Forward Genetic Approaches in Chlamydia trachomatis

1. Chemical Mutagenesis

Note: We found that the replicative RB form is more amenable to chemical mutagenesis than the EB form. At mid-cycle (between 18 to 20 hpi), RBs are at the greatest numbers prior to RB-EB transition. Because Chlamydia trachomatis is an obligate intracellular pathogen, the effects of the mutagen on the host health can limit bacterial recovery. Vero cells were found to be more resistant to the adverse effects of high levels of EMS than other cell lines tested.

Segregation of mutations by recombination requires selection for antibiotic resistant recombinant progeny. We recommend using drug resistant strains (e.g. rifampin, spectinomycin, or trimethoprim) for generating mutants for the ability to perform recombinant analysis and to isolate isogenic strains. Antibiotic resistant strains were generated by a stepwise selection process (15, 16).

Note: Chlamydia trachomatis (strain L2/434/Bu / ATCC VR902B) is BSL2 pathogen. Refer to institutional standard operating procedures (SOP) for handling such pathogens.

1. Preparation of cultured cells and infection:
   1. Seed approximately 1 x 10⁶ Vero cells (ATCC CCL-81) per 25 cm² (T25) flask in 3 ml of Dulbecco Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Incubate at 37 °C, 5% CO₂. Cells should form a confluent monolayer within 24 h.
   2. Aspirate medium. Infect confluent T25 flasks of Vero cells with Chlamydia at a multiplicity of infection (moi) of 5 (~14 x 10⁶) in a total volume of 3 ml of medium (DMEM, 200 ng/ml cyclohexamide, 10% FBS).
2. Begin mutagenesis at 18 hr post infection (hpi):
   1. Prepare 20 mg/ml EMS (Ethyl methanesulfonate) in phosphate buffered saline (PBS) with 0.9 mM calcium chloride and 0.49 mM magnesium chloride in a chemical safety hood as EMS is a volatile liquid.
      Note: EMS is a potent mutagen and carcinogen. Neutralize EMS waste and decontaminate spills, plastic, paper, and glassware with 1 M sodium hydroxide.
   2. Aspirate medium and wash cells once with 3 ml of PBS/MgCl₂/CaCl₂. Incubate cells with 3 ml of 20 mg/ml EMS in PBS/MgCl₂/CaCl₂ for one hour in the fume hood at room temperature. Vero cells are able to survive this concentration of EMS and conditions outside the incubator for short periods. Remember to include a control sample that has not been mutagenized.
   3. Remove medium containing EMS and place it in a container of 1 M NaOH to inactivate the mutagen. Wash infected cells 3 times in 3 ml PBS + MgCl₂/CaCl₂ to remove residual mutagen. Add 6 ml of DMEM/10% FBS with 200 ng/ml cyclohexamide and 25 μg/ml gentamicin, and incubate at 5% CO₂, 37 °C for 48-72 h. Inclusions appear devoid of bacteria about 36 hr after mutagenesis. At 72 h about 10% of inclusions are filled with bacteria while the rest remain empty.
   4. Extract EBs by hypotonic lysis: Completely aspirate medium. Add 1.0 ml H₂O and rock to dislodge cells for 10 min. Lyse cells by pipetting up and down for at least 10 times. Collect lysate in a microfuge and add 0.250 ml 5x sucrose phosphate glutamate buffer (SPG) to bring it to 1x final concentration. Store at -80 °C.
      Note: The level of mutagenesis can be assessed by the induction of rifampin resistance. To assay the frequency of rifampin resistance, plaque 1 x 10⁸ and 7 x 10-fold serial dilutions of bacteria in the presence of 200 ng/ml of rifampin. The frequency of rifampin resistance is defined as the number of rifampin resistant plaques divided by total number of bacteria plated. See section below for plaque instructions.

