the PM/Scl overlap syndrome (PM/Scl). Anti-PM/Scl reactivity is found in 24% of PM/Scl patients and is found in 3–10% of Scl and PM patients. The PM/Scl autoantigen complex comprises 11–16 different polypeptides. Many of those proteins can serve as targets of the anti-PM/Scl B-cell response, but most frequently the PM/Scl-100 and PM/Scl-75 polypeptides are targeted. In the present study we investigated the clinical relevance of a major alpha helical PM/Scl-100 epitope (PM1-α) using a newly developed peptide-based immunoassay and compared the immunological properties of this peptide with native and recombinant PM/Scl antigens. In a technical comparison, we showed that an ELISA based on the PM1-α peptide is more sensitive than common techniques to detect anti-PM/Scl antibodies such as immunoblot, indirect immunofluorescence on HEp-2 cells and ELISA with recombinant PM/Scl polypeptides. We found no statistical evidence of a positive association between anti-PM1-α and other antibodies, with the exception of known PM/Scl components. In our cohort a negative correlation could be found with anti-Scl-70 (topoisomerase I), anti-Jo-1 (histidyl tRNA synthetase) and anti-centromere proteins. In a multicenter evaluation we demonstrated that the PM1-α peptide represents a sensitive and reliable substrate for the detection of a subclass of anti-PM/Scl antibodies. In total, 22/40 (55%) PM/Scl patients, 27/205 (13.2%) Scl patients and 3/40 (7.5%) PM patients, but only 5/288 (1.7%) unrelated controls, tested positive for the anti-PM1-α peptide antibodies. These data indicate that anti-PM1-α antibodies appear to be exclusively present in sera from PM/Scl patients, from Scl patients and, to a lesser extent, from PM patients. The anti-PM1-α ELISA thus offers a new serological marker to diagnose and discriminate different systemic autoimmune disorders.     

Streptococcal mitogenic exotoxin, SmeZ, is the most susceptible M1T1 streptococcal superantigen to degradation by the streptococcal cysteine protease, SpeB

Superantigens (SAgs) play an important role in the pathogenesis of severe invasive infections caused by Group A Streptococcus (GAS). We had shown earlier that the expression of streptococcal cysteine protease SpeB results in partial loss of the immune-stimulating activity of the native secreted GAS SAgs, namely the streptococcal pyrogenic exotoxins produced by the globally disseminated M1T1 GAS strain, associated with invasive infections worldwide. In this study, we examined the susceptibility of each of the M1T1 recombinant SAgs to degradation by rSpeB. Whereas SmeZ was degraded completely within 30 min of incubation with rSpeB, SpeG, and SpeA were more resistant and SpeJ was completely unaffected by the proteolytic effects of this protease. Proteomic analyses demonstrated that the order of susceptibility of the M1T1 SAgs to SpeB proteolysis is unaltered when they are present in a mixture that reflects their native physiological status. As expected, the degradation of SmeZ abolished its immune stimulatory activity. In silico sequence disorder and structural analyses revealed that SmeZ, unlike the three other structurally related SAgs, possesses a putative SpeB cleavage site within an area of the protein likely to be exposed to the surface. The study provides evidence for the effect of subtle structural differences between highly similar SAgs on their biological activity.     

The Crystal Structure of the Streptococcal Collagen-like Protein 2 Globular Domain from Invasive M3-type Group A Streptococcus Shows Significant Similarity to Immunomodulatory HIV Protein gp41

The arsenal of virulence factors deployed by streptococci includes streptococcal collagen-like (Scl) proteins. These proteins, which are characterized by a globular domain and a collagen-like domain, play key roles in host adhesion, host immune defense evasion, and biofilm formation. In this work, we demonstrate that the Scl2.3 protein is expressed on the surface of invasive M3-type strain MGAS315 of Streptococcus pyogenes. We report the crystal structure of Scl2.3 globular domain, the first of any Scl. This structure shows a novel fold among collagen trimerization domains of either bacterial or human origin. Despite there being low sequence identity, we observed that Scl2.3 globular domain structurally resembles the gp41 subunit of the envelope glycoprotein from human immunodeficiency virus type 1, an essential subunit for viral fusion to human T cells. We combined crystallographic data with modeling and molecular dynamics techniques to gather information on the entire lollipop-like Scl2.3 structure. Molecular dynamics data evidence a high flexibility of Scl2.3 with remarkable interdomain motions that are likely instrumental to the protein biological function in mediating adhesive or immune-modulatory functions in host-pathogen interactions. Altogether, our results provide molecular tools for the understanding of Scl-mediated streptococcal pathogenesis and important structural insights for the future design of small molecular inhibitors of streptococcal invasion.     

