Uncovering surface-exposed antigens of Lactobacillus rhamnosus by cell shaving proteomics and two-dimensional immunoblotting

Abstract

The present study reports the identification and comparison of all expressed cell-surface exposed proteins from the well-known probiotic L. rhamnosus GG and a related dairy strain, Lc705. To obtain this information, the cell-surface bound proteins were released from intact cells by trypsin shaving under hypertonic conditions with and without DTT. Liquid chromatography tandem mass spectrometry (LC-MS/MS) analyses of the purified peptides identified a total of 102 and 198 individual proteins from GG and Lc705, respectively. Comparison of both data sets suggested that the Msp-type antigens (Msp1, Msp2) and the serine protease HtrA were uniquely exposed at the cell surface of GG, whereas the Lc705-specific proteins included lactocepin and a wider range of different moonlighting proteins. ImmunoEM analyses with the GG and Lc705 antibodies suggested that the whole-cell immunization yielded antibodies toward surface-bound proteins and proteins that were secreted or released from the cell-surface. One of the detected antigens was a pilus-like structure on the surface of GG cells, which was not detected with Lc705 antibodies. Further 2-DE immunoblotting analysis of GG proteins with both L. rhamnosus antisera revealed that majority of the detected antigens were moonlighting proteins with potential roles in adhesion, pathogen exclusion or immune stimulation. The present study provides the first catalog of surface-exposed proteins from lactobacilli and highlights the importance of the specifically exposed moonlighting proteins for adaptation and probiotic functions of L. rhamnosus.

Keywords: 2-DE immunoblotting; LC−MS/MS; Lactobacillus rhamnosus; immunoEM; surface shaving; surfaceome proteins.