Abstract
Legionella pneumophila is the causative agent of a pneumonia termed Legionnaires' disease. The facultative intracellular bacterium employs the Icm/Dot type IV secretion system (T4SS) and a plethora of translocated "effector" proteins to interfere with host vesicle trafficking pathways and establish a replicative niche, the Legionella-containing vacuole (LCV). Internalization of the pathogen and the events immediately ensuing are accompanied by host cell-mediated phosphoinositide (PI) lipid changes and the Icm/Dot-controlled conversion of the LCV from a PtdIns(3)P-positive vacuole into a PtdIns(4)P-positive replication-permissive compartment, which tightly associates with the endoplasmic reticulum. The source and formation of PtdIns(4)P are ill-defined. Using dually labeled Dictyostelium discoideum amoebae and real-time high-resolution confocal laser scanning microscopy (CLSM), we show here that nascent LCVs continuously capture and accumulate PtdIns(4)P-positive vesicles from the host cell. Trafficking of these PtdIns(4)P-positive vesicles to LCVs occurs independently of the Icm/Dot system, but their sustained association requires a functional T4SS. During the infection, PtdIns(3)P-positive membranes become compacted and segregated from the LCV, and PtdIns(3)P-positive vesicles traffic to the LCV but do not fuse. Moreover, using eukaryotic and prokaryotic PtdIns(4)P probes (2×PHFAPP-green fluorescent protein [2×PHFAPP-GFP] and P4CSidC-GFP, respectively) along with Arf1-GFP, we show that PtdIns(4)P-rich membranes of the trans-Golgi network associate with the LCV. Intriguingly, the interaction dynamics of 2×PHFAPP-GFP and P4CSidC-GFP are spatially separable and reveal the specific PtdIns(4)P pool from which the LCV PI originates. These findings provide high-resolution real-time insights into how L. pneumophila exploits the cellular dynamics of membrane-bound PtdIns(4)P for LCV formation.IMPORTANCE The environmental bacterium Legionella pneumophila causes a life-threatening pneumonia termed Legionnaires' disease. The bacteria grow intracellularly in free-living amoebae as well as in respiratory tract macrophages. To this end, L. pneumophila forms a distinct membrane-bound compartment called the Legionella-containing vacuole (LCV). Phosphoinositide (PI) lipids are crucial regulators of the identity and dynamics of host cell organelles. The PI lipid PtdIns(4)P is a hallmark of the host cell secretory pathway, and decoration of LCVs with this PI is required for pathogen vacuole maturation. The source, dynamics, and mode of accumulation of PtdIns(4)P on LCVs are largely unknown. Using Dictyostelium amoebae producing different fluorescent probes as host cells, we show here that LCVs rapidly acquire PtdIns(4)P through the continuous interaction with PtdIns(4)P-positive host vesicles derived from the Golgi apparatus. Thus, the PI lipid pattern of the secretory pathway contributes to the formation of the replication-permissive pathogen compartment.
Highlights
Legionella pneumophila is the causative agent of a pneumonia termed Legionnaires’ disease
Legionella-containing vacuole (LCV) formation is a complex process depending on the bacterial Icm/Dot type IV secretion system (T4SS) (8), which translocates a plethora of T4SS substrates termed “effector” proteins into host cells, where they subvert critical processes (9–11)
PI lipid, PtdIns(4)P, was previously shown to visibly accumulate on the LCV around 30 min p.i. (25). It is not known whether the PI lipid is formed directly on the LCV by phosphorylation or dephosphorylation of a PI precursor molecule or whether it accumulates on the LCV by interaction with PtdIns(4)P-rich membranes
Summary
Legionella pneumophila is the causative agent of a pneumonia termed Legionnaires’ disease. Internalization of the pathogen and the events immediately ensuing are accompanied by host cell-mediated phosphoinositide (PI) lipid changes and the Icm/Dot-controlled conversion of the LCV from a PtdIns(3)Ppositive vacuole into a PtdIns(4)P-positive replication-permissive compartment, which tightly associates with the endoplasmic reticulum. Using dually labeled Dictyostelium discoideum amoebae and real-time high-resolution confocal laser scanning microscopy (CLSM), we show here that nascent LCVs continuously capture and accumulate PtdIns(4)P-positive vesicles from the host cell Trafficking of these PtdIns(4)P-positive vesicles to LCVs occurs independently of the Icm/Dot system, but their sustained association requires a functional T4SS. The ER tubule-resident large GTPase atlastin[3] (Atl3/Sey1) promotes ER remodeling around LCVs, pathogen vacuole expansion, and intracellular replication (19), and the large dynamin1-like GTPase Dnm1l mediates L. pneumophila-induced mitochondrial fragmentation and inhibition of respiration (20) Another crucial class of regulators of membrane dynamics comprises the phosphoinositide (PI) lipids. PtdIns(4)P is the key PI lipid component of the Golgi apparatus (23) but is present at the plasma membrane and (late) endosomes (22, 24)
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