Leucocyte recruitment and molecular fortification of keratinocytes triggered by streptococcal M1 protein

Streptococcus pyogenes of the M1 serotype is commonly associated with invasive streptococcal infections and development of streptococcal toxic shock syndrome. The M1 protein is a powerful inducer of inflammatory responses for several human cell types, but the reason why M1 protein‐related strains is over‐represented in invasive streptococcal diseases is still not understood. This study was undertaken to investigate if soluble M1 protein can aggravate the severity of streptococcal skin infections in respect to inflammation, leucocyte recruitment, and tissue remodelling as seen in patients with cellulitis and necrotizing fasciitis. We found that HaCaT cells are able to recruit activated leucocytes when encountering M1 protein. Neither the bacterial protein nor activated leucocytes caused cell damage on HaCaT cells, instead HaCaT cells responded to the bacterial virulence factor by releasing several proteins protective against bacterial infection and leucocyte responses. However, although not cytotoxic, M1 protein completely abolished wound healing abilities of HaCaT cells. Taken together, our results demonstrate that M1 protein is a critical virulence factor that can augment streptococcal skin infection suggesting that the protein is an interesting target for drug development.     

Autoantibodies directed to novel components of the PM/Scl complex, the human exosome

The autoantigenic polymyositis/scleroderma (PM/Scl) complex was recently shown to be the human homologue of the yeast exosome, which is an RNA-processing complex. Our aim was to assess whether, in addition to targeting the known autoantigens PM/Scl-100 and PM/Scl-75, autoantibodies also target recently identified components of the PM/Scl complex. The prevalence of autoantibodies directed to six novel human exosome components (hRrp4p, hRrp40p, hRrp41p, hRrp42p, hRrp46p, hCsl4p) was determined in sera from patients with idiopathic inflammatory myopathy (n = 48), scleroderma (n = 11), or the PM/Scl overlap syndrome (n = 10). The sera were analyzed by enzyme-linked immunosorbent assays and western blotting using the affinity-purified recombinant proteins. Our results show that each human exosome component is recognized by autoantibodies. The hRrp4p and hRrp42p components were most frequently targeted. The presence of autoantibodies directed to the novel components of the human exosome was correlated with the presence of the anti-PM/Scl-100 autoantibody in the sera of patients with idiopathic inflammatory myopathy (IIM), as was previously found for the anti-PM/Scl-75 autoantibody. Other clear associations between autoantibody activities were not found. These results further support the conception that the autoimmune response may initially be directed to PM/Scl-100, whereas intermolecular epitope spreading may have caused the autoantibody response directed to the associated components.     

Serum opacity factor is a major fibronectin-binding protein and a virulence determinant of M type 2 Streptococcus pyogenes

Serum opacity factor (SOF) is a fibronectin-binding protein of group A streptococci that opacifies mammalian sera and is expressed by some strains that cause impetigo, pharyngitis and acute glomerulonephritis. Although SOF is expressed by approximately 35% of known serotypes, its role in the pathogenesis of group A streptococcal infections has not been previously investigated. The sof genes from M types 2, 28 and 49 Streptococcus pyogenes were cloned, sequenced, and their deduced amino acid sequences were compared. The gene for FnBA, a fibronectin-binding protein from Streptococcus dysgalactiae, was also cloned and found to express an opacity factor. The leader sequences, the fibronectin-binding domains, and the membrane anchor regions of these proteins were highly conserved. Short spans of conserved sequences were interspersed throughout the remaining parts of the proteins. The sof2 gene was insertionally inactivated in an M type 2 S. pyogenes strain, T2MR. The resultant SOF-negative mutant (YL3) did not express SOF or opacify serum, and exhibited a 71% reduction in binding fibronectin. Complementation of the SOF-negative defect with sof28 in the recombinant strain YL3(pNZ28) fully restored fibronectin-binding activity and the ability to opacify serum. To determine whether sof plays a role in virulence, mice were challenged intraperitoneally with these strains. None of the 10 mice infected with YL3(pNZ28) survived and only 1 out of 15 mice challenged with T2MR survived, whereas 12 out of 15 mice infected with YL3 survived. These data clearly indicate that SOF is a virulence factor, and they provide the first direct evidence that a fibronectin-binding protein contributes to the pathogenesis of group A streptococcal infections in vivo.