Pathogen effector protein screening in yeast identifies Legionella factors that interfere with membrane trafficking

Abstract

Legionella pneumophila invades and replicates intracellularly in human and protozoan hosts. The bacteria use the Icm/Dot type IVB secretion system to translocate effectors that inhibit phagosome maturation and modulate host vesicle trafficking pathways. To understand how L. pneumophila modulates organelle trafficking in host cells, we carried out pathogen effector protein screening in yeast, identifying L. pneumophila genes that produced membrane trafficking [vacuole protein sorting (VPS)] defects in yeast. We identified four L. pneumophila DNA fragments that perturb sorting of vacuolar proteins. Three encode ORFs of unknown function that are translocated via the Icm/Dot transporter from Legionella into macrophages. VPS inhibitor protein (Vip) A is a coiled-coil protein, VipD is a patatin domain-containing protein, and VipF contains an acetyltransferase domain. Processing studies in yeast indicate that VipA, VipD, and VipF inhibit lysosomal protein trafficking by different mechanisms; overexpressing VipA has an effect on carboxypeptidase Y trafficking, whereas VipD interferes with multivesicular body formation at the late endosome and endoplasmic reticulum-to-Golgi body transport. Such differences highlight the multiple strategies L. pneumophila effectors use to subvert host trafficking processes. Using yeast as an effector gene discovery tool allows for a powerful, genetic approach to both the identification of virulence factors and the study of their function.

Fig. 2.

vip gene expression inhibits different trafficking pathways in yeast. (A) Golgi membrane-to-vacuole sorting pathways in S. cerevisiae. The CPY and MVB (CPS) pathways diverge at the late endosome, where the latter involves internal vesicle formation. After fusion with the vacuole, MVBs are degraded in the lumen by various enzymes. ALP is sorted via an AP3-dependent pathway that bypasses endosomes. (B and C) Immunoprecipitation of vacuolar proteins in vip-expressing strains. Cells were [³⁵S] methionine-labeled at 26°C with a 10-min pulse, followed by a chase to the indicated time points as described in Materials and Methods. Intracellular CPY (B) and CPS (C) were immunoprecipitated by using the appropriate antibodies, and samples were resolved by SDS/PAGE and visualized by autoradiography. Precursor (p) and mature (m) forms are indicated to the left. (B) For CPY, the ER-modified (p1), Golgi membrane-modified (p2), and mature (m) forms are shown. (C) For CPS, the Golgi membrane-modified (p) and mature (m) forms are shown.

Fig. 1.

vip gene expression causes CPY-Inv secretion in yeast. (A) Qualitative Inv assay on NSY01 strains transformed with plasmids carrying empty vector, VPS4^E233Q, vipA, vipD, vipE, and vipF clones. A SC-Ura/fructose plate was overlaid with top agar containing chromogenic reagents that resulted in a brown precipitate upon secretion of the CPY-Inv hybrid protein. (B) Quantitative Inv assay on vip clones. Inv activity in total cultures and in culture supernatants was measured as described in Materials and Methods. Values for exogenous and total Inv activity were obtained and used to calculate the percent secreted. Assays were done in triplicate at least twice.

Fig. 3.

Steady-state localization of a GFP-CPS fusion in vip-expressing strains. Cells were harvested in mid-log phase and labeled with N-[3-triethylammoniumpropyl]-4-[p-diethylaminophenylhexatrienyl] pyridinium dibromide (FM4-64) to visualize the vacuole membrane (as described in Materials and Methods). Fluorescence was observed and imaged by using the deltavision system (Applied Precision). DIC, differential interference contrast microscopy (Nomarski optics).

Fig. 4.

Cyclase translocation assays. J774 macrophages were seeded into 96-well tissue culture plates and infected with L. pneumophila strains for 30 min. After the removal of the supernatant, cells were lysed with cold 2.5% perchloric acid and neutralized with KOH. cAMP levels measured as in previous studies (9) by using the cAMP Biotrak Enzymeimmunoassay System (Amersham Pharmacia Biosciences).