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&#160;(strain L2/434/Bu / ATCC VR902B) is BSL2 pathogen. Refer to institutional standard operating procedures (SOP) for handling such pathogens.
<ol>
	<li>Preparation of cultured cells and infection:
	<ol>
		<li>Seed approximately 1 x 106&#160;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 &#176;C, 5% CO2. Cells should form a confluent monolayer within 24 h.</li>
		<li>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&#160;of medium (DMEM, 200 ng/ml&#160;cyclohexamide, 10% FBS).</li>
	</ol>
	</li>
	<li>Begin mutagenesis at 18 hr&#160;post infection (hpi):
	<ol>
		<li>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.</li>
		<li>Aspirate medium and wash cells once with 3 ml&#160;of PBS/MgCl2/CaCl2. Incubate cells with 3 ml of 20 mg/ml EMS in PBS/MgCl2/CaCl2&#160;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.</li>
		<li>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&#160;to remove residual mutagen. Add 6 ml of DMEM/10% FBS with 200 ng/ml cyclohexamide and 25 &#956;g/ml gentamicin, and incubate at 5% CO2, 37 &#176;C for 48-72 h. Inclusions appear devoid of bacteria about 36 hr&#160;after mutagenesis. At 72 h about 10% of inclusions are filled with bacteria while the rest remain empty.</li>
		<li>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 &#176;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&#160;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.</li>
	</ol>
	</li>
</ol>
2. Clonal Isolation of Chlamydia trachomatis&#160;Mutant Strains by Plaque Purification
<ol>
	<li>Set confluent monolayers of Vero cells in 6 well plates: Seed ~0.4 x 106 cells in 3 ml&#160;DMEM/10% FBS per well. Allow cells to form a homogenous monolayer over the next 24 h.</li>
	<li>Thaw stock solution of mutagenized Chlamydia on ice. Perform 6 x 10-fold serial dilutions in a total volume of 200 &#956;l 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.</li>
	<li>Wash Vero cells twice with 3 ml&#160;PBS per well.</li>
	<li>Add 3 ml&#160;DMEM to each well for 6-well plates and 100 &#956;l of the bacterial dilutions in duplicate. Swirl the plate to ensure even mixture.</li>
	<li>Spin infected plates at 2,700 x g for 30 min at 15 &#176;C then incubate at 37 &#176;C in a humidified, 5% CO2 incubator for 1-2 hr.</li>
	<li>Prepare 0.54% agarose in DMEM (for 6 wells):
	<ol>
		<li>Add the following ingredients: 18.9 ml&#160;cold 2x DMEM, 4.2 ml&#160;FBS, 0.42 ml 100x NEAA, 0.042 ml 200 &#956;g/ml cyclohexamide, 0.021 ml 50mg/ml gentamicin. Mix well.</li>
		<li>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 &#176;C before adding to infected cells.</li>
	</ol>
	</li>
	<li>Aspirate bacterial suspension from dishes and apply 6 ml&#160;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.</li>
	<li>Incubate at 37&#176; 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.)</li>
	<li>A day prior to isolating mutants, seed 1 x 104&#160;Vero cells in 100 &#956;l per well in a 96-well plate. Cells will be confluent within 24 h.</li>
	<li>Using a dissection microscope, mark plaques to be picked. Collect plugs of plaques using a sterile 1.0 ml barrier pipette tip.</li>
	<li>Resuspend plaques in 100 &#956;l of DMEM supplemented with 10% FBS, 400 ng/ml cyclohexamide, 50 &#956;g/ml gentamicin.</li>
	<li>To expand mutant strains, overlay the suspension onto confluent monolayers of Vero cells. Spin plates at 2,700 x g, 15&#176; C for 30 min.</li>
	<li>Incubate at 37&#176;C/5% CO2 in a humidified incubator. Harvest EBs when &#62;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.</li>
	<li>Extract EBs by hypotonic lysis of infected cells:
	<ol>
		<li>Completely aspirate medium.</li>
		<li>Add 160 &#956;l of sterile water. Incubate at room temperature for 10 min.</li>
		<li>Disrupt cells by pipetting up and down several times to ensure complete lysis, use barrier tips to avoid cross contamination.</li>
