Proteomic Analysis and Identification of Streptococcus pyogenes Surface-Associated Proteins

Abstract

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...

Conserved anchorless surface proteins as group A streptococcal vaccine candidates

“…Conjecture exists in the scientific literature regarding the biological relevance and certitude of surface expression of anchorless proteins in GAS [5,30]. To validate our findings and to test protective vaccine efficacy of the selected anchorless surface proteins, full-length M1 protein, ADI, TF and FBA were adjuvanted with Complete Freund's Adjuvant (CFA) and used to subcutaneously immunize BALB/c mice and the survival recorded following lethal intraperitoneal M1 GAS challenge.…”

“…These authors also demonstrated considerable cross-protection of the 30-valent vaccine against non-vaccine M serotypes [4]. Recently, a series of independent studies have documented a class of GAS cell wall-associated or secreted metabolic enzymes that contain neither N-terminal leader sequences nor C-terminal cell wall anchors [5][6][7][8][9]. Perhaps because these molecules do not fit common categories of bacterial vaccine antigen, e.g.…”

The cell-envelope proteome of Bifidobacterium longum in an in vitro bile environment

“…In total, taking into account the membrane protein fraction and the envelope-associated soluble protein fraction, one of the most remarkable findings of this work was the large amount of ribosomal proteins detected: 41 out of the 52 ribosomal proteins annotated in the B. longum NCC 2705 genome were identified. Ribosomal proteins are very often found associated with the cell surface in bacteria, and although they are thought to have a certain affinity for the inner leaflet of the membrane, they have also been detected on the bacterial surface exposed to the external medium (Severin et al, 2007;Tjalsma et al, 2008). In some cases it has even been demonstrated that ribosomal proteins are immunomodulatory for humans (Spence & Clark, 2000).…”

“…In relation to this, with the aim of determining the protein profile of the B. longum envelope, and as a first approach to undertaking deeper functional studies, we analysed different subcellular fractions. The application of gel-based and gel-free technologies, combined with high-throughput techniques, allowed us to identify 218 proteins; about 70 % of them were predicted to be, or were previously described as being, in the cell envelope of Gram-positive bacteria (Antikainen et al, 2007;Candela et al, 2007;Eymann et al, 2004;Granato et al, 2004;Jang & Hanash 2003;Kelly et al, 2005b;Nandakumar et al, 2005;Rivera-Amill et al, 2001;Rodríguez-Ortega et al, 2006;Schaumburg et al, 2004;Severin et al, 2007;Silveira et al, 2004;Tjalsma et al, 2008;Wolff et al, 2007). Furthermore, 48 of them are predicted to be integral membrane proteins (contain hypothetical transmembrane segments) and 30 of them have different extracytoplasmic sorting signals.…”

Surface proteins of group G Streptococcus in different phases of growth: patterns of production and implications for the host–bacteria relationship

“…For intracellular proteome preparation (Malmström et al, 2012a), the bacterial cells were lysed using a bead-beater and centrifuged, and the supernatant was prepared for MS analysis, as subsequently described. For surface proteome preparation (Severin et al, 2007;Solis et al, 2010), surface-associated proteins were released by trypsination of the bacterial cells in a hypotonic solution. The trypsinated bacteria were gently centrifuged and the supernatant was prepared for MS analysis, as subsequently described.…”