Legionella hijacks the host Golgi-to-ER retrograde pathway for the association of Legionella-Containing vacuole with the ER

Legionella pneumophila (L. pneumophila) is a gram-negative bacterium that replicates in a compartment that resembles the host endoplasmic reticulum (ER). To create its replicative niche, L. pneumophila manipulates host membrane traffic and fusion machineries. Bacterial proteins called Legionella effectors are translocated into the host cytosol and play a crucial role in these processes. In an early stage of infection, Legionella subverts ER-derived vesicles (ERDVs) by manipulating GTPase Rab1 to facilitate remodeling of the Legionella-containing vacuole (LCV). Subsequently, the LCV associates with the ER in a mechanism that remains elusive. In this study, we show that L. pneumophila recruits GTPases Rab33B and Rab6, which regulate vesicle trafficking from the Golgi to the ER, to the LCV to promote the association of LCV with the ER. We found that recruitment of Rab6 to the LCV depends on Rab33B. Legionella effector SidE family proteins, which phosphoribosyl-ubiquitinate Rab33B, were found to be necessary for the recruitment of Rab33B to the LCV. Immunoprecipitation experiments revealed that L. pneumophila facilitates the interaction of Rab6 with ER-resident SNAREs comprising syntaxin 18, p31, and BNIP1, but not tethering factors including NAG, RINT-1, and ZW10, which are normally required for syntaxin 18-mediated fusion of Golgi-derived vesicles with the ER. Our results identified a Rab33B-Rab6 cascade on the LCV and the interaction of Rab6 with ER-resident SNARE proteins for the association of LCV with the ER and disclosed the unidentified physiological role of SidE family proteins. Author summary Legionella pneumophila causes a sever pneumonia called Legionnaires' disease and a threat of this disease has increased on a world-wide scale. As a feature of L. pneumophila, it secrets over 300 bacterial effectors to adapt and survive inside the host and many of effectors modify the host proteins in a unique manner. L. pneumophila is known to travel inside the host and final destination of this pathogens is the host ER. In the initial step of this travel, L. pneumophila subverts host early vesicular trafficking to remodel the membrane composition of Legionella-containing vacuole (LCV). Although this remodeling process has been well characterized, the molecular machinery of association of remodeled vacuoles with the ER is still obscure. This paper shows that the host GTPases Rab6 and Rab33B, both of which control Golgi-to-ER traffic, are recruited to the LCV in a cascade manner and are required for the association of LCVs with the ER through the interaction between Rab6 and ER-resident t-SNARE proteins. Of note, we demonstrate that a bacteria-specific Rab33B modification called phosphoribosyl-ubiquitination by Legionella effectors proteins of the SidE family is essential for the recruitment of Rab33B to the LCV.

Automated summary

The study revealed that Legionella pneumophila recruits GTPases Rab33B and Rab6 to promote the association of Legionella-containing vacuole (LCV) with the host endoplasmic reticulum (ER), with the involvement of SidE family proteins. The interaction between Rab6 and ER-resident SNARE proteins was found to be crucial for this association.