Name: live-cell forward genetic approach to identify and isolate developmental mutants in chlamydia trachomatis
Uploaded: 2020-06-10T14:00:00
Description: Watch this Scientific Journal Video about Live-Cell Forward Genetic Approach to Identify and Isolate Developmental Mutants in Chlamydia trachomatis at JoVE.com

We thank Dr. Anders Omsland at Washington State University for supplying the CIP-1 axenic media. This work was supported by NIH grant R01AI130072, R21AI135691 and R21AI113617. Additional support was provided by startup funds from the University of Idaho and the Center for Modeling Complex Interactions through their NIH grant P20GM104420.

All Python scripts used in this protocol are available on Github https://github.com/SGrasshopper/Live-cell-data-processing
1. Mutagenize Reporter&#160;&#160;Chlamydia
NOTE: Ctr-L2-hctBprom-mKate2/euoprom-Clover EBs were directly mutagenized using ethyl methanesulfonate (EMS) in the axenic media CIP-1 as this media supports EB metabolism and maintenance of EB infectivity12.
<ol>
	<li>Thaw a chlamydial stock on ice ...

<ol>
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Y., Tan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#963;28+RNA+polymerase+regulates+hctB,+a+late+developmental+gene+in+Chlamydia.">&#963;28 RNA polymerase regulates hctB, a late developmental gene in Chlamydia.</a> Molecular Microbiology. 50 (2), 577-584 (2003).</li><li>Koo, I., Stephens, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Developmentally+Regulated+Two-component+Signal+Transduction+System+in+Chlamydia.">A Developmentally Regulated Two-component Signal Transduction System in Chlamydia.</a> Journal of Biological Chemistry. 278 (19), 17314-17319 (2003).</li><li>Grieshaber, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+Active+Metabolism+on+Elementary+Body+Transcript+Profile+and+Infectivity.">Impact of Active Metabolism on Elementary Body Transcript Profile and Infectivity.</a> Journal of Bacteriology. 200 (14), 00065 (2018).</li><li>Belland, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+transcriptional+profiling+of+the+developmental+cycle+of.">Genomic transcriptional profiling of the developmental cycle of.</a> Proceedings of the National Academy of Sciences U. 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A. 100 (14), 8478-8483 (2003).</li><li>Wang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+transformation+system+for+Chlamydia+trachomatis:+restoration+of+glycogen+biosynthesis+by+acquisition+of+a+plasmid+shuttle+vector.">Development of a transformation system for Chlamydia trachomatis: restoration of glycogen biosynthesis by acquisition of a plasmid shuttle vector.</a> PLoS Pathogens. 7 (9), 1002258 (2011).</li><li>Nguyen, B., Valdivia, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virulence+determinants+in+the+obligate+intracellular+pathogen+Chlamydia+trachomatis+revealed+by+forward+genetic+approaches.">Virulence determinants in the obligate intracellular pathogen Chlamydia trachomatis revealed by forward genetic approaches.</a> Proceedings of the National Academy of Sciences U. S. A. 109 (4), 1263-1268 (2012).</li><li>Mueller, K., Wolf, K., Fields, K., Maurelli, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+Deletion+by+Fluorescence-Reported+Allelic+Exchange+Mutagenesis+in+Chlamydia+trachomatis.">Gene Deletion by Fluorescence-Reported Allelic Exchange Mutagenesis in Chlamydia trachomatis.</a> MBio. 7 (1), 01817 (2016).</li><li>Rosario, C. J., Tan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+early+gene+product+EUO+is+a+transcriptional+repressor+that+selectively+regulates+promoters+of+Chlamydia+late+genes.">The early gene product EUO is a transcriptional repressor that selectively regulates promoters of Chlamydia late genes.</a> Mol Microbiol. 84 (6), 1097-1107 (2012).</li><li>Brickman, T., Barry, C., Hackstadt, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+cloning+and+expression+of+hctB+encoding+a+strain-variant+chlamydial+histone-like+protein+with+DNA-binding+activity.">Molecular cloning and expression of hctB encoding a strain-variant chlamydial histone-like protein with DNA-binding activity.</a> Journal of Bacteriology. 175 (14), 4274-4281 (1993).</li><li>Omsland, A., Sager, J., Nair, V., Sturdevant, D., Hackstadt, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+stage-specific+metabolic+and+transcriptional+activity+of+Chlamydia+trachomatis+in+an+axenic+medium.">Developmental stage-specific metabolic and transcriptional activity of Chlamydia trachomatis in an axenic medium.</a> Proceedings of the National Academy of Sciences U. S. A. 109 (48), 19781-19785 (2012).</li><li>Su, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+recombinant+Chlamydia+trachomatis+major+outer+membrane+protein+binds+to+heparan+sulfate+receptors+on+epithelial+cells.">A recombinant Chlamydia trachomatis major outer membrane protein binds to heparan sulfate receptors on epithelial cells.</a> Proceedings of the National Academy of Sciences U.S.A. 93 (20), 11143-11148 (1996).</li><li>Edelstein, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computer+control+of+microscopes+using+&#181;Manager.">Computer control of microscopes using &#181;Manager.</a> Current Protocols in Molecular Biology. , (2010).</li><li>Tinevez, J. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TrackMate:+An+open+and+extensible+platform+for+single-particle+tracking.">TrackMate: An open and extensible platform for single-particle tracking.</a> Methods. 115, 80-90 (2017).</li><li>Plotly Technologies Inc. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Collaborative data science. , (2015).</li><li>Lam, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+FRET+dynamic+range+with+bright+green+and+red+fluorescent+proteins.">Improving FRET dynamic range with bright green and red fluorescent proteins.</a> Nature Methods. 9 (10), 1005-1012 (2012).</li><li>Shcherbo, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Far-red+fluorescent+tags+for+protein+imaging+in+living+tissues.">Far-red fluorescent tags for protein imaging in living tissues.</a> Biochemical Journal. 418 (3), 567-574 (2009).</li><li>Sega, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+the+genetic+effects+of+ethyl+methanesulfonate.">A review of the genetic effects of ethyl methanesulfonate.</a> Mutation Research. 134 (2-3), 113-142 (1984).</li><li>Ferguson, L., Denny, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Frameshift+mutagenesis+by+acridines+and+other+reversibly+binding+DNA+ligands.">Frameshift mutagenesis by acridines and other reversibly binding DNA ligands.</a> Mutagenesis. 5 (6), 529-540 (1990).</li></ol>

