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.
