Name: applying live cell imaging and cryo-electron tomography to resolve spatiotemporal features of the legionella pneumophila dot/icm secretion system
Uploaded: 2020-03-10T13:30:00
Description: Watch this Scientific Journal Video about Applying Live Cell Imaging and Cryo-Electron Tomography to Resolve Spatiotemporal Features of the Legionella pneumophila Dot/Icm Secretion System at JoVE.com

D.C. and C.R.R. were supported by the NIH (R37AI041699 and R21AI130671). D.P., B.H., and J.L were supported by the National Institutes of Health (R01AI087946 and R01GM107629).

NOTE: All procedures involving the growth, manipulation, and imaging of L. pneumophila should be performed in a biological safety level 2 laboratory in compliance with local guidelines.
1. Insertion of sfGFP into L. pneumophila Chromosome Using Allelic Exchange and Double Selection Strategy (Figure 2, Figure 3)
<ol>
	<li>Clone into the gene replacement vector pSR47S11 the following sequence:...

<ol>
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Elucidating the functions of bacterial secretion systems is key to a complete understanding of host-pathogen interactions. Secretion systems are complex machines that can inject effectors proteins into host cells, and in some cases promote establishment of a subcellular niche that supports bacterial replication. The above method provides important new tools for studying the Dot/Icm secretion system of the respiratory bacterial pathogen Legionella pneumophila, yielding clues to the mechanisms of effector transloc...

Legionella pneumophila (L. pneumophila), the etiological agent of Legionnaires&#39; disease, inhabits freshwater reservoirs, where the bacteria propagate by infecting and replicating within aquatic free-swimming protozoa. L. pneumophila causes disease outbreaks in humans when inhalation of aerosolized bacteria from potable water sources occurs. In infected cells, subversion of host pathways allows L. pneumophila to delay endocytic maturation of the vacuole in which it resides and to promote biogenesis of a cellular compartment that supports bacterial replication. This process is driven by a specialized bacterial type IVB secretion system (T4BSS) known as Dot/Icm and its repertoire of over 300 &#34;effector&#34; proteins that are translocated into the host cytosol during infection to facilitate manipulation of cellular functions1,2,3,4,5. Mutants lacking a functional Dot/Icm apparatus fail to deliver effectors into the host cytosol, are defective for intracellular replication, and are avirulent in animal models of disease6,7.
Many bacterial species have developed extremely complex and dynamic multicomponent machines that are required for infection processes. Other T4BSS like the Dot/Icm system are also essential for intracellular replication of bacterial pathogens such as Coxiella burnetii and Rickettsiella grylli. Although T4BSS are evolutionarily related to prototypical type IVA systems, which mediate DNA transfer and can deliver a limited repertoire of effector proteins, the Dot/Icm system has nearly twice as many machine components and delivers a wide variety of effectors. Presumably, this expansion in the number of components has enabled the Dot/Icm apparatus to accommodate and integrate new effectors easily8,9.
We recently used cryo-electron tomography (cryo-ET) to solve the structure of the Dot/Icm apparatus in situ and showed that it forms a cell envelope-spanning channel that connects to a cytoplasmic complex. Further analysis revealed that the cytosolic ATPase DotB associates with the Dot/Icm system at the L. pneumophila cell pole through interactions with the cytosolic ATPase DotO. We have discovered that DotB displays a cytosolic movement in most bacterial cells, indicating that this ATPase is present in a dynamic cytosolic population but also associates with the polar Dot/Icm complexes. In addition, DotO forms a hexameric assembly of DotO dimers associated with the inner membrane complex, and a DotB hexamer joins to the base of this cytoplasmic complex. The assembly of the DotB-DotO energy complex creates a cytoplasmic channel that directs the translocation of substrates through the T4SS (Figure 1)10.
Despite these recent advances, little is known about how the Dot/Icm system functions and how each protein assembles to form an active apparatus8. Uncovering the regulatory circuitry of the Dot/Icm T4SS is fundamental to understanding the molecular mechanisms of host-pathogen interactions. Therefore, we discuss how to use live cell microscopy and cryo-ET to detect and characterize essential L. pneumophila Dot/Icm system components that are tagged with super-folder GFP (sfGFP). Using quantitative fluorescence microscopy, the polar localization of DotB will be defined in a wild type background or when the type IV system is deleted. Time-lapse microscopy will be used to quantify differences in localization and dynamics between the Dot/Icm cytosolic ATPases.
The combined application of two complementary approaches such as live imaging and cryo-ET provides an advantage compared to other in vitro systems. Both methods are performed in intact cells and preserve the natural environment of the T4BSS, thus minimizing disruption of the native structure during sample preparation. Because overexpression of proteins may impair the stoichiometry of the secretion apparatus, sfGFP fusions are returned via allelic exchange to the Legionella chromosome so that each fusion is encoded in single copy and the expression is driven by the endogenous promoter. Visualization of chromosomally-encoded fusions enables quantification of the exact level of protein being expressed at a defined time point. Cryo-ET also has many advantages for determining the structure of secretion systems. The most notable advantage is that cryo-ET samples are comprised of frozen intact cells that preserve native complexes in the context of bacterial cell architecture. Consequently, cryo-ET may be preferable to biochemical purification approaches, which extract membrane complexes and may strip peripheral proteins from the core apparatus or modify the overall structure. In addition, tagging a protein of interest with a bulky protein such as sfGFP adds a mass that is detectable by cryo-ET and can assist with mapping the different subcomplexes of the Dot/Icm apparatus onto the structure obtained by cryo-ET.
This approach is a powerful tool for uncovering structural information about multimolecular complexes that assemble in the bacterial cell membrane. The interpretation of structures elucidated using these techniques will help the field understand how T4BSS components function, why so many components are required for function, how the components interact within the greater complex, and what functions these subassemblies perform.

