Methods in Molecular Biology Volume 954, 2013, pp 323-332

Methods for Determining Protein Translocation by the Legionella pneumophila Dot/Icm Type IV Secretion System

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Abstract

Legionella pneumophila the causative agent of Legionnaires’ disease, actively manipulates host cell ­processes to establish a membrane-bound replication vacuole permissive for its replication. Establishment of such replication niche requires the Dot/Icm type IV secretion system which translocates a plethora of effectors into host cells. Determining whether a particular protein is a substrate of the transporter is a prerequisite for subsequent functional studies. Thus, a variety of methods have been developed in the last decade to measure Dot/Icm-dependent delivery of protein into the host cell. The combination of these methods and the appropriate screening strategies has allowed for the identification of more than 270 translocated proteins. These efforts have laid a solid foundation for further study of the roles of these proteins in the interactions between L. pneumophila and its host. Here, we summarized the experimental details of these methods.