Proteome analysis of the Gram-positive fish pathogen Renibacterium salmoninarum reveals putative role of membrane vesicles in virulence

Abstract

Bacterial kidney disease (BKD) is a chronic bacterial disease affecting both wild and farmed salmonids. The causative agent for BKD is the Gram-positive fish pathogen Renibacterium salmoninarum. As treatment and prevention of BKD have proven to be difficult, it is important to know and identify the key bacterial proteins that interact with the host. We used subcellular fractionation to report semi-quantitative data for the cytosolic, membrane, extracellular, and membrane vesicle (MV) proteome of R. salmoninarum. These data can aid as a backbone for more targeted experiments regarding the development of new drugs for the treatment of BKD. Further analysis was focused on the MV proteome, where both major immunosuppressive proteins P57/Msa and P22 and proteins involved in bacterial adhesion were found in high abundance. Interestingly, the P22 protein was relatively enriched only in the extracellular and MV fraction, implicating that MVs may play a role in host-pathogen interaction. Compared to the other subcellular fractions, the MVs were also relatively enriched in lipoproteins and all four cell wall hydrolases belonging to the New Lipoprotein C/Protein of 60 kDa (NlpC/P60) family were detected, suggesting an involvement in the formation of the MVs.

Figure 1

Overview of the protein identifications, predicted protein localizations, and predicted signal peptides for all enriched subcellular fractions: Protein abundances were only used for this figure when a protein was quantified in at least 3 out of 4 replicates in one or more of the four subcellular fractions. (a) Principal component analysis of the enriched subcellular fractions. Missing values were imputed from the normal distribution of each replicate (MV = membrane vesicle); (b) Overview of the overlap and the exclusively quantified proteins between the four enriched subcellular fractions (MV = membrane vesicle); (c) Illustration of the summed LFQ intensities of quantified proteins in the four subcellular fractions assigned to the respective predicted PSORTb localizations. The number of quantified proteins with the respective PSORTb prediction in each of the subcellular fractions is indicated above the bars; (d) Illustration of the summed LFQ intensities of quantified proteins in the four subcellular fractions assigned to the predicted SignalP protein signal peptides. The number of quantified proteins with the respective SignalP signal peptide prediction within the subcellular fractions is indicated above the bars.

Figure 2

Schematic visualization of 25 high abundant proteins that are involved in virulence processes of R. salmoninarum and their experimental subcellular localization. Proteins are colored depending on their high abundance (higher than a log2 transformed median normalized LFQ intensity of 2) in one or more of the enriched subcellular fractions. Proteins are marked with their UniProt identifier.

Figure 3

Transmission electron micrographs of isolated and purified membrane vesicles of R. salmoninarum.