Очистка Возбудитель вакуоли с<em> Legionella</em>-Инфицированных Фагоциты
Summary
В этой статье описывается метод выделения и очистки интактных<em> Legionella</em> Содержащего вакуоли (LCV) от амебы и макрофагов. Двухступенчатый протокол включает в себя LCV обогащения иммуно-магнитной сепарации с использованием антитела против бактериальных маркеров LCV и дальнейшей очистки центрифугирования в градиенте плотности.
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 organelles2-4.
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 centrifugation12,13 (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
Discussion
В отличие от ранее опубликованных методов, этот протокол основан на два этапа, первый разделения легких коммерческих автомобилей на иммуно-магнитного подход, а во-вторых, очищение легких коммерческих автомобилей на центрифугирования в градиенте плотности. Изоляция LCV можно легко прос…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Исследования в нашей лаборатории было поддержано Макс фон Петтенкофер Института Людвига-Максимилиана университета Мюнхена, Германское научно-исследовательское общество (DFG, HI 1511/3-1) и Bundesministerium für Bildung унд Forschung (BMBF) "Медицинская геномика инфекция" инициативы ( 0315834C).
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