		<li>Transfer 160 &#956;l of lysates to microfuge tubes.</li>
		<li>Mix in 40 &#956;l of 5x SPG. Store at -80 &#176;C.</li>
	</ol>
	</li>
	<li>Assay and screen mutants for phenotype(s) of interest.</li>
</ol>
3. Whole Genome Sequencing of Selected Chlamydia&#160;Mutants
<ol>
	<li>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.</li>
	<li>Use a fluorometer (Qubit, Invitrogen cat. Q32866) to accurately measure the amount of DNA. 1-5 &#956;g 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&#160;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.</li>
	<li>Fragment DNA to appropriate size (300-400 base pair for Illumina sequencing) using an Adaptive Focused Acoustics S220 instrument (Covaris) according to manufacturer&#8217;s suggested settings. Note: DNA fragmentation by nebulization results in greater loss of sample due to aerosolization of sample.</li>
	<li>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.</li>
	<li>Run sequencing samples. This step is usually performed by commercial services or core facilities operated by dedicated technical staff. Follow their procedures and recommendations.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
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 &#34;progeny&#34; 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 (rifR) H471Y in CTL0567 (rpoB)) such that they can be crossed to wild type strain bearing a different antibiotic resistant allele (e.g. spectinomycin resistance (spcR), G1197A in r01/r02 (16SRNA copies 1 and 2)). For details regarding DNA exchange and recombination in Chlamydia, refer to references (12, 13).
<ol>
	<li>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.</li>
	<li>At 44 hpi, harvest EBs by hypotonic lysis of infected cells:
	<ol>
		<li>Completely aspirate medium.</li>
		<li>Add 0.4 ml of sterile water and let stand for 10 min.</li>
		<li>Lyse cells by vigorous pipetting up &#38; down for at least 10 times. Transfer lysates to a microfuge tube.</li>
		<li>Mix in 0.1 ml of 5x SPG for a final concentration of 1x SPG.</li>
		<li>Crude lysates can be used immediately or stored at -80 &#176;C.</li>
	</ol>
	</li>
	<li>Plaque 50 &#956;l 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.)</li>
	<li>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.</li>
	<li>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.</li>
</ol>
Table 1: Antibiotic concentrations for selection of recombinants:
<table border="1">
	<tbody>
		<tr>
			<th>Final Concentration</th>
			<th>Preparation instructions</th>
		</tr>
		<tr>
			<td>200 ng/ml rifampin</td>
			<td>Make 25 mg/ml storage stock in dimethyl sulfoxide (DMSO). Make up 200 &#956;g/ml working solution by diluting the storage stock with H2O. Store at -20 &#176;C in the dark.</td>
		</tr>
		<tr>
			<td>200 &#956;g/ml trimethoprim</td>
			<td>Make a 100 mg/ml stock in DMSO. Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>200 &#956;g/ml spectinomycin</td>
			<td>Make a 100 mg/ml stock in water in 100 &#956;l aliquots. Store at -20 &#176;C. Avoid repeated freeze thaw.</td>
		</tr>
	</tbody>
</table>
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.
<ol>
	<li>Infect 1.2 x 105&#160;chlamydiae onto monolayers of Vero cells grown on 12-well plates.</li>
	<li>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&#160;microfuge tube.</li>
	<li>Precipitate DNA by the addition of 0.188 ml of 100% ethanol. Mix by inversion.</li>
	<li>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&#160;at room temperature or 4 &#176;C, and decant supernatant.</li>
	<li>Wash DNA precipitate twice with 1.0 ml&#160;of 75% ethanol.</li>
	<li>Air-dry DNA after removing the ethanol for 15 sec.</li>
	<li>Solubilize DNA with 0.2 ml&#160;8 mM NaOH then neutralize to pH 8.0 with 20.2 &#956;l&#160;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.).</li>
	<li>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.</li>
</ol>