Dissecting the mechanisms that control the chlamydial developmental cycle has been hindered by the limitations of the currently available genetic tools. Employing our promoter-reporter Chlamydia in conjunction with live-cell automated microscopy, a system was built which enables monitoring of cell-type development in individual inclusions over a 24 h period. This system, in combination with chemical mutagenesis and direct inclusion isolation has established a method to rapidly and clonally select Chlamydia</...

Chlamydia trachomatis (Ctr) is an obligate intracellular pathogen that progresses through a biphasic developmental cycle that is essential for its survival and proliferation1. This cycle consists of two developmental forms, the elementary body (EB) and the reticulate body (RB). The EB is replication incompetent but mediates cell invasion through effector induced endocytosis2. Once in the host, the EB matures to the replicative RB. The RB carries out multiple rounds of replication prior to converting back to the EB in order to initiate subsequent rounds of infection.
The limited array of genetic tools has restricted most of the chlamydial research to biochemical studies or the use of surrogate systems. As a consequence, elucidation of gene regulation and control of the developmental cycle has been difficult3,4. One of the more important challenges in the chlamydial field is the high resolution temporal tracking of the chlamydial developmental cycle and the identification of the proteins involved in its regulation. Gene expression during the chlamydial developmental cycle has traditionally been performed by destructive &#8220;end point&#8221; methods including RNAseq, qPCR, and fixed cell microscopy5,6. Although these methods have provided invaluable information, the techniques employed are laborious and have low temporal resolution5,6.
Within the last decade, genetic manipulation of Ctr has progressed with the introduction of plasmid transformation and methods for mutagenesis7,8,9. For this study, a plasmid-based system was developed to monitor chlamydial development in individual inclusions in real time over the course of an infection. A chlamydial transformant was created that expressed both an RB and EB cell-type specific promoter-reporter. The RB specific reporter was constructed by fusing the promoter of the early RB gene euo upstream of the fluorescent protein Clover. EUO is a transcriptional regulator that represses a subset of late EB associated genes10. The promoter of hctB, which encodes a histone-like protein involved in EB nucleoid condensation, was cloned directly upstream of mKate2 (RFP) to create the EB specific reporter11. The backbone for hctBprom-mKate2/euoprom-Clover was p2TK2SW27. The hctB and euo promoters were amplified from Ctr-L2 genomic DNA. Each promoter sequence consisted of ~100 base pairs upstream of the predicted transcription start site for the specified chlamydial gene plus the first 30 nucleotide (10 amino acids) of the respective ORF. The fluorescent FP variants were commercially obtained as Ctr codon optimized gene blocks and cloned in frame with the first 30 nucleotide of each chlamydial gene and promoter. The incD terminator was cloned directly downstream of mKate2. The second promoter-reporter was inserted downstream of the incD terminator. The ampicillin resistance gene (bla) in p2TK2SW2 was replaced with the aadA gene (Spectinomycin resistance) from pBam4. This resulted in the final construct p2TK2-hctBprom-mKate2/euoprom-Clover (Figure 1A) that was transformed into Ctr-L27. This RB/EB reporter strain allowed for the observation of the developmental cycle within single inclusions using live-cell microscopy (Figure 1B,C).
Employing our promoter-reporter construct in combination with chemical mutagenesis, a protocol was devised to track and isolate individual clones that exhibited developmental abnormalities from mutagenized populations of Ctr serovar L2. This protocol allows for the direct monitoring of individual chlamydial inclusions, tracking of the gene expression profiles over time, identifying chlamydial clones that express an altered developmental gene expression pattern, and clonal isolation of Chlamydia from individual inclusions.
Although this protocol has been created specifically for the identification of genes involved in chlamydial development, it could be easily adapted to interrogate any number of chlamydial genetic pathways.

