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

The widespread, obligate intracellular, protozoan parasite Toxoplasma gondii causes opportunistic disease in immuno-compromised patients and causes birth defects upon congenital infection. The lytic replication cycle is characterized by three stages: 1. active invasion of a nucleated host cell; 2. replication inside the host cell; 3. active egress from the host cell. The mechanism of egress is increasingly being appreciated as a unique, highly regulated process, which is still poorly understood at the molecular level. The signaling pathways underlying egress have been characterized through the use of pharmacological agents acting on different aspects of the pathways1-5. As such, several independent triggers of egress have been identified which all converge on the release of intracellular Ca2+, a signal that is also critical for host cell invasion6-8. This insight informed a candidate gene approach which led to the identification of plant like calcium dependent protein kinase (CDPK) involved in egress9. In addition, several recent breakthroughs in understanding egress have been made using (chemical) genetic approaches10-12. To combine the wealth of pharmacological information with the increasing genetic accessibility of Toxoplasma we recently established a screen permitting the enrichment for parasite mutants with a defect in host cell egress13. Although chemical mutagenesis using N-ethyl-N-nitrosourea (ENU) or ethyl methanesulfonate (EMS) has been used for decades in the study of Toxoplasma biology11,14,15, only recently has genetic mapping of mutations underlying the phenotypes become routine16-18. Furthermore, by generating temperature-sensitive mutants, essential processes can be dissected and the underlying genes directly identified. These mutants behave as wild-type under the permissive temperature (35 &deg;C), but fail to proliferate at the restrictive temperature (40 &deg;C) as a result of the mutation in question. Here we illustrate a new phenotypic screening method to isolate mutants with a temperature-sensitive egress phenotype13. The challenge for egress screens is to separate egressed from non-egressed parasites, which is complicated by fast re-invasion and general stickiness of the parasites to host cells. A previously established egress screen was based on a cumbersome series of biotinylation steps to separate intracellular from extracellular parasites11. This method also did not generate conditional mutants resulting in weak phenotypes. The method described here overcomes the strong attachment of egressing parasites by including a glycan competitor, dextran sulfate (DS), that prevents parasites from sticking to the host cell19. Moreover, extracellular parasites are specifically killed off by pyrrolidine dithiocarbamate (PDTC), which leaves intracellular parasites unharmed20. Therefore, with a new phenotypic screen to specifically isolate parasite mutants with defects in induced egress, the power of genetics can now be fully deployed to unravel the molecular mechanisms underlying host cell egress.

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

Forward genetics is a powerful method to unravel the molecular level of how Toxoplasma egresses from its host cell. Protocols are provided to chemically mutagenize parasites, enrich for mutants with defects in induced egress, and validate the phenotype of cloned mutants.

The widespread, obligate intracellular, protozoan parasite Toxoplasma gondii causes opportunistic disease in immuno-compromised patients and causes birth ...

Forward Genetic Approaches in Chlamydia trachomatis

Chlamydia trachomatis, the etiological agent of sexually transmitted diseases and ocular infections, remains poorly characterized due to its intractability to experimental transformation with recombinant DNA. We developed an approach to perform genetic analysis in C. trachomatis&#160;despite the lack of molecular genetic tools. Our method involves: i.) chemical mutagenesis to rapidly generate comprehensive libraries of genetically-defined mutants with distinct phenotypes; ii.) whole-genome sequencing (WGS) to map the underlying genetic lesions and to find associations between mutated gene(s) and a common phenotype; iii.) generation of recombinant strains through co-infection of mammalian cells with mutant and wild type bacteria. Accordingly, we were able to establish causal relationships between genotypes and phenotypes. The coupling of chemically-induced gene variation and WGS to establish correlative genotype&#8211;phenotype associations should be broadly applicable to the large list of medically and environmentally important microorganisms currently intractable to genetic analysis.

Forward Genetic Approaches in Chlamydia trachomatis

We describe a methodology to perform genetic analysis in Chlamydia based on chemical mutagenesis and whole genome sequencing. In addition, a system for DNA exchange within infected cells is described that can be used for genetic mapping. This method may be broadly applicable to microbial systems lacking transformation systems and molecular genetic tools.

Chlamydia trachomatis, the etiological agent of sexually transmitted diseases and ocular infections, remains poorly characterized due to its ...

Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

The obligate intracellular bacterium Chlamydia elicits a great burden on global public health. C.&#160;trachomatis is the leading bacterial cause of sexually transmitted infection and also the primary cause of preventable blindness in the world. An essential determinant for successful infection of host cells by Chlamydia is the bacterium's ability to manipulate host cell signaling from within a novel, vacuolar compartment called the inclusion. From within the inclusion, Chlamydia acquire nutrients required for their 2-3 day developmental growth, and they additionally secrete a panel of effector proteins onto the cytosolic face of the vacuole membrane and into the host cytosol. Gaps in our understanding of Chlamydia biology, however, present significant challenges for visualizing and analyzing this intracellular compartment. Recently, a reverse-imaging strategy for visualizing the inclusion using GFP expressing host cells was described. This approach rationally exploits the intrinsic impermeability of the inclusion membrane to large molecules such as GFP. In this work, we describe how GFP- or mCherry-expressing host cells are generated for subsequent visualization of chlamydial inclusions. Furthermore, this method is shown to effectively substitute for costly antibody-based enumeration methods, can be used in tandem with other fluorescent labels, such as GFP-expressing Chlamydia, and can be exploited to derive key quantitative data about inclusion membrane growth from a range of Chlamydia species and strains.

Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

A live cell fluorescent protein based method for illuminating cellular vacuoles (inclusions) containing Chlamydia is described. This strategy enables rapid, automated determination of Chlamydia infectivity in samples and can be used to quantitatively investigate inclusion growth dynamics.

The obligate intracellular bacterium Chlamydia elicits a great burden on global public health. C.&#160;trachomatis is the leading bacterial cause of ...

Forward Genetics Screens Using Macrophages to Identify Toxoplasma gondii Genes Important for Resistance to IFN-&#947;-Dependent Cell Autonomous Immunity

Toxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular protozoan pathogen. The parasite invades and replicates within virtually any warm blooded vertebrate cell type. During parasite invasion of a host cell, the parasite creates a parasitophorous vacuole (PV) that originates from the host cell membrane independent of phagocytosis within which the parasite replicates. While IFN-dependent-innate and cell mediated immunity is important for eventual control of infection, innate immune cells, including neutrophils, monocytes and dendritic cells, can also serve as vehicles for systemic dissemination of the parasite early in infection. An approach is described that utilizes the host innate immune response, in this case macrophages, in a forward genetic screen to identify parasite mutants with a fitness defect in infected macrophages following activation but normal invasion and replication in na&#239;ve macrophages. Thus, the screen isolates parasite mutants that have a specific defect in their ability to resist the effects of macrophage activation. The paper describes two broad phenotypes of mutant parasites following activation of infected macrophages: parasite stasis versus parasite degradation, often in amorphous vacuoles. The parasite mutants are then analyzed to identify the responsible parasite genes specifically important for resistance to induced mediators of cell autonomous immunity. The paper presents a general approach for the forward genetics screen that, in theory, can be modified to target parasite genes important for resistance to specific antimicrobial mediators. It also describes an approach to evaluate the specific macrophage antimicrobial mediators to which the parasite mutant is susceptible. Activation of infected macrophages can also promote parasite differentiation from the tachyzoite to bradyzoite stage that maintains chronic infection. Therefore, methodology is presented to evaluate the importance of the identified parasite gene to establishment of chronic infection.

Forward Genetics Screens Using Macrophages to Identify Toxoplasma gondii Genes Important for Resistance to IFN-&#947;-Dependent Cell Autonomous Immunity

Forward genetics is a powerful approach to identify genes in intracellular pathogens important for resistance to cell autonomous immunity. The current approach uses innate immune cells, specifically macrophages, to identify novel Toxoplasma gondii genes important for immune evasion.

Toxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular protozoan pathogen. The parasite invades and replicates within ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.

Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

Retroviral Transduction of Helper T Cells as a Genetic Approach to Study Mechanisms Controlling their Differentiation and Function

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents autoimmunity. Many approaches involving different model organisms have been utilized to understand the mechanisms controlling helper T cell development and function. However, studies using mouse models have proven to be highly informative due to the availability of genetic, cellular, and biochemical systems. One genetic approach in mice used by many labs involves retroviral transduction of primary helper T cells. This is a powerful approach due to its relative ease, making it accessible to almost any laboratory with basic skills in molecular biology and immunology. Therefore, multiple genes in wild type or mutant forms can readily be tested for function in helper T cells to understand their importance and mechanisms of action. We have optimized this approach and describe here the protocols for production of high titer retroviruses, isolation of primary murine helper T cells, and their transduction by retroviruses and differentiation toward the different helper subsets. Finally, the use of this approach is described in uncovering mechanisms utilized by microRNAs (miRNAs) to regulate pathways controlling helper T cell development and function.

Many experimental systems have been utilized to understand the mechanisms regulating T cell development and function in an immune response. Here a genetic approach using retroviral transduction is described, which is economic, time efficient, and most importantly, highly informative in identifying regulatory pathways.

