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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.
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.
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 ....
All Python scripts used in this protocol are available on Github https://github.com/SGrasshopper/Live-cell-data-processing
1. Mutagenize Reporter  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.
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.......
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
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.
....Name | Company | Catalog Number | Comments |
24-well polystyrene plates | Corning | 3524 | Cell culture growth for reinfection of isolates |
6-well glass bottom plates | Cellvis | P06-1.5H-N | Cell culture growth for imaging |
96-well glass bottom plates | Nunc | 165305 | Cell culture growth for imaging |
Bold line CO2 Unit | OKO Labs | CO2 UNIT BL | Stage incubator CO2 control |
Bold line T Unit | OKO Labs | H301-T-UNIT-BL-PLUS | Stage incubator temperature control |
Borosilicate glass capillary tubes | Sutter Instrument | B1005010 | Capillary tubes |
BrightLine bandpass emissions filter (514/30nm) | Semrock | FF01-514/30-25 | Fluoescent filter cube |
BrightLine bandpass emissions filter (641/75nm) | Semrock | FF02-641/75-25 | Fluoescent filter cube |
CellTram Vario | Eppendorf | 5196000030 | Microinjector |
Chlamydia trachmatis serovar L2 | ATCC | VR-577 | Chlamydia trachomatis |
CIP-1 media | In house | NA | Axenic media. IPB supplemented with 1% FBS, 25 μM amino acids, 0.5 mM G6P, 1.0 mM ATP, 0.5 mM DTT, and 50 μM GTP, UTP, and CTP. (Omsland, A. 2012) made in-house. |
Cos-7 cells (ATCC) | ATCC | CRL-1651 | African green monkey kidney cell (host cells) |
Cycloheximide | MP Biomedicals | 194527 | Host cell growth inhibitor |
Ethyl methanesulfonate, 99% | Acros Organics | AC205260100 | Mutagen |
Fetal Plex | Gemini Bio-Products | 100-602 | Supplement for base growth media |
Fiji/ImageJ | https://imagej.net/Fiji | NA | Open sourse Image analysis software. https://imagej.net/Fiji |
Galaxy 170 S CO2 incubator | Eppendorf | CO1700100X | Cell culture incubation |
gblocks (Fluorescent FP variants: Clover and mKate2) | Integrated DNA Technologies | NA | gblock ORFs of Ctr optimized FP varients for cloning into p2TK2SW2 |
Gentamycin 10mg/ml | Gibco | 15710-064 | Antibiotic for growth media |
HBSS (Hank's Balanced Salt Solution) | Corning | 21-020-CM | Host cells rinse |
Heparin sodium | Amersham Life Science | 16920 | inhibits and reverses the early electrostatic interactions between the host cell and EBs |
HEPES 1M | GE Life Sciences | SH30237.01 | pH buffer for growth media |
InjectMan | Eppendorf | 5179 000.018 | Micromanipulator |
Jupyter Notebook | https://jupyter.org/ | NA | Visualization of inclusion traces. https://jupyter.org/ |
Lambda 10-3 | Sutter Instrument | LB10-3 | Filter wheel controler |
Oko Touch | OKO Labs | Oko Touch | Interface to control the Bold line T and CO2 Unit |
Prior XY stage | Prior | H107 | Motorized XY microscope stage |
PrismR Centrifuge | Labnet | C2500-R | Temperature controlled microcentrifuge |
Problot Hybridization oven | Labnet | H1200A | Rocking Incubator for infection with Chlamydia |
Proscan II | Prior | H30V4 | XYZ microscope stage controler |
Purifier Class 2 Biosafety Cabinet | Labconco | 362804 | Cell culture work |
RPMI-1640 (no phenol red) | Gibco | 11835-030 | Base growth media for imaging |
RPMI-1640 (phenol red) | GE Life Sciences | SH30027.01 | Base growth media |
scopeLED excitation LEDs (470nm,595nm) | scopeLED | F140 | Excitation light |
Sonic Dismembrator Model 500 | Fisher Scientific | 15-338-550 | Sonicator, resuspending chlamydial pellet |
Stage incubator | OKO Labs | H301-K-FRAME | Cluster well plate incubation chamber |
sucrose-phosphate-glutamate buffer 1X (SPG) | In house | NA | 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) |
T-75 Flasks | Thermo Scientific | 156499 | Cell culture growth |
TE 300 inverted microscope | Nikon | 16724 | microscope |
THOR LED | Thor Labs | LEDD1B | White light |
Trypsin | Corning | 25-052-CI | Dislodges host cells from flask for seeding into plates |
Zyla sCMOS | Andor | ZYLA-5.5-USB3 | imaging camera |
µManager 2.0gamma | https://github.com/micro-manager/micro-manager | NA | Open sourse automated microscope control software package |
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