Isolation of pathogen-containing vacuoles.

Methods in molecular biology (Clifton, N.J.) (2013-03-16)
Olga Shevchuk, Michael Steinert

Dictyostelium discoideum cells are "professional phagocytes," as they ingest a large variety of bacteria, yeast, and inert particles. Several bacterial pathogens are able to survive intracellularly within specialized vacuoles of D. discoideum by interfering with host signaling pathways. To better understand the molecular mechanisms underlying these evolutionary conserved processes we have established a method for the isolation of pathogen-containing vacuoles (PCVs). The isolation protocol describes the infection of D. discoideum cells with the intracellular pathogen Legionella pneumophila, loading of the lysosomal compartment with colloidal iron, mechanical lysis of host cells, iodophenylnitrophenyltetrazolium (INT) heavy labeling of mitochondria, removal of nucleic acid by Benzonase treatment, separation of nuclei by low-speed centrifugation, and the magnetic removal of lysosomes. The subcellular fractionation in a discontinuous sucrose density OptiPrep gradient allows the separation of mitochondria and to prepare PCVs with high purity. The proteins isolated from PCVs have been successfully subjected to mass spectrometry and allowed to analyze pathogen-directed maturation processes of vacuoles. The method can also be applied for subsequent protein modification analyses and lipidome comparisons.

Product Number
Product Description

Iron(III) chloride, sublimed grade, ≥99.9% trace metals basis
Iron(III) chloride, anhydrous, powder, ≥99.99% trace metals basis
Iron(III) chloride hexahydrate, puriss. p.a., ACS reagent, crystallized, 98.0-102% (RT)
Iron(III) chloride hexahydrate, ACS reagent, 97%
Iron(III) chloride hexahydrate, puriss. p.a., reag. Ph. Eur., ≥99%
Iron(III) chloride hexahydrate, reagent grade, ≥98%, chunks
Iron(III) chloride solution, 0.2 M in 2-methyltetrahydrofuran
TDA Reagent, suitable for microbiology
Iron(III) chloride solution, purum, 45% FeCl3 basis