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There is nothing to disclose.

This methodology meets the basic requirements for genetic analysis as it establishes linkage between genotypes and phenotypes. Importantly, this is achieved without the aid of conventional molecular tools for recombinant DNA transformation and insertional inactivation of genes in bacteria, which is often a rate-limiting step in the analysis of gene function in non-model microbes.
One critical step is to ensure clonality of plaque-purified mutants. Cross contamination with wild type or &#34;fit...

The obligate intracellular bacterium Chlamydia trachomatis accounts for an estimated 2.8 million genital tract infections per year in the United States (Center for Disease Control) with associated sequel&#230; such as pelvic inflammatory disease, ectopic pregnancies, and infertility (1).&#160;Chlamydia&#160;spp have a unique physiology with a biphasic developmental cycle consisting of two forms: the infectious but non-replicating elementary body (EB) and the noninfectious but replicative reticulate body (RB). Infection begins with the attachment of EBs to epithelial cells followed by endoctyotosis (2). Within a membrane-bound vacuole termed an inclusion, EBs differentiate into the RB form, which then replicates by binary fission. At mid-cycle, RBs transitions back into EBs, which are then expelled into the extracellular space to initiate new rounds of infection when the host cell lyses (3).
C. trachomatis is refractory to routine manipulation with standard molecular genetic tools, such as targeted gene replacement, transposons, and transducing phages, which have been central to most studies in bacterial genetics, It is unclear the extent to which individual Chlamydia genes contribute to the evasion of innate immunity, nutrient acquisition, developmental transitions, or other processes important for the pathogen&#8217;s survival within a eukaryotic host (4). Consequently, this pathogen remains poorly characterized despite its clinical importance.
The genomes of Chlamydia spp. are relatively small (~1 Mb) (5) with multiple species and biovars sequenced using Next Generation sequencing technologies. Comparative genome analysis by WGS has provided unique insights into the evolution of chlamydial species and their adaptation to humans (6-8) and to some extent has provided some information as to the potential function of virulence factors (9, 10). The genetic diversity displayed by clinical isolates does not provide the resolution required to systematically map the function of most virulence factors, presumably because mutations in such genes would have been readily selected against. Without confounding effects from natural selection, mutagen-induced gene variation, coupled with defined assays that measure defects in virulence, can expand the spectrum of mutations that can be surveyed. Chemical mutagens, in particular, are useful as they can generate null, conditional, hypomorphic (reduced function), and hypermorphic (gain of function) alleles. With the arrival of robust next-generation genome-sequencing technologies, such mutations can be readily identified and mapped. In this manner, strong associations can be made between mutations in a gene or genetic pathway and a common phenotype, enabling the application of forward genetic approaches.
The genome sequences of clinical strains revealed mosaicism between serovars and loci of frequent recombination (11). Empirical evidence of recombination was demonstrated through the co-infection of two different antibiotic resistant strains and selection of dual resistant recombinant progeny, which was revealed to have genetic contributions from both strains (12, 13). Thus, genetic exchange between wild type and mutant strains in a co-infection setting allows segregation of chemically-induced mutations to pinpoint the affected gene that leads to the observed phenotype.
Here we describe a methodology to perform genetic analysis in Chlamydia&#160;based on chemical mutagenesis, WGS, and a system for DNA exchange within infected cells (14) (Figure 1).

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	<colgroup width="144">
	</colgroup>
	<colgroup width="84">
	</colgroup>
	<tbody>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle Medium (DMEM)</td>
			<td>Life Technologies</td>
			<td>11995-073</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Cellgro</td>
			<td>35-010-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl methanesulfonate (EMS)</td>
			<td>Sigma</td>
			<td>M0880</td>
			<td></td>
		</tr>
		<tr>
			<td>Cyclohexamide</td>
			<td>Sigma</td>
			<td>C4859-1ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Life Technologies</td>
			<td>15750-060</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Life Technologies</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS) solution with 0.493 mM MgCl2 and 0.901 mM CaCl2 (PBS+MgCl2/CaCl2)</td>
			<td>Life Technologies</td>
			<td>14040-133</td>
			<td></td>
		</tr>
		<tr>
			<td>1 M NaOH</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5x SPG buffer (1.25 M sucrose, 50 mM sodium phosphate, 25 mM glutamic acid)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SPG buffer (0.25 M sucrose, 10 mM sodium phosphate, 5 mM glutamic acid)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Water (sterile, tissue culture grade)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2x DMEM (prepared from powder, buffered with 7.4 g/L Sodium Bicarbonate</td>
			<td>Sigma</td>
			<td>D7777</td>
			<td></td>
		</tr>
		<tr>
			<td>Nonessential amino acids (NEAA)</td>
			<td>Life Technologies</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr>
			<td>1.2% GTG Agarose , autoclaved</td>
			<td>Lonzo</td>
			<td>50070</td>
			<td></td>
		</tr>
		<tr>
			<td>Genomic DNA purification kits</td>
			<td>Qiagen</td>
			<td>69504</td>
			<td></td>
		</tr>
		<tr>
			<td>DNAzol</td>
			<td>Life Technologies</td>
			<td>10503-027</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol (molecular biology grade)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>8 mM NaOH</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.1 M HEPES ( 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>25 cm2 tissue culture flasks (T25 flasks)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>6-well tissue culture plates</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>12-well tissue culture plates</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96-well tissue culture plates</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chemical safety hood</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Biological safety hood</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#62;Centrifuge and adaptors for spinning tissue plates</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#62;Dissection microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorometer (Qubit)</td>
			<td>Invitrogen</td>
			<td>Q32866</td>
			<td></td>
		</tr>
		<tr>
			<td>Adaptive Focused Acoustics S220 instrument</td>
			<td>Covaris</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Exposure to mutagen leads to inclusions that appear devoid of bacteria,presumably due to bacterial cell death. Typically,serovar LGV-L2 will completely lyse infected cells within 48 h post infection, but treatment with mutagens can extend this cycle to &#62;90 h. Roughly 10% of inclusions are expected to recover. In our experiments, infected Vero cells treated with 20 mg/ml EMS led to a 99% decrease in the recovery of infectious progeny compared to untreated controls. Mutagen treatment also led to the emergence of plaque...

