Surfome analysis as a fast track to vaccine discovery: identification of a novel protective antigen for Group B Streptococcus hypervirulent strain COH1

Abstract

Safe recombinant vaccines, based on a small number of antigenic proteins, are emerging as the most attractive, cost-effective solution against infectious diseases. In the present work, we confirmed previous data from our laboratory showing that whole viable bacterial cell treatment with proteases followed by the identification of released peptides by mass spectrometry is the method of choice for the rapid and reliable identification of vaccine candidates in Gram-positive bacteria. When applied to the Group B Streptococcus COH1 strain, 43 surface-associated proteins were identified, including all the protective antigens described in the literature as well as a new protective antigen, the cell wall-anchored protein SAN_1485 belonging to the serine-rich repeat protein family. This strategy overcomes the difficulties so far encountered in the identification of novel vaccine candidates and speeds up the entire vaccine discovery process by reducing the number of recombinant proteins to be tested in the animal model.

Fig. 1.

Representation of the proteomics strategy used to identify surface-exposed proteins. Peptides and polypeptides released into the supernatant by surface digestion were either directly analyzed by LC/MS/MS (single digestion) or subjected to a second step of digestion with trypsin after a denaturing and reducing treatment (double digestion). MS/MS spectra were then searched against a database containing protein sequence data derived from the eight completely sequenced GBS strains and from the S. agalactiae section of the NCBInr database for protein identification.

Fig. 2.

Schematic topological representation of each protein identified on the surface of COH1 GBS strain. Topological organization of the identified proteins was predicted using PSORTb information. The orange line represents the membrane, and the red line represents the cell wall. For lipoproteins, the lipoyl anchor is represented as a black segment embedded within the membrane, whereas for the cell wall-anchored proteins, the last C-terminal blue residues are the LPXTG signature anchored to the cell wall. The identified peptides are marked in red.

Fig. 3.

COH1 GBS strain surface proteome. The 43 proteins belonging to the COH1 surface proteome were grouped into families based on their predicted cellular location. Red areas of each pie indicate the number of PSORTb-predicted proteins that were not found in the surface proteome, whereas the yellow areas represent the number of identified proteins belonging to each protein family.

Fig. 4.

Amino acid sequence of the novel protective antigen SAN_1485. The portions of the protein that were identified by surface digestion and mass spectrometry are marked in red. The gray background defines the region of the protein that was cloned and used for immunization. The LPXTG cell wall-anchoring motif is highlighted in the yellow box