Enolase, a key glycolytic enzyme, belongs to a novel class of surface proteins which do not possess classical machinery for surface transport, yet through an unknown mechanism are transported on the cell surface. Enolase is a multifunctional protein, and its ability to serve as a plasminogen receptor on the surface of a variety of hematopoetic, epithelial and endothelial cells suggests that it may play an important role in the intravascular and pericellular fibrinolytic system. Its role in systemic and invasive autoimmune disorders was recognized only very recently. In addition to this property, its ability to function as a heat-shock protein and to bind cytoskeletal and chromatin structures indicate that enolase may play a crucial role in transcription and a variety of pathophysiological processes.

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 ␣-enolase, a key glycolytic enzyme. Dose-dependent ␣-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 SENbound plasmin and SEN-specific immune responses, provide evidence for an important role of SEN in the disease process and post-streptococcal autoimmune diseases.Streptococcus pyogenes is responsible for a wide variety of human diseases that range from suppurative infections of the throat (pharyngitis), skin (impetigo), and underlying tissues (necrotizing fasciitis), to an often fatal toxic shock syndrome, and the post-streptococcal sequelae, rheumatic fever, and acute glomerulonephritis. Bacterial surface proteins play a major role in these disease processes by exhibiting a wide range of functions. As data have become available, it is clear that most surface proteins found on Gram-positive bacteria, particularly those on group A streptococci, have a great deal of structural similarities (1, 2). Proteins for which the function(s) has been defined have been found to be multifunctional, whereas in others a function has only been attributed to one of two or more domains (2, 3). Thus, the multifunctional characteristics of these surface proteins increase the complexity of the Grampositive surface beyond what has been previously imagined.We recently described one such multifunctional protein, streptococcal surface dehydrogenase (SDH), 1 as a major surface protein on group A streptococci and other streptococcal groups which is structurally and functionally related to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (4). SDH also has an ADP-ribosylating activity (5) and exhibits multiple binding activities to several mammalian proteins such as fibronectin and cytoskeletal proteins (4). A structurally and enzymatically similar streptococcal protein, Plr, was also identified on group A streptococci, based on its ability to bind plasmin (6). SDH, how...

SummaryThe surface of streptococci presents an array of different proteins, each designed to perform a specific function. In an attempt to understand the early events in group A streptococci infection, we have identified and purified a major surface protein from group A type 6 streptococci that has both an enzymatic activity and a binding capacity for a variety of proteins. Mass spectrometric analysis of the purified molecule revealed a monomer of 35.8 kD. Molecular sieve chromatography and sodium dodecyl sulfate (SDS)-gel dectrophoresis suggest that the native conformation of the protein is likely to be a tetramer of 156 kD. NH2-terminal amino acid sequence analysis revealed 83% homology in the first 18 residues and about 56% in the first 39 residues with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of eukaryotic or bacterial origin. This streptococcal surface GAPDH (SDH) exhibits a dose-dependent dehydrogenase activity on glyceraldehyde-3-phosphate in the presence of fl-nicotinamide adenine dinucleotide both in its pure form and on the streptococcal surface. Its sensitivity to trypsin on whole organism and its inability to be removed with 2 M NaCI or 2% SDS support its surface location and tight attachment to the streptococcal cell. Affinity-purified antibodies to SDH detected the presence of this protein on the surface of all M serotypes of group A streptococcal tested. Purified SDH was found to bind to fibronectin, lysozyme, as well as the cytoskeletal proteins myosin and actin. The binding activity to myosin was found to be localized to the globular heavy meromyosin domain. SDH did not bind to streptococcal M protein, tropomyosin, or the coiled-coil domain of myosin. The multiple binding capacity of the SDH in conjunction with its GAPDH activity may play a role in the colonization, internalization, and the subsequent proliferation of group A streptococci.

A highly conserved hexapeptide sequence (both at the protein- and DNA level) has been identified within the C-terminal end of all 11 known surface proteins from Gram-positive cocci. The hexapeptide, with the consensus sequence LPXTGE, is located about 9 amino acids N-terminal from the C-terminal hydrophobic domain which is found in all these surface molecules. The conservation of the hexapeptide, despite sequence variation within the regions flanking it, suggests that it is important for the attachment of these proteins within the cell.