2. Clonal Isolation of Chlamydia trachomatis Mutant Strains by Plaque Purification

1. Set confluent monolayers of Vero cells in 6 well plates: Seed ~0.4 x 10⁶ cells in 3 ml DMEM/10% FBS per well. Allow cells to form a homogenous monolayer over the next 24 h.
2. Thaw stock solution of mutagenized Chlamydia on ice. Perform 6 x 10-fold serial dilutions in a total volume of 200 μl per dilution in DMEM/10% FBS and set them on ice.
   Note: Bacterial titer in inclusion forming units (IFU) is a good estimate of plaque forming units (pfu). Aim to plaque 10, 100, and 1000 pfu.
3. Wash Vero cells twice with 3 ml PBS per well.
4. Add 3 ml DMEM to each well for 6-well plates and 100 μl of the bacterial dilutions in duplicate. Swirl the plate to ensure even mixture.
5. Spin infected plates at 2,700 x g for 30 min at 15 °C then incubate at 37 °C in a humidified, 5% CO₂ incubator for 1-2 hr.
6. Prepare 0.54% agarose in DMEM (for 6 wells):
   1. Add the following ingredients: 18.9 ml cold 2x DMEM, 4.2 ml FBS, 0.42 ml 100x NEAA, 0.042 ml 200 μg/ml cyclohexamide, 0.021 ml 50mg/ml gentamicin. Mix well.
   2. Mix in 18.9 ml of hot sterile 1.2% agarose/H₂O, stirring to prevent clumps. Mixture should be warm to touch. Keep in a warm water bath at 55 °C before adding to infected cells.
7. Aspirate bacterial suspension from dishes and apply 6 ml 0.54% agarose/DMEM per well. Allow agarose to solidify completely at room temperature outside the hood for 15 min. Dry the plates with the lids removed, in the biosafety hood for 15 min.
   Note: Vibration from the biological containment hood can distort the solidifying agarose.
8. Incubate at 37° C in CO₂ incubator for 7-10 days. Plaques should be visible with the aid of a dissecting microscope. (Refer to Figure 3 for a typical image of plaques.)
9. A day prior to isolating mutants, seed 1 x 10⁴ Vero cells in 100 μl per well in a 96-well plate. Cells will be confluent within 24 h.
10. Using a dissection microscope, mark plaques to be picked. Collect plugs of plaques using a sterile 1.0 ml barrier pipette tip.
11. Resuspend plaques in 100 μl of DMEM supplemented with 10% FBS, 400 ng/ml cyclohexamide, 50 μg/ml gentamicin.
12. To expand mutant strains, overlay the suspension onto confluent monolayers of Vero cells. Spin plates at 2,700 x g, 15° C for 30 min.
13. Incubate at 37°C/5% CO₂ in a humidified incubator. Harvest EBs when >50% of the cells are infected, which can occur as early as 48 hpi and as late as 14 days. This amount of infected cells allows enough viable bacteria to be stored. Mutant strains differ in growth rates and infectivity. Allow slow growing strains to naturally re-infect neighboring cells until enough cells are infected.
14. Extract EBs by hypotonic lysis of infected cells:
    1. Completely aspirate medium.
    2. Add 160 μl of sterile water. Incubate at room temperature for 10 min.
    3. Disrupt cells by pipetting up and down several times to ensure complete lysis, use barrier tips to avoid cross contamination.
    4. Transfer 160 μl of lysates to microfuge tubes.
    5. Mix in 40 μl of 5x SPG. Store at -80 °C.
15. Assay and screen mutants for phenotype(s) of interest.

3. Whole Genome Sequencing of Selected Chlamydia Mutants

1. Extract genomic DNA from gradient-purified EBs, which are largely free of contaminating host DNA. Protocols for large scale culture of Chlamydia and purification of EBs can be found in ref. (17). Use commercial genomic DNA purification kits (e.g. Qiagen cat. 69504) to extract DNA from 2 x 10⁹ bacteria following manufacturer instructions.
2. Use a fluorometer (Qubit, Invitrogen cat. Q32866) to accurately measure the amount of DNA. 1-5 μg of purified DNA is required for the Illumina sequencing platform.
   Note: Several platforms can be used to sequence bacterial genomes, including Illumina/Solexa- and SoliD-based systems. We chose to use HiSeq (Illumina) as it produces high density of short, but high quality sequencing reads. Smaller scale genome sequencers, such as IonTorrent (Life Technologies) and MySeq (Illumina) are suitable too and more than sufficient for multiplexing these quencing of up to 4 Chlamydia genomes at a time, which exceeds the minimal coverage of 25X for adequate genome assembly. Preparation of DNA sequencing libraries for the Illumina platform are described below.
3. Fragment DNA to appropriate size (300-400 base pair for Illumina sequencing) using an Adaptive Focused Acoustics S220 instrument (Covaris) according to manufacturer’s suggested settings. Note: DNA fragmentation by nebulization results in greater loss of sample due to aerosolization of sample.
4. Prepare the genomic sequencing libraries using commercial construction kits (Illumina FC-102-1001), following the instructions of the manufacturer. Libraries can be indexed using barcoded primers (Illumina FC-121-1003) such that multiple samples can be pooled and sequenced simultaneously.
5. Run sequencing samples. This step is usually performed by commercial services or core facilities operated by dedicated technical staff. Follow their procedures and recommendations.
6. Illumina reads (FASTQ format) can be assembled to a reference genome using user friendly graphic user interface software, such as Geneious (Biomatters), which can be also used for SNP/mutation identification. To map mutations, set parameters to 90% for minimum variant frequency and 50X for minimum coverage. Open source genome assemblers, such as MAQ (18) and BWA (19), can also be used.
7. Confirm mutations by Sanger sequencing: use genomic DNA as template for PCR amplification of 300-500 bp regions flanking identified mutations and sequence purified or diluted (1:20) PCR products by Sanger sequencing.