<table>
	<tbody>
		<tr>
			<td>24-well polystyrene plates</td>
			<td>Corning</td>
			<td>3524</td>
			<td>Cell culture growth for reinfection of isolates</td>
		</tr>
		<tr>
			<td>6-well glass bottom plates</td>
			<td>Cellvis</td>
			<td>P06-1.5H-N</td>
			<td>Cell culture growth for imaging</td>
		</tr>
		<tr>
			<td>96-well glass bottom plates</td>
			<td>Nunc</td>
			<td>165305</td>
			<td>Cell culture growth for imaging</td>
		</tr>
		<tr>
			<td>Bold line CO2 Unit</td>
			<td>OKO Labs</td>
			<td>CO2 UNIT BL</td>
			<td>Stage incubator CO2 control</td>
		</tr>
		<tr>
			<td>Bold line T Unit</td>
			<td>OKO Labs</td>
			<td>H301-T-UNIT-BL-PLUS</td>
			<td>Stage incubator temperature control</td>
		</tr>
		<tr>
			<td>Borosilicate glass capillary tubes</td>
			<td>Sutter Instrument</td>
			<td>B1005010</td>
			<td>Capillary tubes</td>
		</tr>
		<tr>
			<td>BrightLine bandpass emissions filter (514/30nm)</td>
			<td>Semrock</td>
			<td>FF01-514/30-25</td>
			<td>Fluoescent filter cube</td>
		</tr>
		<tr>
			<td>BrightLine bandpass emissions filter (641/75nm)</td>
			<td>Semrock</td>
			<td>FF02-641/75-25</td>
			<td>Fluoescent filter cube</td>
		</tr>
		<tr>
			<td>CellTram Vario</td>
			<td>Eppendorf</td>
			<td>5196000030</td>
			<td>Microinjector</td>
		</tr>
		<tr>
			<td>Chlamydia trachmatis serovar L2</td>
			<td>ATCC</td>
			<td>VR-577</td>
			<td>Chlamydia trachomatis</td>
		</tr>
		<tr>
			<td>CIP-1 media</td>
			<td>In house</td>
			<td>NA</td>
			<td>Axenic media. IPB supplemented with 1% FBS, 25 &#956;M amino acids, 0.5 
			mM G6P, 1.0 mM ATP, 0.5 mM DTT, and 50 &#956;M GTP, UTP, and 
			CTP. (Omsland, A. 2012) made in-house.</td>
		</tr>
		<tr>
			<td>Cos-7 cells (ATCC)</td>
			<td>ATCC</td>
			<td>CRL-1651</td>
			<td>African green monkey kidney cell (host cells)</td>
		</tr>
		<tr>
			<td>Cycloheximide</td>
			<td>MP Biomedicals</td>
			<td>194527</td>
			<td>Host cell growth inhibitor</td>
		</tr>
		<tr>
			<td>Ethyl methanesulfonate, 99%</td>
			<td>Acros Organics</td>
			<td>AC205260100</td>
			<td>Mutagen</td>
		</tr>
		<tr>
			<td>Fetal Plex</td>
			<td>Gemini Bio-Products</td>
			<td>100-602</td>
			<td>Supplement for base growth media</td>
		</tr>
		<tr>
			<td>Fiji/ImageJ</td>
			<td>https://imagej.net/Fiji</td>
			<td>NA</td>
			<td>Open sourse Image analysis software. https://imagej.net/Fiji</td>
		</tr>
		<tr>
			<td>Galaxy 170 S CO2 incubator</td>
			<td>Eppendorf</td>
			<td>CO1700100X</td>
			<td>Cell culture incubation</td>
		</tr>
		<tr>
			<td>gblocks (Fluorescent FP variants: Clover and mKate2)</td>
			<td>Integrated DNA Technologies</td>
			<td>NA</td>
			<td>gblock ORFs of Ctr optimized FP varients for cloning into p2TK2SW2</td>
		</tr>
		<tr>
			<td>Gentamycin 10mg/ml</td>
			<td>Gibco</td>
			<td>15710-064</td>
			<td>Antibiotic for growth media</td>
		</tr>
		<tr>
			<td>HBSS (Hank&#39;s Balanced Salt Solution)</td>
			<td>Corning</td>
			<td>21-020-CM</td>
			<td>Host cells rinse</td>
		</tr>
		<tr>
			<td>Heparin sodium</td>
			<td>Amersham Life Science</td>
			<td>16920</td>
			<td>inhibits and reverses the early electrostatic interactions between the host cell and EBs</td>
