Abstract
Legionella pneumophila is an opportunistic bacterial pathogen that causes a severe lung infection termed "Legionnaires' disease." The pathogen replicates in environmental protozoa as well as in macrophages within a unique membrane-bound compartment, the Legionella-containing-vacuole (LCV). LCV formation requires the bacterial Icm/Dot type IV secretion system, which translocates ca. 300 "effector proteins" into host cells, where they target distinct host factors. The L. pneumophila "pentuple" mutant (Δpentuple) lacks 5 gene clusters (31% of the effector proteins) and replicates in macrophages but not in Dictyostelium discoideum amoeba. To elucidate the host factors defining a replication-permissive compartment, we compare here the proteomes of intact LCVs isolated from D. discoideum or macrophages infected with Δpentuple or the parental strain Lp02. This analysis revealed that the majority of host proteins are shared in D. discoideum or macrophage LCVs containing the mutant or the parental strain, respectively, whereas some proteins preferentially localize to distinct LCVs. The small GTPase Rap1 was identified on D. discoideum LCVs containing strain Lp02 but not the Δpentuple mutant and on macrophage LCVs containing either strain. The localization pattern of active Rap1 on D. discoideum or macrophage LCVs was confirmed by fluorescence microscopy and imaging flow cytometry, and the depletion of Rap1 by RNA interference significantly reduced the intracellular growth of L. pneumophila Thus, comparative proteomics identified Rap1 as a novel LCV host component implicated in intracellular replication of L. pneumophila.
Highlights
The causative agent of Legionnaires’ disease, Legionella pneumophila, is a ubiquitous environmental bacterium that naturally colonizes complex aquatic biofilms, where the bacteria preferentially parasitize free-living protozoa (1–3)
The pentuple mutant strain is defective for intracellular replication in D. discoideum and other amoeba (Acanthamoeba castellanii, Hartmanella vermiformis), but grows in bone marrowderived macrophages (BMM) derived from the A/J mouse strain
Enrichment of Legionella-containing vacuole” (LCV) from these phagocytes by immuno-affinity separation requires the presence of the bacterial effector protein SidC, which is missing from the ⌬pentuple mutant
Summary
The causative agent of Legionnaires’ disease, Legionella pneumophila, is a ubiquitous environmental bacterium that naturally colonizes complex aquatic biofilms, where the bacteria preferentially parasitize free-living protozoa (1–3). The opportunistic pathogen survives and replicates both in extracellular and intracellular niches. To this end, L. pneumophila adopts a biphasic life style and switches between a transmissive (virulent) and a replicative (nonvirulent) phase (4). 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, and identified a set of virulence factors (“effector proteins”) exclusively produced in the exponential phase (8, 9). Through an evolutionary conserved mechanism the pathogen replicates within LCVs in human alveolar macrophages, causing the potentially fatal Legionnaires’ pneumonia (13). PMMB207C-Ptac-RBS-gfp-RBS-MCS-sidC pMMB207C-Ptac-RBS-dsred-RBS-MCS-sidC GFP-Rap[1] (alias RapA) expression construct, pDM317 RalGDSRBD-GFP expression construct, pDM115 pMMB207C, gfp (constitutive) pMMB207C, dsRed-express (constitutive)
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