Proteomic comparison of membrane and extracellular proteins from invasive (PAO1) and cytotoxic (6206) strains of Pseudomonas aeruginosa

Abstract

Strains of Pseudomonas aeruginosa can be phenotypically classified by their mode of pathogenicity as either invasive, where the bacterium is internalised by host cells, or cytotoxic, where the host cell is killed without internalisation through the expression of cytotoxicity factors. These phenotypes are thought to depend primarily on the interactions of pseudomonal membrane and secreted proteins with host cells. We report here comparisons of outer membrane and extracellular protein-enriched fractions from invasive (PAO1) and cytotoxic (6206) strains of P. aeruginosa separated by two-dimensional (2-D) gel electrophoresis. Gel image comparisons revealed the two strains express essentially identical membrane protein profiles under the conditions investigated. Membrane protein strain differences were typically the result of minor amino acid sequence variations resulting in small mass and isoelectric point shifts visible on 2-D gels. Analysis of extracellular proteins from stationary phase growth, however, revealed significantly different protein profiles. Extracellular fractions from the invasive PAO1 strain were dominated by extracellular proteases including elastase (LasB), LasA protease and chitin-binding protein, as well as several previously designated 'hypothetical' proteins. LasB appeared to be highly processed with 28 discrete mass and isoelectric point forms detected in this study. The significant number of active extracellular proteases (including LasB itself) may account for this processing. Conversely, extracellular fractions from strain 6206 consisted mainly of cellular and membrane exposed proteins including GroEL, DnaK and flagellar subunits. These are thought to result from cellular turnover during growth and the reliance on the secretory mechanisms of this strain to produce high levels of cytotoxicity factors, such as ExoU, which may be produced only upon specific interactions with host cells. These studies will aid in elucidating the differences between invasive and cytotoxic P. aeruginosa at the proteome level.

Proteome analysis of extracellular proteins regulated by the las and rhl quorum sensing systems in Pseudomonas aeruginosa PAO1

“…This is consistent with previous studies showing that both the las and rhl systems regulate Xcp-mediated type II secretion in P. aeruginosa (Chapon-Herve et al, 1997), and that loss of QS results in reduced expression of several secreted proteins, including elastase (lasB) (Passador et al, 1993;Pearson et al, 1997). Since LasB is the major constituent of PAO1 culture supernatants (accounting for approximately 60 % of the total visible protein abundance in this strain; Nouwens et al, 2002), a reduction in lasB expression combined with inhibited secretion, both caused by the loss of QS, may explain the dramatic reduction in extracellular protein concentration seen here.…”

Proteomic Characterization of the Whole Secretome of Legionella pneumophila and Functional Analysis of Outer Membrane Vesicles

“…So far, extracellular proteomes of various gram-positive and gram-negative bacterial pathogens have been characterized (17,60,84). A proteomic analysis of type II secreted effector proteins of L. pneumophila was recently described (26).…”

Expression of the quorum-sensing regulatory protein LasR is strongly affected by iron and oxygen concentrations in cultures of Pseudomonas aeruginosa irrespective of cell density

“…Several large-scale transcriptomic and proteomic studies of QS and iron limitation in P. aeruginosa have been published recently Arevalo-Ferro et al, 2003;Nouwens et al, 2002Nouwens et al, , 2003Palma et al, 2003;Wagner et al, 2003;Schuster et al, 2003Schuster et al, , 2004Juhas et al, 2004;Wolfgang et al, 2004;Cornelis & Aendekerk, 2004). We compared the regulation of secreted proteins detected in our work by proteomic analysis with those from the transcriptomic and proteomic studies reported in the literature (Table 1).…”