		</tr>
		<tr>
			<td>HEPES 1M</td>
			<td>GE Life Sciences</td>
			<td>SH30237.01</td>
			<td>pH buffer for growth media</td>
		</tr>
		<tr>
			<td>InjectMan</td>
			<td>Eppendorf</td>
			<td>5179 000.018</td>
			<td>Micromanipulator</td>
		</tr>
		<tr>
			<td>Jupyter Notebook</td>
			<td>https://jupyter.org/</td>
			<td>NA</td>
			<td>Visualization of inclusion traces. https://jupyter.org/</td>
		</tr>
		<tr>
			<td>Lambda 10-3</td>
			<td>Sutter Instrument</td>
			<td>LB10-3</td>
			<td>Filter wheel controler</td>
		</tr>
		<tr>
			<td>Oko Touch</td>
			<td>OKO Labs</td>
			<td>Oko Touch</td>
			<td>Interface to control the Bold line T and CO2 Unit</td>
		</tr>
		<tr>
			<td>Prior XY stage</td>
			<td>Prior</td>
			<td>H107</td>
			<td>Motorized XY microscope stage</td>
		</tr>
		<tr>
			<td>PrismR Centrifuge</td>
			<td>Labnet</td>
			<td>C2500-R</td>
			<td>Temperature controlled microcentrifuge</td>
		</tr>
		<tr>
			<td>Problot Hybridization oven</td>
			<td>Labnet</td>
			<td>H1200A</td>
			<td>Rocking Incubator for infection with Chlamydia</td>
		</tr>
		<tr>
			<td>Proscan II</td>
			<td>Prior</td>
			<td>H30V4</td>
			<td>XYZ microscope stage controler</td>
		</tr>
		<tr>
			<td>Purifier Class 2 Biosafety Cabinet</td>
			<td>Labconco</td>
			<td>362804</td>
			<td>Cell culture work</td>
		</tr>
		<tr>
			<td>RPMI-1640 (no phenol red)</td>
			<td>Gibco</td>
			<td>11835-030</td>
			<td>Base growth media for imaging</td>
		</tr>
		<tr>
			<td>RPMI-1640 (phenol red)</td>
			<td>GE Life Sciences</td>
			<td>SH30027.01</td>
			<td>Base growth media</td>
		</tr>
		<tr>
			<td>scopeLED excitation LEDs (470nm,595nm)</td>
			<td>scopeLED</td>
			<td>F140</td>
			<td>Excitation light</td>
		</tr>
		<tr>
			<td>Sonic Dismembrator Model 500</td>
			<td>Fisher Scientific</td>
			<td>15-338-550</td>
			<td>Sonicator, resuspending chlamydial pellet</td>
		</tr>
		<tr>
			<td>Stage incubator</td>
			<td>OKO Labs</td>
			<td>H301-K-FRAME</td>
			<td>Cluster well plate incubation chamber</td>
		</tr>
		<tr>
			<td>sucrose-phosphate-glutamate buffer 1X (SPG)</td>
			<td>In house</td>
			<td>NA</td>
			<td>Chlamydial storage buffer. (10 mM sodium phosphate [8 mM K 2HPO 4, 2 mM KH 2PO 4], 220 mM sucrose, 0.50 mM L-glutamic acid; pH 7.4)</td>
		</tr>
		<tr>
			<td>T-75 Flasks</td>
			<td>Thermo Scientific</td>
			<td>156499</td>
			<td>Cell culture growth</td>
		</tr>
		<tr>
			<td>TE 300 inverted microscope</td>
			<td>Nikon</td>
			<td>16724</td>
			<td>microscope</td>
		</tr>
		<tr>
			<td>THOR LED</td>
			<td>Thor Labs</td>
			<td>LEDD1B</td>
			<td>White light</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Corning</td>
			<td>25-052-CI</td>
			<td>Dislodges host cells from flask for seeding into plates</td>
		</tr>
		<tr>
			<td>Zyla sCMOS</td>
			<td>Andor</td>
			<td>ZYLA-5.5-USB3</td>
			<td>imaging camera</td>
		</tr>
		<tr>
			<td>&#181;Manager 2.0gamma</td>
			<td>https://github.com/micro-manager/micro-manager</td>
			<td>NA</td>
			<td>Open sourse automated microscope control software package</td>
		</tr>
	</tbody>
</table>

