Influence of Impaired Lipoprotein Biogenesis on Surface and Exoproteome of Streptococcus pneumoniae

Abstract

Surface proteins are important for the fitness and virulence of the Gram-positive pathogen Streptococcus pneumoniae. They are crucial for interaction of the pathogen with its human host during infection. Therefore, the analysis of the pneumococcal surface proteome is an important task that requires powerful tools. In this study, two different methods, an optimized biotinylation approach and shaving with trypsin beads, were applied to study the pneumococcal surface proteome and to identify surface-exposed protein domains, respectively. The identification of nearly 95% of the predicted lipoproteins and 75% of the predicted sortase substrates reflects the high coverage of the two classical surface protein classes accomplished in this study. Furthermore, the biotinylation approach was applied to study the impact of an impaired lipoprotein maturation pathway on the cell envelope proteome and exoproteome. Loss of the lipoprotein diacylglyceryl transferase Lgt leads to striking changes in the lipoprotein distribution. Many lipoproteins disappear from the surface proteome and accumulate in the exoproteome. Further insights into lipoprotein processing in pneumococci are provided by immunoblot analyses of bacterial lysates and corresponding supernatant fractions. Taken together, the first comprehensive overview of the pneumococcal surface and exoproteome is presented, and a model for lipoprotein processing in S. pneumoniae is proposed.

Structure of the pneumococcal l,d‐carboxypeptidase DacB and pathophysiological effects of disabled cell wall hydrolases DacA and DacB

“…Proper anchoring of lipoproteins requires both, the lipoprotein‐specific signal peptidase Lsp and the diacylglyceryl transferase Lgt. Loss of the Lgt leads to striking changes in the maturation and distribution of lipoproteins (Pribyl et al ., ). To assess the functional consequences of loss‐of‐function of the Lgt or Dac proteins on surface localization, PGN turnover and virulence, pneumococcal mutants deficient in DacA, DacB, DacA and DacB, or Lgt were generated in WT S. pneumoniae D39 and its isogenic non‐encapsulated strain D39Δ cps by insertion‐deletion mutagenesis (Fig.…”

Modular Architecture and Unique Teichoic Acid Recognition Features of Choline-Binding Protein L (CbpL) Contributing to Pneumococcal Pathogenesis

“…This enzyme, together with the lipoprotein specific signal peptidase Lsp, is essential to anchor lipoproteins properly in the membrane. The deficiency of the Lgt was shown to alter the molecular weight and localization of lipoproteins25. The Ltp_Lipoprotein in CbpL, however, is 48 aa in length (TIGR4) and lacks the recognition lipobox motif LxxC.…”

Non-Adhesive Surface Proteins of Streptococcus pneumoniae

“…Increasingly in these types of experiments, surface proteins are identified by liquid chromatographyÀmass spectrometry following "surface-shaving" of live cells using proteases [1,2] or, alternatively, by characterization of membrane-derived extracellular vesicles [3]. With the advent of the genomics era, bioinformatic approaches have been introduced, usually relying on the identification of specific recognition sites in the predicted amino acid sequence of a given protein.…”

“…To detect these non-classical surface proteins, high-throughput proteomics approaches can be used whereby proteinaceous structures on the surface of the pneumococcus are cleaved off using a protease treatment, purified, and analyzed by LC/MS analysis [1,2]. These markers obviously do not function for moonlighting proteins that have an intracellular role but are also found on the surface.…”