4. Generation of Chlamydia Recombinants

Note: Chlamydia can exchange DNA during infection, which allows for the generation of recombinant strains (12, 13, 20). After co-infection of cells with two unique antibiotic resistant strains, recombinant "progeny" can be selected for dual antibiotic resistance (12, 13). We took advantage of this phenomenon to segregate mutations and to generate co-isogenic strains. Hence, mutant strains were generated in antibiotic resistant background (e.g. rifampin resistance (rif^R) H471Y in CTL0567 (rpoB)) such that they can be crossed to wild type strain bearing a different antibiotic resistant allele (e.g. spectinomycin resistance (spc^R), G1197A in r01/r02 (16SRNA copies 1 and 2)). For details regarding DNA exchange and recombination in Chlamydia, refer to references (12, 13).

1. Co-infect wild type and clonal mutant strains by centrifuging6 x 10⁵ bacteria from each strain onto confluent monolayers of Vero cells grown on a 24-well plate. In parallel, infect monolayers with each strain alone.
2. At 44 hpi, harvest EBs by hypotonic lysis of infected cells:
   1. Completely aspirate medium.
   2. Add 0.4 ml of sterile water and let stand for 10 min.
   3. Lyse cells by vigorous pipetting up & down for at least 10 times. Transfer lysates to a microfuge tube.
   4. Mix in 0.1 ml of 5x SPG for a final concentration of 1x SPG.
   5. Crude lysates can be used immediately or stored at -80 °C.
3. Plaque 50 μl of the crude EB preps and 5 x 1:10 serial dilutions in agarose/DMEM supplemented with appropriate antibiotics to select for recombinants. (See table for concentrations of typical antibiotics for selection of recombinants.)
4. Plaque purify and enrich recombinant strains as above. Score recombinants for presence or absence of the desired phenotype. Extract DNA from recombinant strains by DNAzol treatment (next section) for the purpose of genotyping for the presence or absence of mutations.
5. Crosses can be repeated with strains bearing other antibiotic resistance markers to further segregate mutations, and generate isogenic strains. Co-infect selected recombinants to another wild type strain bearing a different antibiotic marker.

Table 1: Antibiotic concentrations for selection of recombinants:

5. Extraction of Genomic DNA from Recombinant Strains for Genotyping

Note: Column-based purification kits can also be used. An advantage of DNA extraction by DNAzol treatment is cost.

1. Infect 1.2 x 10⁵ chlamydiae onto monolayers of Vero cells grown on 12-well plates.
2. At 36-48 hpi, aspirate medium and add 0.375 ml of DNAzol (Invitrogen 10503-027). Gently agitate the cells and pipet the lysates into a 1.5 ml microfuge tube.
3. Precipitate DNA by the addition of 0.188 ml of 100% ethanol. Mix by inversion.
4. Extract DNA by spooling with a pipette tip and place it in a clean tube. Alternatively, pellet the DNA by centrifugation at top speed for 2 min at room temperature or 4 °C, and decant supernatant.
5. Wash DNA precipitate twice with 1.0 ml of 75% ethanol.
6. Air-dry DNA after removing the ethanol for 15 sec.
7. Solubilize DNA with 0.2 ml 8 mM NaOH then neutralize to pH 8.0 with 20.2 μl of 0.1 M HEPES. Bring up to a final volume of 0.5 ml with water or TE. Alternatively, resuspend DNA in TE buffer (DNA pellet will not solubilized completely.).
8. Use DNAzol-extracted DNA as template for PCR amplification of 300-500 bp regions flanking identified mutations. Sequence purified or diluted (1:20) PCR products by Sanger sequencing.