live-cell forward genetic approach to identify and isolate developmental mutants in chlamydia trachomatis

The intracellular bacterial pathogen Chlamydia trachomatis undergoes a developmental cycle consisting of two morphologically discrete developmental forms. The non-replicative elementary body (EB) initiates infection of the host. Once inside, the EB differentiates into the reticulate body (RB). The RB then undergoes multiple rounds of replication, before differentiating back to the infectious EB form. This cycle is essential for chlamydial survival as failure to switch between cell types prevents either host invasion or replication.
Limitations in genetic techniques due to the obligate intracellular nature of Chlamydia have hampered identification of the molecular mechanisms involved in the cell-type development. We designed a novel dual promoter-reporter plasmid system that, in conjunction with live-cell microscopy, allows for the visualization of cell type switching in real time. To identify genes involved in the regulation of cell-type development, the live-cell promoter-reporter system was leveraged for the development of a forward genetic approach by combining chemical mutagenesis of the dual reporter strain, imaging and tracking of Chlamydia with altered developmental kinetics, followed by clonal isolation of mutants. This forward genetic workflow is a flexible tool that can be modified for directed interrogation into a wide range of genetic pathways.

Direct EMS mutagenesis of our promoter-reporter chlamydial strain resulted in an ~75% reduction in infectivity. Using the described live-cell imaging protocol, ~600 inclusions were imaged and tracked over a 24 h period. The fluorescent expression kinetics of both reporters in each inclusion was visualized using custom Python notebook scripts. Two visualization approaches were implemented to identify candidate mutagenized Chlamydia for isolation. The first methodology (step 3.3.8) visualizes the time to half-maxi...

