Rapid Isolation of intact Salmonella-containing vacuoles using paramagnetic nanoparticles.
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Authors
Singh, Vikash
Schwerk, Peter
Tedin, Karsten
Publication Date
2018Journal Title
Gut Pathog
ISSN
1757-4749
Publisher
Springer Science and Business Media LLC
Volume
10
Pages
33
Language
eng
Type
Article
Physical Medium
Electronic-eCollection
Metadata
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Singh, V., Schwerk, P., & Tedin, K. (2018). Rapid Isolation of intact Salmonella-containing vacuoles using paramagnetic nanoparticles.. Gut Pathog, 10 33. https://doi.org/10.1186/s13099-018-0256-7
Abstract
BACKGROUND: Both typhoidal and non-typhoidal Salmonella infections remain a considerable cause of morbidity and mortality globally, and impose a major socio-economic burden worldwide. A key property of all pathogenic Salmonella strains is the ability to invade host cells and reside within an intracellular, vacuolar compartment called the Salmonella-containing vacuole (SCV). Although the SCV is involved in both immune-evasion and intracellular replication and spread within the host, information about the host:pathogen interactions at this interface are limited, in part due to the technical difficulties involved in purification of these vacuoles. While a number of column- or gradient-based methods have been applied, cross-contamination with other host cell organelles or rupture of the labile SCV membrane has further complicated efforts to successfully isolate SCVs. RESULTS: Here, we report the isolation of intact SCVs using carbon-coated, paramagnetic nanoparticles. The approach permits rapid isolation of intact SCVs from human macrophages in vitro without involving numerous purification steps. Bacteria are pre-labeled with modified nanoparticles prior to infection, and at various times post-infection, host cells are lysed and intact pathogen-containing phagosomes are recovered after application of a mild magnetic field. Purified, intact SCVs isolated using this method were shown to display high levels of co-association of internalized Salmonella with the standard SCV markers Rab5 and LAMP-1 using both microscopic and protein based methods. CONCLUSION: The method described is highly efficient, robust and permits rapid isolation of intact SCVs from human macrophages without involving numerous purification steps. The method can also be applied to other intracellular pathogens that reside within a vacuole-like compartment within host cells. Future work using the approach should aid in identification and characterization of host factors associated with the membranes of such intracellular pathogens, which could potentially serve as pharmaceutical targets against intracellular pathogens residing within vacuoles.
Identifiers
External DOI: https://doi.org/10.1186/s13099-018-0256-7
This record's URL: https://www.repository.cam.ac.uk/handle/1810/285375
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