Outer-membrane proteomic maps and surface-exposed proteins of Legionella pneumophila using cellular fractionation and fluorescent labelling

Abstract

Bacterial surface-associated proteins play crucial roles in host-pathogen interactions and pathogenesis. The identification of these proteins represents an important goal of bacterial proteomics for vaccine development, but also for environmental concerns such as microbial biosensing. Here, we developed such an approach for Legionella pneumophila, a bacterium that causes severe pneumonia. We propose a complementary strategy consisting of (1) a fluorescent labelling of surface-exposed proteins in parallel with (2) a fractionation of the outer-membrane protein extract. These two distinct protein populations were subsequently separated using two-dimensional gel electrophoresis and characterised by mass spectrometry. Within these populations, we found proteins which were expected for the compartments studied, but also a great number of proteins never experimentally described, and also a non-negligible fraction of proteins never described in these fractions. These data provided new routes of inspection for transport and host recognition for Legionella pneumophila. In addition, these results on the membranome and surfaceome show that Legionella in the stationary phase of growth possesses the major determinants to infect host cells.

Proteomics and Transcriptomics Characterization of Bile Stress Response in Probiotic Lactobacillus rhamnosus GG

“…surfome) of GG using DIGE labeling of intact GG cells (wholecell DIGE labeling). This methodology has been used previously for labeling of bacterial surface-exposed proteins in bacterial species such as Porphyromonas gingivalis (48) and Legionella pneumophila (49). Possible cell lysis occurring during CyDye labeling was tested by plating GG cells incubated in the DIGE labeling buffer onto MRS agar.…”

Immunoproteomic Analysis of the Development of Natural Immunity in Subjects Colonized by Neisseria meningitidis Reveals Potential Vaccine Candidates

“…Alternatively, the serum antibody reactivity to these proteins observed on Western blots may be the result of the disruption of meningococcal cells by complement-mediated bacteriolysis (14) occurring following meningococcal colonization. Although the presence of cytoplasmic proteins in OM preparations may be disputed, several proteomic studies have shown them to be present in OM preparations from other gram-negative bacteria, including those from Escherichia coli (29), Legionella pneumophila (27), N. lactamica (53), and N. meningitidis (9,37,59). Indeed, a fluorescence-activated cell sorting analysis of whole meningococcal cells using polyclonal antibodies against corresponding recombinant proteins has shown the presence of predicted cytoplasmic proteins on the external surface of the OM (9).…”

A method to map changes in bacterial surface composition induced by regulatory RNAs in Escherichia coli and Staphylococcus aureus

“…Two previous studies in human [4] and Legionella pneumophila [5] have shown that the three Cy Dyes specifically label membrane proteins. However, using two complementary approaches we have verified that the three dyes did not efficiently penetrate into the Gram-negative E. coli and the Gram-positive S. aureus cells (Supplementary Information).…”

“…Here we have adapted a method that allows specific labeling of exposed lysine residues of surface proteins from living bacteria followed by 2D gel electrophoresis [4,5]. This method was applied to Escherichia coli and Staphylococcus aureus to follow the dynamics of cell surface composition that could be controlled by the expression of regulatory RNAs.…”