Watch this Scientific Journal Video about Live-Cell Forward Genetic Approach to Identify and Isolate Developmental Mutants in Chlamydia trachomatis at JoVE.com

Live-Cell Forward Genetic Approach to Identify and Isolate Developmental Mutants in Chlamydia trachomatis

This protocol utilizes fluorescent promoter-reporters, live-cell microscopy, and individual inclusion extraction in a directed forward genetic approach to identify and isolate developmental mutants of Chlamydia trachomatis.

Live-Cell Forward Genetic Approach to Identify and Isolate Developmental Mutants in Chlamydia trachomatis

The intracellular bacterial pathogen Chlamydia trachomatis undergoes a developmental cycle consisting of two morphologically discrete developmental forms. ...

This protocol provides a unique tool for the generation and isolation of Chlamydia with altered developmental profiles, ultimately allowing for the identification of the genes that regulate the developmental cycle. The main advantages of this method are the ability to track chlamydia cell type development in real time and the ability to isolate chlamydia, with alter developmental programs. To mutagenize a chlamydia trachomatis reporter, saw a chlamydial stock containing approximately three times 10th to the seventh elementary bodies.Transformed with the reporter plasma of interest and pellet, the thought is stocked by centrofugation. use sonication at 10%power for 10 seconds, to resuspend the elementary bodies in 100 microliters of accidental metabolism buffer and split the resulting elementary body suspension equally, between two 1.5 milliliter micro centrifuge tubes. Add 50 microliters of freshly prepared EMS metabolism solution into the chlamydia aliquots for mutagenesis and 50 microliters of vaccinic metabolism buffer to the chlamydial aliquot only for stock mutagenesis and incubate both samples for 20 minutes, at room temperature.EMS is a known carcinogen wear gloves during handling, and soak all EMS contaminated equipment and medium, in one molar sodium hydroxide for 24 hours before disposal. To set up a wholesale culture, for the imaging and isolation of the mutigenized chlamydia trachometers. Seed six well glass bottom plates, with six times 10 to the fifth cost seven cells, in two milliliters of complete medium per well and seed a 24 well polystyrene plate with 10 to the fifth cause seven cells in one milliliter of complete medium per well.To infect the host cell culture with mutagenized denies chlamydia trachomatis infect, five Wells of a glass bottomed plate with approximately six times 10 to the fifth, of mutagenized elementary bodies. In 1.5 milliliters of ice cold HBSS per well. and infect the remaining well, with approximately two times 10 to the fifth mock mutagenized elementary bodies in 1.5 milliliters of ice cold HBSS per well, after a 15 minute incubation at 37 degrees Celsius, with rocking, wash the infected cells with 37 degrees Celsius HBSS supplemented with one milligram per milliliter of heparin, followed immediately by an HBSS rinse.wash the cells with heparin again, followed immediately by two rinses with HBSS alone to ensure that all of the heparin is removed. After the last wash, add four milliliters of 37 degrees Celsius imaging medium, to each well. And fill the inter well spaces with 37 degrees Celsius deionized water then place the plates in the cell culture incubator for 10 hours.At the end of the incubation set the microscope stage incubator to 5%carbon dioxide, and 37 degrees Celsius. And place the six well glass bottom plate, into the stage incubator. In the imaging software, Use the high content screening plugin to select the six well plate template, and generate an imaging position list consisting of 12 fields of view per well use the autofocus option to set the focus for the automated imaging and set the exposure to 250 milliseconds at a 4%intensity in the GFB channel and an 18%in density in the RFP channel.So at the imaging cycle for 24 hours, with 30 minute intervals, to capture the kinetics of the developmental cycle and set the image capture to include multiple Z-stacks with a range of focus that ends on either side of the InFocus slice, then select relative Z for imaging, multiple slices in the acquisition window and use the image stack file option. to set the images to be saved as TIFF stack files. To identify inclusion tracks, with alter developmental profiles, use the import cell in the EMS screen, markdown Python notebook, to import the inclusion track data from the spots in track statistics, CSV files into the Panda's data frame.Use the calculate cell to calculate the time to have maximum expression for both the early and late reporters for