Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

Summary

This article describes a method for the isolation and purification of intact Legionella-containing vacuoles (LCVs) from amoeba and macrophages. The two-step protocol comprises LCV enrichment by immuno-magnetic separation using an antibody against a bacterial LCV marker and further purification by density gradient centrifugation.

Abstract

The opportunistic pathogen Legionella pneumophila is an amoeba-resistant bacterium, which also replicates in alveolar macrophages thus causing the severe pneumonia "Legionnaires' disease"¹. In protozoan and mammalian phagocytes, L. pneumophila employs a conserved mechanism to form a specific, replication-permissive compartment, the "Legionella-containing vacuole" (LCV). LCV formation requires the bacterial Icm/Dot type IV secretion system (T4SS), which translocates as many as 275 "effector" proteins into host cells. The effectors manipulate host proteins as well as lipids and communicate with secretory, endosomal and mitochondrial organelles^2-4.

The formation of LCVs represents a complex, robust and redundant process, which is difficult to grasp in a reductionist manner. An integrative approach is required to comprehensively understand LCV formation, including a global analysis of pathogen-host factor interactions and their temporal and spatial dynamics. As a first step towards this goal, intact LCVs are purified and analyzed by proteomics and lipidomics.

The composition and formation of pathogen-containing vacuoles has been investigated by proteomic analysis using liquid chromatography or 2-D gel electrophoresis coupled to mass-spectrometry. Vacuoles isolated from either the social soil amoeba Dictyostelium discoideum or mammalian phagocytes harboured Leishmania⁵, Listeria⁶, Mycobacterium⁷, Rhodococcus⁸, Salmonella⁹ or Legionella spp.¹⁰. However, the purification protocols employed in these studies are time-consuming and tedious, as they require e.g. electron microscopy to analyse LCV morphology, integrity and purity. Additionally, these protocols do not exploit specific features of the pathogen vacuole for enrichment.

The method presented here overcomes these limitations by employing D. discoideum producing a fluorescent LCV marker and by targeting the bacterial effector protein SidC, which selectively anchors to the LCV membrane by binding to phosphatidylinositol 4-phosphate (PtdIns(4)P)^3,11 . LCVs are enriched in a first step by immuno-magnetic separation using an affinity-purified primary antibody against SidC and a secondary antibody coupled to magnetic beads, followed in a second step by a classical Histodenz density gradient centrifugation^12,13 (Fig. 1).

A proteome study of isolated LCVs from D. discoideum revealed more than 560 host cell proteins, including proteins associated with phagocytic vesicles, mitochondria, ER and Golgi, as well as several GTPases, which have not been implicated in LCV formation before¹³. LCVs enriched and purified with the protocol outlined here can be further analyzed by microscopy (immunofluorescence, electron microscopy), biochemical methods (Western blot) and proteomic or lipidomic approaches.

Protocol

1. Preparations for LCV Isolation

1. Streak out Legionella pneumophila producing DsRed-Express^13,14 from glycerol stocks on CYE agar plates with 5 μg/mL chloramphenicol (Cam) four days before LCV isolation. Incubate the bacteria at 37 °C.
2. Seed out 1 x 10⁷ D. discoideum producing GFP-calnexin or RAW264.7 murine macrophages in 75 cm² tissue culture flasks one day prior infection. Use 10 ml HL5 medium with 20 μg/mL G418 for D. discoideum and incubate the amoebae at 23 °C. For RAW264.7 macrophages use 10 ml RPMI 1640 medium supplemented with 10% FCS (heat inactivated) and 1% glutamine, and grow....

Discussion

In contrast to previously published methods, this protocol is based on two steps, first the separation of LCVs by an immuno-magnetic approach, and second, the purification of LCVs by density gradient centrifugation. The LCV isolation can be easily followed by fluorescence microscopy, when either fluorescently labeled bacteria and GFP-producing cells or antibody staining is used. The protocol described here is a simple, straight-forward method, which results in the enrichment of LCVs with high purity. Importantly, the pro.......