Abstract
Legionella possesses a pivotal secretion machinery to deliver virulence factors to eukaryotic host cells. In this protocol, we describe the procedure for isolation of the native core complex of the Dot/Icm type IV secretion system from L. pneumophila aiming to perform biochemical and transmission electron microscopy analyses.
Keywords: Legionella, Type IV secretion, Core complex, Isolation, Electron microscopy, Bacteria, Nanomachine, Structure
Background
Legionella pneumophila is a Gram-negative bacterial pathogen that causes lung infection known as Legionnaires’ disease (Fields et al., 2002). L. pneumophila utilizes a type IV secretion system (T4SS) encoded by the dot/icm genes to transport about 300 bacterial proteins into the cytosol of their eukaryotic host to hijack cellular processes (Hubber and Roy, 2010). Composed of more than 20 proteins, the T4SS is a nanomachine built on the bacterial inner and outer membranes (Nagai and Kubori 2011; Kubori and Nagai 2016). The core complex of Dot/Icm T4SS is a biochemically stable part of the system and forms a transport conduit bridging the inner and outer membrane (Kubori et al. 2014). The core complex is composed of at least five proteins; three outer membrane-associated proteins, DotC, DotD and DotH, and two inner membrane proteins, DotF and DotG (Vincent et al., 2006). Based on the procedure for biochemical isolation of another bacterial nanomachine, the type III secretion system, from Salmonella typhimurium (Kubori et al., 1998; Marlovits et al., 2004), we modified the protocol to adapt it to the purification of the T4SS of L. pneumophla. In this protocol, we present the procedure to isolate the native core complex of the T4SS from detergent lysed wild-type L. pneumophila based on separation by ultracentrifugation. T4SS isolated using this procedure can be used to perform biochemical and transmission electron microscopy analyses described previously (Kubori et al., 2014).
Materials and Reagents
Equipment
Procedure
Note: Procedure below should be done at 4 °C.
Data analysis
The second round of ultracentrifugation enhances the purity of the isolated complex, and the following column chromatography further removes the background contaminations (Figure 1). Figure 1. Example of isolated Dot/Icm T4SS core complex. A. SDS-PAGE analysis of the isolated complex in comparison between 1st and 2nd rounds of ultracentrifugation (steps 21 and 25, respectively). Whole cell: Whole cell lysate (step 20); cfg1 ppt: 1st ultracentrifugation pellet (step 22); cfg2 ppt: 2nd ultracentrifugation pellet (step 26). As a negative control, a L. pneumophila strain lacking all dot/icm genes (∆T4SS) was also submitted to the isolation procedure (the rightmost lane). B. The electron micrograph of the fraction obtained by the 2nd ultracentrifugation; C. SDS-PAGE analysis of the fractions obtained by a size exclusion column chromatography (step 27); D. The electron micrograph of the fraction A10 of (C). Molecular weight markers are shown in kDa. MOMP: Major Outer membrane Protein. The images are adapted from Kubori et al. (2014).
Recipes
Acknowledgments
This work has been financially supported by MEXT/JSPS KAKENHI Grants 15H01322 (to TK). The protocol presented here has been adapted from Kubori et al. (2014).
References
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