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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10 nm colloidal gold particles</td>
			<td>Aurion</td>
			<td>25486</td>
			<td></td>
		</tr>
		<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>
		</tr>
		<tr>
			<td>CoolSNAP EZ 20 MHz digital monochrome camera</td>
			<td>Photometrics</td>
			<td></td>
			<td></td>
		</tr>
		<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>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Spectra X light engine</td>
			<td>Lumencor</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Taq 2X Master Mix</td>
			<td>New England BioLabs</td>
			<td>M0270</td>
			<td></td>
		</tr>
		<tr>
			<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>
</table>

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 ...

This protocol is useful for characterizing the assembling functions of bacterial secretion complexes and can lead to a fundamental understanding of the molecular mechanisms that are required for host pathogen interactions. This technique integrates complementary approaches that preserve the native structure of the sample and allows and see to visualization of proteins complexes in intact bacterial cells. It increases our understanding of the structural function relationships of bacterial secretion systems.This method can be adapted to study other types of secretion systems or other cellular complexes in a wide variety of bacterial species. Start by making agarose pads for live cell imaging. Prepare about 30 mL of 1%low melt agarose solution in water and microwave it in a glass flask for about 90 seconds swirling it occasionally until the agarose is completely dissolved.Place two 22 by 22 by 0.15mm glass slides on the edge of a 25 by 75 by 1.1mm glass slide one on top of the other. Then, stack two more of the smaller slides on the other edge. Pipette about 1 mL of the molten agarose into the center slide between the two upper glass slides and place another large slide on top of the molten agarose making sure to avoid formation of air bubbles.Cool the slides at four degrees celsius for 15 minutes then use a scalpel or razor blade to gently cut the pad into small squares. Fix a double-sided adhesive frame on a 25 by 75 by 1.1mm glass slide and place several pads on the slide. Dissolve a heavy patch of Legionella Pneumophila in 1 mL of double-distilled water.Then vortex and pipette two to three microliters of the dilution onto the pads. Gently place a 58 by 24 by 0.15 mm cover slip over the adhesive frame. Select cells for imaging using bright field lighting.Then, in the capture window of the microscope's software, Adjust the ND to 180 and the binning to two by two. Use the 488 nanometer channel to expose the sample for 500 to 1000 milliseconds and validate the specificity of the fluorescence by imaging untagged Legionella Pneumophila with the same parameters. To quantify polarity, adjust the image contrast so the bacteria are clearly visible.Then zoom in on the region of interest and use the region tool to place a 0.25 by 1.3 micrometer rectangle starting at the pole and extending into the cytoplasm making sure that the rectangle remains within the bacterial borders. Mark at least 200 bacteria and create masks by using the region to mask button in the analyze menu. Click on mask statistics and choose object under mask scope.Then mark mean intensity and variance under features and intensity. Export the data as a text file and use the spreadsheet application to calculate the polarity scores of each bacterium as the ratio between the variance and the mean intensity. For high throughput applications use face contrast microscopy to acquire dual channel images.Make sure to choose fields of view where the bacteria are fully separated. Next, adjust the face channel contrast of the image to a level where the bacteria are clearly visible. Open the dual channel image and launch the create segment mask window.Adjust the appropriate threshold, then remove the small objects by clicking the define objects button and adjusting the minimum size to 100 pixels. Under refine mask, select remove edges objects and separate masks of bacteria that are adjacent to each other. To quantify the dynamics of the fusion proteins, open the image capture window and mark time lapse.Enter two in the number of time points box. Acquire two successful images of Legionella Pneumophila expressing the fluorescent protein of interest. Adjust the image contrast until the cells are clearly visible.Then use the region tool to place a 0.25 by 0.25 micrometer square in the middle of at least 400 cells. Next, use the region to mask button in the analyze menu to create a mask of the squares of interest. Create a new empty mask and use the pixel tool to mark the entire cell area of at least 25 random cells.Create another mask and use the large brush tool to mark areas between the cells. Finally, select mask statistics and make sure object is selected under mask scope for mask one. Under features and intensity, choose mean intensity.Export the data and repeat the process for mask 2. For mask 3, select the entire mask and export the mean intensity. To prepare the electron cryotomography sample, grow a heavy patch of Legionella Pneumophila on CYE Agrastreptomisum plates for 48 hours at 37 degrees Celsius.Resuspend the cells in water to an OD 600 of about 0.7. Then add five microliters of colloidal gold particles to 20 microliters of the cell suspension. Glow discharge a holey carbon grid and pipette five microliters of the cell mixture on it.Let it stand for one minute. Blot the grid with filter paper and freeze it in liquid ethane using a gravity-driven plunger apparatus. After inserting superfolder GFP into the Legionella Pneumophila chromosome, live cell fluorescent microscopy was performed to compare the fluorescent signal with that of the parental superfolder GFP negative strain.In addition, bright field microscopy was used to examine the proportion of cells with a specific fluorescent signal. Only a fraction of the cells that produced DotB superfolder GFP displayed polar localization of the fusion protein. When characterizing the distribution of DotB fluorescent signal, it was found that the intensity of DotB superfolder GFP at the poles was about two times higher than in the cytosol.To investigate if DotB superfolder GFP polar localisation was dependent on a fully assembled type four secretion system, polarity scores of DotB superfolder GFP were determined for individual cells in a wild type, and in a type four secretion system mutant. Indeed, polar positioning of DotB superfolder GFP disappeared when the Dot/ICM system was deleted. Dynamic patterns indicated that the DotB ATPase was present in a dynamic cytosolic population and was recruited to the polar localized Dot/ICM type four secretion system at a late stage assembly reaction.High throughput cryo-ET was used to visualize the intact type four secretion system apparatus in a Legionella Pneumophila strain expressing DotB superfolder GFP. Reconstruction of the cell revealed multiple Dot/ICM machines embedded in the cell envelope. The overall positioning of DotB and superfolder GFP in relation to the intact type four secretion system machine was also determined.Three-dimensional surface renderings indicated that superfolder GFP is positioned below the DotB hexamer, which assembles as a disc at the base of the cytoplasmic ATPase complex. When attempting this technique, make sure to assess the stability of the fusion proteins and the functionality of the tag secretion system before proceeding to image acquisition. Modeling and fitting can be effective ways to analyze the dynamic patterns and structural densities as well as for evaluation of conformational changes or localisation of subunits into the structure.This approach can help in understanding how different type four secret system components function, how they interact within the greater complex, and what functions different subassemblies perform.

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Research

JoVE Journal

Immunology and Infection

6.7K 조회수.  Yale University School of Medicine. 이 프로토콜은 세균 분비 복합체의 조립 기능을 특성화하는 데 유용하며 숙주 병원체 상호 작용에 필요한 분자 메커니즘의 근본적인 이해로 이어질 수 있습니다. 이 기술은 샘플의 기본 구조를 보존하고 그대로 세균 세포에서 단백질 복합체의 시각화를 허용하고 볼 수있는 보완적 인 접근 방식을 통합합니다. 그것은 세균 성 분비 시스템의 구조적 기능 관계에 대한 우리의 ?...