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It is well established that prokaryotes and eukaryotes alike utilize phosphotransfer to regulate cellular functions. One method by which this occurs is via eukaryote-like serine/threonine kinase (ESTK)-and phosphatase (ESTP)-regulated pathways. The role of these enzymes in Staphylococcus aureushas not yet been examined. This resilient organism is a common cause of hospital-acquired and community-associated infections, infecting immunocompromised and immunocompetent hosts alike. In this study, we have characterized a major functional ESTK (STK) and ESTP (STP) in S. aureus and found them to be critical modulators of cell wall structure and susceptibility to cell wall-acting ␤-lactam antibiotics. By utilizing gene knockout strategies, we created S. aureus N315 mutants lacking STP and/or STK. The strain lacking both STP and STK displayed notable cell division defects, including multiple and incomplete septa, bulging, and irregular cell size, as observed by transmission electron microscopy. Mutants lacking STP alone displayed thickened cell walls and increased resistance to the peptidoglycan-targeting glycylglycine endopeptidase lysostaphin, compared to the wild type. Additionally, mutant strains lacking STK or both STK and STP displayed increased sensitivity to cell wall-acting cephalosporin and carbapenem antibiotics. Together, these results indicate that S. aureus STK-and STP-mediated reversible phosphorylation reactions play a critical role in proper cell wall architecture, and thus the modulation of antimicrobial resistance, in S. aureus.Staphylococcus aureus constitutes a major public health threat, as it is the most common hospital-associated pathogen in the world and its prevalence in community-acquired infections is on the rise (18). This gram-positive coccus is armed with a wide variety of virulence factors that contribute to diseases ranging from mild food poisoning, skin lesions, and boils to severe and often fatal endocarditis, osteomyelitis, pneumonia, and toxic shock syndrome (28). Staphylococci are known for their evolving mechanisms of antimicrobial resistance, which have resulted in the spread of methicillin-resistant and even vancomycin-resistant S. aureus, severely limiting treatment options for those infected (41). The signaling cascades which enable the staphylococcus to evolve such resistance mechanisms and cause infection remain a major field of study.A recent comparative analysis of several prokaryotic genomes suggests that one-component regulatory systems (in contrast to the conventional paradigm of two-component regulatory systems) are, in fact, the most abundant signaling systems in prokaryotes (43). These one-component systems include eukaryote-like serine/threonine kinases (ESTKs) and phosphatases (ESTPs), which have emerged as critical signaling molecules in prokaryotes over the past decade (5). Since the first characterization of an ESTK in soil bacteria (Myxococcus xanthus Currently, no information is available on the role of ESTKmediated signaling in S. aureus. In the present investi...

Streptococcal surface enolase (SEN) is a major plasminogen-binding protein of group A streptococci. Our earlier biochemical studies have suggested that the region responsible for this property is likely located at the C-terminal end of the SEN molecule. In the present study, the gene encoding SEN was cloned from group A streptococci M6 isolate D471. A series of mutations in the sen gene corresponding to the C-terminal region ( 428 KSFYNLKK 435 ) of the SEN molecule were created by either deleting one or more terminal lysine residues or replacing them with leucine. All purified recombinant SEN proteins with altered C-terminal ends were found to be enzymatically active and were analyzed for their Glu-and Lys-plasminogen-binding activities. Wild-type SEN bound to Lys-plasminogen with almost three times more affinity than to Glu-plasminogen. However, the recombinant mutant SEN proteins with a deletion of Lys434-435 or with K435L and K434-435L replacements showed a significant decrease in Glu-and Lys-plasminogen-binding activities. Accordingly, a streptococcal mutant expressing SEN-K434-435L showed a significant decrease in Glu-and Lys-plasminogen-binding activities. Biochemical and functional analyses of the isogenic mutant strain revealed a significant decrease in its abilities to cleave a chromogenic tripeptide substrate, acquire plasminogen from human plasma, and penetrate the extracellular matrix. Together, these data indicate that the last two C-terminal lysine residues of surfaceexposed SEN contribute significantly to the plasminogen-binding activity of intact group A streptococci and hence to their ability to exploit host properties to their own advantage in tissue invasion.