Vijaykumar Pancholi

alpha-enolase, a novel strong plasmin(ogen) binding protein on the surface of pathogenic streptococci.

The plasmin(ogen) binding property of group A streptococci is incriminated in tissue invasion processes. We have characterized a novel 45-kDa protein displaying strong plasmin(ogen) binding activity from the streptococcal surface. Based on its biochemical properties, we confirmed the identity of this protein as α-enolase, a key glycolytic enzyme. Dose-dependent α-enolase activity, immune electron microscopy of whole streptococci using specific antibodies, and the opsonic nature of polyclonal and monoclonal antibodies concluded the presence of this protein on the streptococcal surface. We, henceforth, termed the 45-kDa protein, SEN (streptococcal surface enolase). SEN is found ubiquitously on the surface of most streptococcal groups and serotypes and showed significantly greater plasmin(ogen) binding affinity compared with previously reported streptococcal plasminogen binding proteins. Both the C-terminal lysine residue of SEN and a region N-terminal to it play a critical role in plasminogen binding. Results from competitive plasminogen binding inhibition assays and cross-linking studies with intact streptococci indicate that SEN contributes significantly to the overall streptococcal ability to bind plasmin(ogen). Our findings, showing both the protected protease activity of SEN-bound plasmin and SEN-specific immune responses, provide evidence for an important role of SEN in the disease process and post-streptococcal autoimmune diseases.

Antibodies to Streptococcal Surface Enolase React with Human α-Enolase: Implications in Poststreptococcal Sequelae

The pathogenic mechanisms for developing acute rheumatic fever after group A streptococcal pharyngitis are still poorly understood. The glycolytic enzyme enolase is one of the major proteins on the surface of group A streptococci. Herein, significant cross-reactivity was shown between streptococcal enolase and human enolase. Fluorocytometric analysis revealed that antistreptococcal enolase antibodies react with the enolase expressed on the surface of hematopoietic cells. Furthermore, the enolase on the leukocyte surface was found to be up-regulated by inflammatory stimuli. Evaluation of antibody titers indicated that serum samples from patients with acute rheumatic fever have higher levels of antibodies that react with the human and bacterial enolases than do serum samples from patients with streptococcal pharyngitis or healthy control subjects. These results show that streptococcal enolase is a novel cross-reactive antigen that may play an important role in the initiation of the autoimmune diseases related to streptococcal infection.

Induction of lysogenic bacteriophage and phage-associated toxin from group a streptococci during coculture with human pharyngeal cells.

ABSTRACT We found that when group A streptococci are cocultured with human pharyngeal cells, they upregulate and secrete a 25-kDa toxin, determined to be the bacteriophage-encoded streptococcal pyrogenic exotoxin C (SpeC). This prompted us to determine if the bacteriophage themselves are induced during coculture conditions. We found that bacteriophage induction does occur, resulting in the release of ∼105 phage particles during the 3-h coculture. Furthermore, we show that the bacteriophage induction event is mediated by a pharyngeal cell soluble factor for which we provide an initial characterization.

Group A Streptococci Bind to Mucin and Human Pharyngeal Cells through Sialic Acid-Containing Receptors

ABSTRACT The first step in the colonization of group A streptococci (Streptococcus pyogenes) is adherence to pharyngeal epithelial cells. Prior to adherence to their target tissue, the first barrier that the streptococci encounter is the mucous layer of the respiratory tract. The present study was undertaken to characterize the interaction between mucin, the major glycoprotein component of mucus, and streptococci. We report here that S. pyogenes is able to bind to bovine submaxillary mucin in solid-phase microtiter plate assays. Western blots probed with 125I-labeled mucin and a panel of monoclonal antibodies revealed that the streptococcal M protein is one of two cell wall-associated proteins responsible for this binding. The binding was further localized to the N-terminal portion of the M molecule. Further analysis revealed that the M protein binds to the sialic acid moieties on mucin, and this interaction seems to be based on M-protein conformation rather than specific amino acid sequences. We found that sialic acid also plays a critical role in the adherence of an M6 streptococcal strain to the Detroit 562 human pharyngeal cell line and have identified α2-6-linked sialic acid as an important sialylated linkage for M-protein recognition. Western blot analysis of extracted pharyngeal cell membrane proteins identified three potential sialic acid-containing receptors for the M protein. The results are the first to show that sialic acid not only is involved in the binding of the streptococci to mucin but also plays an important role in adherence of group A streptococci to the pharyngeal cell surface.

The In Vitro Interaction of Streptococcus pyogenes with Human Pharyngeal Cells Induces a Phage-Encoded Extracellular DNase

ABSTRACT The role lysogenic bacteriophage play in the pathogenesis of the host bacterium is poorly understood. In a previous study, we found that streptococcal coculture with human pharyngeal cells resulted in the induction of lysogenic bacteriophage as well as the phage-associated streptococcal pyrogenic exotoxin C (SpeC). In this study, we have determined that in addition to SpeC induction, a number of other streptococcal proteins are also released by the bacteria during coculture with pharyngeal cells. Among these, we identified and characterized a novel 27-kDa secreted protein. Sequence analysis of this novel protein demonstrated it to be encoded by the same lysogenic bacteriophage which harbors speC. Protein sequence analysis revealed varied homologies with several streptococcal DNases. Further biochemical characterization of the recombinantly expressed protein verified it to be a divalent cation-dependent streptococcal phage-encoded DNase (Spd1). Although functionally distinct, SpeC and Spd1 are associated by a number of parameters, including genetic proximity and transcriptional regulation. Finally, we speculate on the induction of phage-encoded DNase (Spd1) enhancing the fitness of both bacteria and phage.

A Novel Plasminogen/Plasmin Binding Protein on the Surface of Group A Streptococci

The increasing occurence of invasive group A streptococcal infections is of special public concern. The invasive property of group A streptococci is attributed in part to its ability to bind plasmin or plasminogen. We reported earlier of a novel protein, streptococcal surface dehydrogenase (SDH) having glyceraldehyde-3-phosphate dehydrogenase activity, as a multifunctional major protein on the surface of group A streptococci3. A structurally and functionally (having GAPDH activity) similar protein, Plamin receptor Plr, has been reported to have a strong plasmin binding activity1. Unlike Plr, SDH binds very weakly to plasmin3. In addition, we reported earlier that SDH binds strongly to various mammalian proteins such as actin, myosin, fibronectin and lysozyme3. Since SDH is a weak plasmin(ogen) binding protein, we searched for a protein other than SDH that accounts for strong plasmin(ogen) binding activity of streptococci. We here report that group A streptococci that express SDH on the surface also show a plasmin/plasminogen binding activity directed to a unique 45 kDa protein.