each track and use the bokeh plotting package, in the half max plot cell, to visualize the time two half max expression to half maximal expression against that of hctB prom. Use the bokeh interactive track ID Explorer, to identify inclusions from the mutant population, that fall outside of the mock treated scatter cloud. To visualize changes in the promoter expression kinetics dynamically in the animated blood cell graph the expression intensities of EOU prom against hctB prom.Then visualize a snapshot from the animated graph, in the inclusion locator cell. To isolate the developmental Newtons, from the inclusions of interest, position the field of view over a well with an identified inclusion, and pass the needle over the phase differential interference contrast white light channel light source, to visualize the needle, use the 595 nanometer, excitation channel, to visualize the elementary bodies for extraction. And use the fine adjustment and the micro manipulator, to maneuver the capillary needle to the inclusion, rupture the inclusion with the needle, and use the micro injector to draw the elementary bodies into the capillary needle tip.Expel the extracted elementary bodies, from the capillary needle into a single well, of the prepared at 24 well polystyrene plate, and use a new capillary needle for the next extraction. After a sufficient expansion of each isolate, disrupt the infected monolayer with a one milliliter micropipet tip and transfer the medium cell debris and released chlamydia into a new 1.5 milliliter microcentrifuge tube. Pellet the harvested chlamydia by centrifugation, and resuspend the pellet in 75 microliters of ice cold, sucrose phosphate glutamate buffer, then split the inclusion suspension equally, between three new 1.5 milliliter screw cap micro centrifuge tubes, and store the tubes at minus 80 to read Celsius.To set up a host cell culture for the imaging of mutagen ISED isolates seed a 96 well glass bottom plate with 1.6 times 10 to the fourth cost seven cells, in 100 microliters of complete medium per well. After the host cell monolayer reaches confluency thaw the harvested mutant clones and wild-type chlamydia stocks on ice and use one column per isolate to perform, a two-fold serial dilution of each mutant isolate. Infect, the remaining column with wild-type chlamydia, at a multiplicity of infection of approximately 0.5, and place a plate in the cell culture incubator for 10 hours at the end of the incubation, select three wells per mutant isolate that correspond to a multiplicity of infection of less than one, and generate an imaging position list.consisting of two fields of view per well. In this scatterplot, the time to half maximal expression of the EOU and hctB promoters from individual chlamydial isolates can be observed clones A3667 and B3858 was elected for isolation as they produced shorter times to have maximal expression, from the EOU promoter and longer times four hctB. Inclusions with altered kinetics can be identified based on the visualization, of the dynamic gene expression of the two promoters, and a snapshot of the animated graph can be used, to identify the location of the inclusions of interest.In these representative visualizations, a total of 24 inclusions were identified for isolation, 10 of which exhibited differential kinetics, upon retesting in three different phenotypic categories, eight isolates exhibited decreased EOU promoter expression at approximately 24 hours post-infection. As demonstrated by the A3667 clone, the B3858 isolate, also exhibited a decreased EOU promoter expression, at about 24 hours post-infection. But an overall increase in hctB promoter expression, whereas the B3662 isolate, expressed increased levels of florescence from the EOU promoter followed by a sudden loss of expression in both promoters.Analysis of the live cell micrographs for mutant B3662, reveals that host cell lysis occurred, in cells infected with this mutant, much earlier than in wild type infected cells. Cell type development deficient chlamydia recovery, may not be possible, as these cells can not reinfect the host. Genome wide association studies can be used after isolation, to identify developmental genes, through statistical correlation, with the incorporation of alternative promoter reporters.This method can be extended, to the probing of numerous genetic pathways, into the dissection of genetic circuit mechanisms, and other intracellular pathogens.

live-cell-forward-genetic-approach-to-identify-isolate-developmental

Research

JoVE Journal

Immunology and Infection

A Genetic Screen to Isolate Toxoplasma gondii Host-cell Egress Mutants

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