TY - JOUR
T1 - The Legionella longbeachae Icm/Dot substrate SidC selectively binds phosphatidylinositol 4-phosphate with nanomolar affinity and promotes pathogen vacuole-endoplasmic reticulum interactions
AU - Dolinsky, Stephanie
AU - Haneburger, Ina
AU - Cichy, Adam
AU - Hannemann, Mandy
AU - Itzen, Aymelt
AU - Hilbi, Hubert
N1 - Publisher Copyright:
© 2014, American Society for Microbiology.
PY - 2014
Y1 - 2014
N2 - Legionella spp. cause the severe pneumonia Legionnaires' disease. The environmental bacteria replicate intracellularly in freeliving amoebae and human alveolar macrophages within a distinct, endoplasmic reticulum (ER)-derived compartment termed the Legionella-containing vacuole (LCV). LCV formation requires the bacterial Icm/Dot type IV secretion system (T4SS) that translocates into host cells a plethora of different "effector" proteins, some of which anchor to the pathogen vacuole by binding to phosphoinositide (PI) lipids. Here, we identified by unbiased pulldown assays in Legionella longbeachae lysates a 111-kDa SidC homologue as the major phosphatidylinositol 4-phosphate [PtdIns(4)P]-binding protein. The PI-binding domain was mapped to a 20-kDa P4C [PtdIns(4)P binding of SidC] fragment. Isothermal titration calorimetry revealed that SidC of L. longbeachae (SidCLlo) binds PtdIns(4)P with a Kd (dissociation constant) of 71nM, which is 3 to 4 times lower than that of the SidC orthologue of Legionella pneumophila (SidCLpn). Upon infection of RAW 264.7 macrophages with L. longbeachae, endogenous SidCLlo or ectopically produced SidCLpn localized in an Icm/Dot-dependent manner to the PtdIns(4)P-positive LCVs. An L. longbeachae ΔsidC deletion mutant was impaired for calnexin recruitment to LCVs in Dictyostelium discoideum amoebae and outcompeted by wild-type bacteria in Acanthamoeba castellanii. Calnexin recruitment was restored by SidCLlo or its orthologues SidCLpn and SdcALpn. Conversely, calnexin recruitment was restored by SidCLlo in L. pneumophila lacking sidC and sdcA. Together, biochemical, genetic, and cell biological data indicate that SidCLlo is an L. longbeachae effector that binds through a P4C domain with high affinity to PtdIns(4)P on LCVs, promotes ER recruitment to the LCV, and thus plays a role in pathogen-host interactions.
AB - Legionella spp. cause the severe pneumonia Legionnaires' disease. The environmental bacteria replicate intracellularly in freeliving amoebae and human alveolar macrophages within a distinct, endoplasmic reticulum (ER)-derived compartment termed the Legionella-containing vacuole (LCV). LCV formation requires the bacterial Icm/Dot type IV secretion system (T4SS) that translocates into host cells a plethora of different "effector" proteins, some of which anchor to the pathogen vacuole by binding to phosphoinositide (PI) lipids. Here, we identified by unbiased pulldown assays in Legionella longbeachae lysates a 111-kDa SidC homologue as the major phosphatidylinositol 4-phosphate [PtdIns(4)P]-binding protein. The PI-binding domain was mapped to a 20-kDa P4C [PtdIns(4)P binding of SidC] fragment. Isothermal titration calorimetry revealed that SidC of L. longbeachae (SidCLlo) binds PtdIns(4)P with a Kd (dissociation constant) of 71nM, which is 3 to 4 times lower than that of the SidC orthologue of Legionella pneumophila (SidCLpn). Upon infection of RAW 264.7 macrophages with L. longbeachae, endogenous SidCLlo or ectopically produced SidCLpn localized in an Icm/Dot-dependent manner to the PtdIns(4)P-positive LCVs. An L. longbeachae ΔsidC deletion mutant was impaired for calnexin recruitment to LCVs in Dictyostelium discoideum amoebae and outcompeted by wild-type bacteria in Acanthamoeba castellanii. Calnexin recruitment was restored by SidCLlo or its orthologues SidCLpn and SdcALpn. Conversely, calnexin recruitment was restored by SidCLlo in L. pneumophila lacking sidC and sdcA. Together, biochemical, genetic, and cell biological data indicate that SidCLlo is an L. longbeachae effector that binds through a P4C domain with high affinity to PtdIns(4)P on LCVs, promotes ER recruitment to the LCV, and thus plays a role in pathogen-host interactions.
UR - http://www.scopus.com/inward/record.url?scp=84907099490&partnerID=8YFLogxK
U2 - 10.1128/IAI.01685-14
DO - 10.1128/IAI.01685-14
M3 - Article
C2 - 25024371
AN - SCOPUS:84907099490
SN - 0019-9567
VL - 82
SP - 4021
EP - 4033
JO - Infection and Immunity
JF - Infection and Immunity
IS - 10
ER -