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			<td>10 nm colloidal gold particles</td>
			<td>Aurion</td>
			<td>25486</td>
			<td></td>
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		<tr>
			<td>100x Plan Apo objective (1.4 NA)</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ACES</td>
			<td>Sigma-Aldrich</td>
			<td>A9758</td>
			<td></td>
		</tr>
		<tr>
			<td>Activated charcoal</td>
			<td>Sigma-Aldrich</td>
			<td>C5510</td>
			<td></td>
		</tr>
		<tr>
			<td>Agaroze GPG/LMP, low melt</td>
			<td>American bioanalytical</td>
			<td>AB00981</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto dehydrated agar</td>
			<td>BD</td>
			<td>214010</td>
			<td></td>
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			<td>CoolSNAP EZ 20 MHz digital monochrome camera</td>
			<td>Photometrics</td>
			<td></td>
			<td></td>
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		<tr>
			<td>Gene Frame, 1.7x2.8 cm, 125 &#181;L</td>
			<td>Fisher Scientific</td>
			<td>AB-0578</td>
			<td></td>
		</tr>
		<tr>
			<td>Holey Carbon grid R 2/1 Cu 200 mesh</td>
			<td>Quantifoil</td>
			<td>Q225-CR1</td>
			<td></td>
		</tr>
		<tr>
			<td>Iron(III) nitrate nonahydrate</td>
			<td>Sigma-Aldrich</td>
			<td>216828</td>
			<td></td>
		</tr>
		<tr>
			<td>K2 Summit camera for cryo-EM</td>
			<td>GATAN</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>L-Cysteine</td>
			<td>Sigma-Aldrich</td>
			<td>C7352</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope cover slides 22x22 mm</td>
			<td>Fisher Scientific</td>
			<td>12-542B</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope cover slides 24x50 mm</td>
			<td>Fisher Scientific</td>
			<td>12-545K</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slides 25x75x1 mm</td>
			<td>Globe Scientific</td>
			<td>1380</td>
			<td></td>
		</tr>
		<tr>
			<td>SlideBook 6.0</td>
			<td>Intelligent Imaging Innovations</td>
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			<td>Spectra X light engine</td>
			<td>Lumencor</td>
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			<td>Taq 2X Master Mix</td>
			<td>New England BioLabs</td>
			<td>M0270</td>
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			<td>Titan Krios</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast Extract</td>
			<td>BD</td>
			<td>212750</td>
			<td></td>
		</tr>
	</tbody>
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applying live cell imaging and cryo-electron tomography to resolve spatiotemporal features of the legionella pneumophila dot/icm secretion system

The Dot/Icm secretion system of Legionella pneumophila is a complex type IV secretion system (T4SS) nanomachine that localizes at the bacterial pole and mediates the delivery of protein and DNA substrates to target cells, a process generally requiring direct cell-to-cell contact. We have recently solved the structure of the Dot/Icm apparatus by cryo-electron tomography (cryo-ET) and showed that it forms a cell envelope-spanning channel that connects to a cytoplasmic complex. Applying two complementary approaches that preserve the native structure of the specimen, fluorescent microscopy in living cells and cryo-ET, allows in situ visualization of proteins and assimilation of the stoichiometry and timing of production of each machine component relative to other Dot/Icm subunits. To investigate the requirements for polar positioning and to characterize dynamic features associated with T4SS machine biogenesis, we have fused a gene encoding superfolder green fluorescent protein to Dot/Icm ATPase genes at their native positions on the chromosome. The following method integrates quantitative fluorescence microscopy of living cells and cryo-ET to quantify polar localization, dynamics, and structure of these proteins in intact bacterial cells. Applying these approaches for studying the Legionella pneumophila T4SS is useful for characterizing the function of the Dot/Icm system and can be adapted to study a wide variety of bacterial pathogens that utilize the T4SS or other types of bacterial secretion complexes.

Homologous recombination with double selection in two steps was used to construct the defined insertion of sfGFP. In the first step, triparental mating was performed, where the pRK600 conjugative plasmid (an IncP plasmid) from the E. coli helper strain MT616 was mobilized to the donor E. coli strain with the suicide vector pSR47S containing the sfGFP gene flanked by the two homologous regions, the origin of transfer oriT and the Bacillus subtilis counterselection gene sacB. Next, the c...

Watch this Scientific Journal Video about Applying Live Cell Imaging and Cryo-Electron Tomography to Resolve Spatiotemporal Features of the Legionella pneumophila Dot/Icm Secretion System at JoVE.com

Applying Live Cell Imaging and Cryo-Electron Tomography to Resolve Spatiotemporal Features of the Legionella pneumophila Dot/Icm Secretion System

Imaging of bacterial cells is an emerging systems biology approach focused on defining static and dynamic processes that dictate the function of large macromolecular machines. Here, integration of quantitative live cell imaging and cryo-electron tomography is used to study Legionella pneumophila type IV secretion system architecture and functions.

Applying Live Cell Imaging and Cryo-Electron Tomography to Resolve Spatiotemporal Features of the Legionella pneumophila Dot/Icm Secretion System

The Dot/Icm secretion system of Legionella pneumophila is a complex type IV secretion system (T4SS) nanomachine that localizes at the bacterial pole and ...

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