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 phase-specific 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 life-stage specific markers for pathogen analysis.
Highlights
Introduction of ChromosomalHA-tags and Western Blot Analysis of Defect in organelle trafficking/Intracellular multiplication (Dot/Icm) Effector Abundance in E and PE L. pneumophila—Hemagglutinin (HA) tags were chromosomally introduced in L. pneumophila JR32 to express N-terminally tagged Dot/Icm effectors for comparison of quantities present in E and PE
For E/PE sample preparation, L. pneumophila was grown in broth until E or PE and enrichment of flagellin FliC was used to confirm entry into PE [12, 63, 64] (Fig. 1A and 1B)
We provide comprehensive maps of a total of 960 different proteins quantified in three life stages, E, PE, and unculturable microcosms (UNC), of L. pneumophila and in different bacterial subfractions (Table I)
Summary
Introduction of ChromosomalHA-tags and Western Blot Analysis of Dot/Icm Effector Abundance in E and PE L. pneumophila—Hemagglutinin (HA) tags were chromosomally introduced in L. pneumophila JR32 to express N-terminally tagged Dot/Icm effectors for comparison of quantities present in E and PE. An ϳ1000bp fragment containing the effector gene start codon in the middle was cloned into pGEMT-Easy (Promega) resulting in plasmids pPA203, pPA205, pPA206, pPA208 to pPA211. The HA sequence was introduced adjacent to the effector start codons by “round-the-horn” site-directed mutagenesis [60] resulting in pPA213, pPA215, pPA216, pPA218 to pPA221 (see supplemental Table S2). The mutagenic fragments were amplified from pGEMT-EZ by primers pGEMT_a1f and pGEMT_b1r and were blunt ligated into the allelic exchange vector pLAW344, yielding pPA233, pPA235, pPA236, pPA238 to pPA241 (see supplemental Table S2) [62]. Allelic exchange was confirmed by PCR with primer HA_f which binds the HA-tag gene sequence, and effector-specific reverse primers localized outside of the homology region used for recombination. Resulting clones were grown comparably to the strains for proteomic analysis (see above) and equal amounts of material from E and PE bacteria were subjected to SDS-PAGE and Western blotting with protein detection via anti HA. mAb (Covance)
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