Watch this Scientific Journal Video about Forward Genetic Approaches in Chlamydia trachomatis at JoVE.com

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.

Forward Genetic Approaches in Chlamydia trachomatis

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

forward-genetic-approaches-in-chlamydia-trachomatis

Research

JoVE Journal

Immunology and Infection

12.8K Views.  Duke University Medical Center. 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.

Video: Forward Genetic Approaches in Chlamydia trachomatis

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 ...

Genetic Manipulation in &Delta;ku80 Strains for Functional Genomic Analysis of Toxoplasma gondii

Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene function and phenotype(s). The development of mutant strains with targeted gene deletions, targeted mutations, complemented gene function, and/or tagged genes provides powerful strategies to address gene function, particularly if these genetic manipulations can be efficiently targeted to the gene locus of interest using integration mediated by double cross over homologous recombination.
Due to very high rates of nonhomologous recombination, functional genomic analysis of Toxoplasma gondii has been previously limited by the absence of efficient methods for targeting gene deletions and gene replacements to specific genetic loci. Recently, we abolished the major pathway of nonhomologous recombination in type I and type II strains of T. gondii by deleting the gene encoding the KU80 protein1,2. The &Delta;ku80 strains behave normally during tachyzoite (acute) and bradyzoite (chronic) stages in vitro and in vivo and exhibit essentially a 100% frequency of homologous recombination. The &Delta;ku80 strains make functional genomic studies feasible on the single gene as well as on the genome scale1-4.
Here, we report methods for using type I and type II &Delta;ku80&Delta;hxgprt strains to advance gene targeting approaches in T. gondii. We outline efficient methods for generating gene deletions, gene replacements, and tagged genes by targeted insertion or deletion of the hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) selectable marker. The described gene targeting protocol can be used in a variety of ways in &Delta;ku80 strains to advance functional analysis of the parasite genome and to develop single strains that carry multiple targeted genetic manipulations. The application of this genetic method and subsequent phenotypic assays will reveal fundamental and unique aspects of the biology of T. gondii and related significant human pathogens that cause malaria (Plasmodium sp.) and cryptosporidiosis (Cryptosporidium).

Genetic Manipulation in &lt;em&gt;&amp;Delta;ku80&lt;/em&gt; Strains for Functional Genomic Analysis of &lt;em&gt;Toxoplasma gondii&lt;/em&gt;

Here we report a method for using type I and type II &Delta;ku80 strains of Toxoplasma gondii to efficiently generate targeted gene deletions and gene replacements for functional genomic analysis.

Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene ...

VIGS-Mediated Forward Genetics Screening for Identification of Genes Involved in Nonhost Resistance

Nonhost disease resistance of plants against bacterial pathogens is controlled by complex defense pathways. Understanding this mechanism is important for developing durable disease-resistant plants against wide range of pathogens. Virus-induced gene silencing (VIGS)-based forward genetics screening is a useful approach for identification of plant defense genes imparting nonhost resistance. Tobacco rattle virus (TRV)-based VIGS vector is the most efficient VIGS vector to date and has been efficiently used to silence endogenous target genes in Nicotiana benthamiana.

	In this manuscript, we demonstrate a forward genetics screening approach for silencing of individual clones from a cDNA library in N. benthamiana and assessing the response of gene silenced plants for compromised nonhost resistance against nonhost pathogens, Pseudomonas syringae pv. tomato T1, P. syringae pv. glycinea, and X. campestris pv. vesicatoria. These bacterial pathogens are engineered to express GFPuv protein and their green fluorescing colonies can be seen by naked eye under UV light in the nonhost pathogen inoculated plants if the silenced target gene is involved in imparting nonhost resistance. This facilitates reliable and faster identification of gene silenced plants susceptible to nonhost pathogens. Further, promising candidate gene information can be known by sequencing the plant gene insert in TRV vector. Here we demonstrate the high throughput capability of VIGS-mediated forward genetics to identify genes involved in nonhost resistance. Approximately, 100 cDNAs can be individually silenced in about two to three weeks and their relevance in nonhost resistance against several nonhost bacterial pathogens can be studied in a week thereafter. In this manuscript, we enumerate the detailed steps involved in this screening. VIGS-mediated forward genetics screening approach can be extended not only to identifying genes involved in nonhost resistance but also to studying genes imparting several biotic and abiotic stress tolerances in various plant species.