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents ...

A Simple Red Blood Cell Lysis Method for the Establishment of B Lymphoblastoid Cell Lines

A number of methods exist for the transformation of B lymphocytes by the Epstein Barr virus in vitro into immortalized cell lines. We have developed a new method with a powerful and simple strategy for the establishment of B-LCLs, the red blood cell lysis method. This method simplified the PBMC separation procedure with red blood cell removal, and used as little as 0.5 mL of whole blood for establishing EBV-immortalized cell lines, which can proliferate to large cell numbers in a relatively short amount time with a 100% success rate. The method is simple, reliable, time saving, and applicable to treating a large number of the clinical samples.

A simple red blood cell lysis method for establishing B-LCLs was developed with high immortalization efficiency, requiring a small amount of blood and saving time from initiation to cryopreservation.

A number of methods exist for the transformation of B lymphocytes by the Epstein Barr virus in vitro into immortalized cell lines. We have developed a new ...

An Efficient and Simple Method to Establish NK and T Cell Lines from Patients with Chronic Active Epstein-Barr Virus Infection

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed feeder cells, purified NK or T cells with as much as 10 mL of blood, or a high-dose of IL-2. This study presents a new method with a powerful and simple strategy to establish NK and T cell lines by culturing the peripheral blood mononuclear cells (PBMC) with the addition of recombinant human IL-2 (rhIL-2), and uses as little as 2 mL of whole blood. The cells can proliferate quickly in two weeks and be maintained for more than 3 months. With this method, 7 NK or T cell lines have been established with a high success rate. This method is simple, reliable, and applicable to establishing cell lines from more cases of CAEBV or NK/T cell lymphoma.

A simple method for obtaining NK and T cell clones from CAEBV patients was developed with high efficiency, a small amount of peripheral blood, and a low-dose of IL-2.

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed ...

Visual and Microscopic Evaluation of Streptomyces Developmental Mutants

Streptomycetes are filamentous soil bacteria belonging to the phylum Actinobacteria that are found throughout the world and produce a wide array of antibiotics and other secondary metabolites. Streptomyces coelicolor is a well-characterized, non-pathogenic species that is amenable to a variety of analyses in the lab. The phenotyping methods described here use S. coelicolor as a model streptomycete; however, the methods are applicable to all members of this large genus as well as some closely related actinomycetes. Phenotyping is necessary to characterize new species of Streptomyces identified in the environment, and it is also a vital first step in characterizing newly isolated mutant strains of Streptomyces. Proficiency in phenotyping is important for the many new researchers who are entering the field of Streptomyces research, which includes the study of bacterial development, cell division, chromosome segregation, and second messenger signaling. The recent crowdsourcing of antibiotic discovery through the isolation of new soil microbes has resulted in an increased need for training in phenotyping for instructors new to the field of Streptomyces research and their college or high school students. This manuscript describes methods for bacterial strain propagation, storage, and characterization through visual and microscopic examination. After reading this article, new researchers (microbiology education laboratories and citizen scientists) should be able to manipulate Streptomyces strains and begin visual characterization experiments.

Visual and Microscopic Evaluation of Streptomyces Developmental Mutants

Here we present protocols for novice researchers to initiate phenotyping for the pharmacologically important bacterial genus Streptomyces.

Streptomycetes are filamentous soil bacteria belonging to the phylum Actinobacteria that are found throughout the world and produce a wide array of ...

Live-Cell Fluorescence Microscopy to Investigate Subcellular Protein Localization and Cell Morphology Changes in Bacteria

Investigations of factors influencing cell division and cell shape in bacteria are commonly performed in conjunction with high-resolution fluorescence microscopy as observations made at a population level may not truly reflect what occurs at a single cell level. Live-cell timelapse microscopy allows investigators to monitor the changes in cell division or cell morphology which provide valuable insights regarding subcellular localization of proteins and timing of gene expression, as it happens, to potentially aid in answering important biological questions. Here, we describe our protocol to monitor phenotypic changes in Bacillus subtilis and Staphylococcus aureus using a high-resolution deconvolution microscope. The objective of this report is to provide a simple and clear protocol that can be adopted by other investigators interested in conducting fluorescence microscopy experiments to study different biological processes in bacteria as well as other organisms.

This article provides a step-by-step guide to investigate protein subcellular localization dynamics and to monitor morphological changes using high-resolution fluorescence microscopy in Bacillus subtilis and Staphylococcus aureus.

Investigations of factors influencing cell division and cell shape in bacteria are commonly performed in conjunction with high-resolution fluorescence ...