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Streptococcus pyogenes is a gram-positive human pathogen that causes a wide spectrum of disease, placing a significant burden on public health. Bacterial surface-associated proteins play crucial roles in host-pathogen interactions and pathogenesis and are important targets for the immune system. The identification of these proteins for vaccine development is an important goal of bacterial proteomics. Here we describe a method of proteolytic digestion of surface-exposed proteins to identify surface antigens of S. pyogenes. Peptides generated by trypsin digestion were analyzed by multidimensional tandem mass spectrometry. This approach allowed the identification of 79 proteins on the bacterial surface, including 14 proteins containing cell wall-anchoring motifs, 12 lipoproteins, 9 secreted proteins, 22 membrane-associated proteins, 1 bacteriophage-associated protein, and 21 proteins commonly identified as cytoplasmic. Thirty-three of these proteins have not been previously identified as cell surface associated in S. pyogenes. Several proteins were expressed in Escherichia coli, and the purified proteins were used to generate specific mouse antisera for use in a whole-cell enzymelinked immunosorbent assay. The immunoreactivity of specific antisera to some of these antigens confirmed their surface localization. The data reported here will provide guidance in the development of a novel vaccine to prevent infections caused by S. pyogenes.Streptococcus pyogenes, also known as group A Streptococcus, is a gram-positive bacterium that causes a wide spectrum of diseases ranging from mild localized infections, such as pharyngitis and impetigo, to severe invasive diseases, such as necrotizing fasciitis and streptococcal toxic shock-like syndrome. Invasive streptococcal disease is associated with high morbidity and mortality rates (37). S. pyogenes is also associated with a variety of autoimmune sequelae such as acute rheumatic fever, which after repeated episodes can result in rheumatic valvular heart disease, the most common cause of pediatric heart disease worldwide (11). In spite of the high mortality and substantial economic losses caused by these diseases, there is currently no licensed vaccine to prevent human S. pyogenes infections.For many years, efforts to develop a vaccine to protect against S. pyogenes infections were focused on the surfaceassociated M protein (19, 38), a major virulence factor of S. pyogenes. However, there are at least two significant limitations for using M protein as a vaccine antigen. First, the M protein contains a highly variable amino-terminal region that determines the S. pyogenes serotype. With over 150 different M serotypes identified, it is difficult to envision using the M protein as a broadly efficacious vaccine. Second, M protein elicits antibodies that are cross-reactive with human cardiac myosin and are associated with the development of acute rheumatic fever (10). To circumvent these issues, we have initiated an alternative strategy to identify other proteins localized to the sur...

Aggregation substance, a plasmid-encoded Enterococcus faecalis surface protein, plays a role in mediating the formation of mating aggregates, resulting in plasmid transfer. The role of aggregation substance in the internalization of E. faecalis by cultured intestinal epithelial cells, namely HT-29 cells, was analyzed. It was associated with a significant increase in endocytosis of E. faecalis by HT-29 cells: Numbers of internalized enterococci were fewer than of an invasive strain of Listeria monocytogenes, similar to Salmonella typhimurium and another L. monocytogenes strain, and greater than relatively noninvasive strains of E. faecalis, Proteus mirabilis, and Escherichia coli. Electron microscopy confirmed aggregation substance on the surface of strains interacting with the enterocyte microvillous surface, and intracellular enterococci were localized within membrane-bound vacuoles in the enterocyte cytoplasm. Thus, aggregation substance may facilitate E. faecalis internalization by host epithelial cells.

Exposure of Enterococcusfaecalis cells carrying the tetracycline resistance plasmid pCF10 to the heptapeptide pheromone cCF10 results in an increase in conjugal transfer frequency by as much as 106-fold. Pheromone-induced donor cells also express at least two plasmid-encoded surface proteins, the 130-kDa Sec 10 protein, which is involved in surface exclusion, and the 150-kDa AsclO protein, which has been associated with the formation of mating aggregates. Previous subcloning and transposon mutagenesis studies indicated that the adjacent EcoRI c (7.5 kb) and e (4.5 kb) fragments of pCF10 encode the structural genes for these proteins and that the EcoRI c fragment also encodes at least two regulatory genes involved in activation of the expression of the genes encoding AsclO and SeclO. In this paper, the results of physical and genetic analysis of this region of pCF10, along with the complete DNA sequences of the EcoRI c and e fragments, are reported. The results of the genetic studies indicate the location of the structural genes for the surface proteins and reveal important features of their transcription. In addition, we provide evidence here and in the accompanying paper (S. B. Olmsted, S.-M. Kao, L. J. van Putte, J. C. Gallo, and G. M. Dunny, J. Bacteriol. 173:7665-7672, 1991) for a role of Ascl0 in mating aggregate formation. The data also reveal a complex positive control system that acts at distances of at least 3 to 6 kb to activate expression of AsclO. DNA sequence analysis presented here reveals the positions of a number of specific genes, termed prg (pheromone-responsive genes) in this region of pCF10. The genes mapped include prgA (encoding SeclO) and prgB (encoding Asc10), as well as four putative regulatory genes, prgX, -R, -S, and -T. Although the predicted amino acid sequences of SeclO and AsclO have some structural features in common with a number of surface proteins of gram-positive cocci, and the AsclO sequence is highly similar to that of a similar protein encoded by the pheromone-inducible plasmid pAD1 (D.Galli, F. Lottspeich, and R. Wirth, Mol. Microbiol. 4:895-904, 1990), the regulatory genes show relatively little resemblance to any previously sequenced genes from either procaryotes or eucaryotes.