Virus-induced gene silencing is an useful tool for identifying genes involved in nonhost resistance of plants. We demonstrate the use of bacterial pathogens expressing GFPuv in identifying gene silenced plants susceptible to nonhost pathogens. This approach is easy, fast and facilitates large scale screening and similar protocol can be applied to studying various other plant-microbe interactions.


	Nonhost disease resistance of plants against bacterial pathogens is controlled by complex defense pathways. Understanding this mechanism is important ...

Discovery of New Intracellular Pathogens by Amoebal Coculture and Amoebal Enrichment Approaches

Intracellular pathogens such as legionella, mycobacteria and Chlamydia-like organisms are difficult to isolate because they often grow poorly or not at all on selective media that are usually used to cultivate bacteria. For this reason, many of these pathogens were discovered only recently or following important outbreaks. These pathogens are often associated with amoebae, which serve as host-cell and allow the survival and growth of the bacteria. We intend here to provide a demonstration of two techniques that allow isolation and characterization of intracellular pathogens present in clinical or environmental samples: the amoebal coculture and the amoebal enrichment. Amoebal coculture allows recovery of intracellular bacteria by inoculating the investigated sample onto an amoebal lawn that can be infected and lysed by the intracellular bacteria present in the sample. Amoebal enrichment allows recovery of amoebae present in a clinical or environmental sample. This can lead to discovery of new amoebal species but also of new intracellular bacteria growing specifically in these amoebae. Together, these two techniques help to discover new intracellular bacteria able to grow in amoebae. Because of their ability to infect amoebae and resist phagocytosis, these intracellular bacteria might also escape phagocytosis by macrophages and thus, be pathogenic for higher eukaryotes.

Amoebal coculture is a cell culture system using adherent amoebae to selectively grow intracellular pathogens able to resist phagocytic cells such as amoebae and macrophages. It thus represents a key tool to discover new infectious agents. Amoebal enrichment allows discovery of new amoebal species and of their specific intracellular bacteria.

Intracellular pathogens such as legionella, mycobacteria and Chlamydia-like organisms are difficult to isolate because they often grow poorly or not at ...

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-&amp;#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 ...

Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysis by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented.

Here, we present phenomic approaches for the functional characterization of putative phage genes. Techniques include a developed assay capable of monitoring host anabolic metabolism, the Multi-phenotype Assay Plates (MAPs), in addition to the established method of metabolomics, capable of measuring effects to catabolic metabolism.

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage ...

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 ...

Markerless Gene Deletion by Floxed Cassette Allelic Exchange Mutagenesis in Chlamydia trachomatis

Chlamydia trachomatis is an obligate intracellular pathogen that has been historically difficult to genetically manipulate. Definitive progress in elucidating the mechanisms that C. trachomatis use to create and maintain a privileged intracellular niche has been limited due to a lack of genetic tools. Fortunately, there have recently been several new advances in genetic manipulation techniques. Among these is the development of fluorescence-reported allelic exchange mutagenesis (FRAEM). This method allows targeted gene deletion coupled with insertion of a selection cassette encoding antibiotic resistance and green fluorescent protein (GFP). Reliance on this strategy can be complicated when targeting genes within polycistronic operons due to the potential of polar effects on downstream genes. Floxed cassette allelic exchange mutagenesis (FLAEM), the protocol for which is described here, was developed to alleviate cassette-induced polar effects. FLAEM utilizes Cre-loxP genome editing to remove the selection cassette after targeted deletion by allelic exchange. The resulting strains contain markerless gene deletions of one or more coding sequences. This technique facilitates direct assessment of gene function and expands the repertoire of tools for genetic manipulation in C. trachomatis.

Markerless Gene Deletion by Floxed Cassette Allelic Exchange Mutagenesis in Chlamydia trachomatis

Described here is a method for targeted, markerless gene deletion in Chlamydia trachomatis using floxed cassette allelic exchange mutagenesis, FLAEM.