Life Stage-specific Proteomes of Legionella pneumophila Reveal a Highly Differential Abundance of Virulence-associated Dot/Icm effectors

Abstract

Major differences in the transcriptional program underlying the phenotypic switch between exponential and post-exponential growth of Legionella pneumophila were formerly described characterizing important alterations in infection capacity. Additionally, a third state is known where the bacteria transform in a viable but nonculturable state under stress, such as starvation. We here describe phase-related proteomic changes in exponential phase (E), postexponential phase (PE) bacteria, and unculturable microcosms (UNC) containing viable but nonculturable state cells, and identify phasespecific proteins. We present data on different bacterial subproteomes of E and PE, such as soluble whole cell proteins, outer membrane-associated proteins, and extracellular proteins. In total, 1368 different proteins were identified, 922 were quantified and 397 showed differential abundance in E/PE. The quantified subproteomes of soluble whole cell proteins, outer membrane-associated proteins, and extracellular proteins; 841, 55, and 77 proteins, respectively, were visualized in Voronoi treemaps. 95 proteins were quantified exclusively in E, such as cell division proteins MreC, FtsN, FtsA, and ZipA; 33 exclusively in PE, such as motility-related proteins of flagellum biogenesis FlgE, FlgK, and FliA; and 9 exclusively in unculturable microcosms soluble whole cell proteins, such as hypothetical, as well as transport/binding-, and metabolism-related proteins. A high frequency of differentially abundant or phase-exclusive proteins was observed among the 91 quantified effectors of the major virulence-associated protein secretion system Dot/Icm (> 60%). 24 were E-exclusive, such as LepA/B, YlfA, MavG, Lpg2271, and 13 were PE-exclusive, such as RalF, VipD, Lem10. The growth phase-related specific abundance of a subset of Dot/Icm virulence effectors was confirmed by means of Western blotting. We therefore conclude that many effectors are predominantly abundant at either E or PE which suggests their phase specific function. The distinct temporal or spatial presence of such proteins might have important implications for functional assignments in the future or for use as lifestage specific markers for pathogen analysis. Molecular

Comparative Proteomics of Purified Pathogen Vacuoles Correlates Intracellular Replication of Legionella pneumophila with the Small GTPase Ras-related protein 1 (Rap1)

“…The life cycle is regulated by the growth phase (5): bacteria in postexponential phase induce virulence, motility, and stress resistance, whereas exponentially growing bacteria repress these traits and upregulate metabolic pathways (6,7). Growth phase-specific proteomic analyses of L. pneumophila grown in broth confirmed the up-regulation of virulence and motility traits in postexponential growth phase, but also identified a set of virulence factors ("effector proteins") exclusively produced in the exponential phase (8,9).…”

Improving Proteome Coverage for Small Sample Amounts: An Advanced Method for Proteomics Approaches with Low Bacterial Cell Numbers

“…Analysis of 5 × 10 6 L. pneumophila bacteria resulted in 58% more peptides (from 5110 to 8071) and 38% more proteins (1043 to 1440) using the SP3* protocol. In comparison, Aurass et al [25] were able to quantify 1368 different L. pneumophila proteins (5% less than in this study) using a fractionation approach and 20 µg protein. Thus, with the optimized SP3* protocol a similar number of proteins is amendable to quantification in less time (without fractionation) and with about 100 times fewer starting material.…”

“…Analysis of 5 × 10 6 L. pneumophila bacteria resulted in 58% more peptides (from 5110 to 8071) and 38% more proteins (1043 to 1440) using the SP3* protocol. In comparison, Aurass et al . were able to quantify 1368 different L. pneumophila proteins (5% less than in this study) using a fractionation approach and 20 µg protein.…”

“…For L. pneumophila to our knowledge only global proteome data obtained with bacterial numbers at least 50 times larger than those from infection settings, are currently available. [25,26] In the present work, on average 835 peptides that represented 303 proteins or 9% of the theoretical proteome could be identified with CD+ISD from 2 × 10 6 L. pneumophila bacteria. In contrast, about 1222 proteins (37% of the theoretical proteome) and 6040 peptides were identified with the SP3* protocol from the same number of bacteria.…”

“…For all three bacterial species tested, the SP3* protocol enabled an increased peptide and protein identification rate compared to the CD+ISD protocol ( Figure ; Figure S1 and , Supporting Information). For L. pneumophila to our knowledge only global proteome data obtained with bacterial numbers at least 50 times larger than those from infection settings, are currently available . In the present work, on average 835 peptides that represented 303 proteins or 9% of the theoretical proteome could be identified with CD+ISD from 2 × 10 6 L. pneumophila bacteria.…”