TheLegionella pneumophilaphosphatidylinositol-4 phosphate-binding type IV substrate SidC recruits endoplasmic reticulum vesicles to a replication-permissive vacuole

Abstract

SummaryLegionella pneumophila, the causative agent of Legionnaires' disease, uses the intracellular multiplication/defective organelle trafficking (Icm/ Dot) type IV secretion system to establish within amoebae and macrophages an endoplasmic reticulum (ER)-derived replication-permissive compartment, the Legionella-containing vacuole (LCV). The Icm/Dot substrate SidC and its paralogue SdcA anchor to LCVs via phosphatidylinositol-4 phosphate [PtdIns(4)P]. Here we identify the unique 20 kDa PtdIns(4)P-binding domain of SidC, which upon heterologous expression in Dictyostelium binds to LCVs and thus is useful as a PtdIns(4)P-specific probe. LCVs harbouring L. pneumophila DsidC-sdcA mutant bacteria recruit ER and ER-derived vesicles less efficiently and carry endosomal but not lysosomal markers. The phenotypes are complemented by supplying sidC on a plasmid. L. pneumophila DsidC-sdcA grows at wild-type rate in calnexin-negative LCVs, suggesting that communication with the ER is dispensable for establishing a replicative compartment. The amount of SidC and calnexin is directly proportional on isolated LCVs, and in a cell-free system, the recruitment of calnexin-positive vesicles to LCVs harbouring DsidC-sdcA mutant bacteria is impaired. Beads coated with purified SidC or its 70 kDa N-terminal fragment recruit ER vesicles in Dictyostelium and macrophage lysates. Our results establish SidC as an L. pneumophila effector protein, which anchors to PtdIns(4)P on LCVs and recruits ER vesicles to a replication-permissive vacuole.

The Legionella effector SidC defines a unique family of ubiquitin ligases important for bacterial phagosomal remodeling

“…The Legionella effector SidC was such an example. SidC and its paralog SdcA were first assigned as vesicle tethering factors for promoting the recruitment and fusion of ER-derived vesicles to the bacterial phagosome (12). Recently, crystal structures of the N-terminal portion of SidC were reported (38,40).…”

“…SidC and its paralog SdcA have been shown to recruit ER vesicles to the LCV (12). We thus investigated the relevance of the E3 ligase activity of SidC in Legionella infection.…”

“…Both proteins were shown to recruit ER vesicles to the LCV while anchored on the LCV via a specific C-terminal phosphatidylinositol-4-phosphate [PI(4)P]-binding domain (3,11,12). Bacterial vacuoles harboring the ΔsidC-sdcA mutant bacteria recruit ER-derived vesicles less efficiently, and in vitro experiments further showed that the N-terminal 70-kDa fragment of SidC binds to ER vesicles in Dictyostelium and macrophage lysates (12). However, the biochemical mechanism for SidCmediated ER recruitment remains unclear.…”

The ubiquitin system: a critical regulator of innate immunity and pathogen–host interactions

“…127 SidC and its paralog SdcA, identified in L. pneumophila, are capable of recruiting the ER-derived vesicles to build LCV. 128 A new study showed that SidC/SdcA is a novel type of bacterial E3 ligase with a special C46-H444-D446 catalytic triad that can form high-molecular-weight polyubiquitin chains. 129 In addition, the effector RavZ specifically deconjugates the lipid-attached LC3 marks and thereby prevents the autophagic pathway by functioning as a cysteine protease.…”

Structural basis for substrate recognition by a unique Legionella phosphoinositide phosphatase

“…It has been suggested that PI(4)P is enriched on the LCV membrane, which among other functions, anchors effectors such as SidM/DrrA, SidC, and SdcA to promote the recruitment and fusion of the endoplasmic reticulum derived vesicles with the LCV (18,19). In the amoebae host Dictyostelium discoideum, the Legionella effector protein LpnE appears to recruit the host PI-5-phosphatase OCRL to the LCV, leading to restriction of intracellular bacterial growth (20).…”