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).

<table><tbody><tr><td>Dulbecco&amp;rsquo;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 MgCl&lt;sub&gt;2&lt;/sub&gt; and 0.901 mM CaCl&lt;sub&gt;2&lt;/sub&gt; (PBS+MgCl&lt;sub&gt;2&lt;/sub&gt;/CaCl&lt;sub&gt;2&lt;/sub&gt;)</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>&gt;Centrifuge and adaptors for spinning tissue plates</td><td></td><td></td><td></td></tr><tr><td>&gt;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 Human Fallopian Tube Model for Investigation of C. trachomatis Infections

Genital tract infections with Chlamydia trachomatis (C. trachomatis) are the most frequent transmitted sexually disease in women worldwide. Inefficient clearance or persistence of the pathogens may lead to ascending infections of the upper genital tract and are supposed to cause chronic inflammatory damage to infected tissues 1,2. As a consequence, severe clinical sequelae like pelvic inflammatory disease (PID), tubal occlusion and infertility may occur 3,4. 
Most of the research with C. trachomatis has been conducted in epithelial cell lines (e.g. HEp-2 cells and HeLa-229) or in mice. However, as with cell- culture based models, they do neither reflect the physiology of native tissue nor the pathophysiology of C. trachomatis genital tract infections in vivo 5. Further limitations are given by the fact that central signaling cascades (e.g. IFN-&gamma; mediated JAK/STAT signaling pathway) that control intracellular chlamydial growth fundamentally differ between mice and humans 6,7. We and others therefore established a whole organ fallopian tube model to investigate direct interactions between C. trachomatis and human fallopian tube cells ex vivo 8,9.
For this purpose, human fallopian tubes from women undergoing hysterectomy were collected and infected with C. trachomatis serovar D. Within 24 h post infection, specimen where analyzed using scanning electron microscopy (SEM) and transmission electron microscopy (TEM) to detect Chlamydia trachomatis mediated epithelial damage as well as C. trachomatis inclusion formation in the fallopian tissue.

A Human Fallopian Tube Model for Investigation of C. trachomatis Infections

We describe an ex vivo infection model for visualisation of direct interactions from bacterial pathogens with human fallopian tube cells. The whole organ tissue model was established to investigate C. trachomatis induced pathology to the female fallopian tube under "life-like" conditions.

Genital tract infections with Chlamydia trachomatis (C. trachomatis) are the most frequent transmitted sexually disease in women worldwide. Inefficient ...

Medicine

Identification of Host Pathways Targeted by Bacterial Effector Proteins using Yeast Toxicity and Suppressor Screens

Intracellular bacteria secrete virulence factors called effector proteins into the host cytosol that act to subvert host proteins and/or their associated biological pathways to the benefit of the bacterium. Identification of putative bacterial effector proteins has become more manageable due to advances in bacterial genome sequencing and the advent of algorithms that allow in silico identification of genes encoding secretion candidates and/or eukaryotic-like domains. However, identification of these important virulence factors is only an initial step. Naturally, the goal is to determine the molecular function of effector proteins and elucidate how they interact with the host. In recent years, techniques like the yeast two-hybrid screen and large-scale immunoprecipitations coupled with mass spectrometry have aided in the identification of protein-protein interactions. Although identification of a host binding partner is the crucial first step toward elucidating the molecular function of a bacterial effector protein, sometimes the host protein is found to have multiple biological functions (e.g., actin, clathrin, tubulin), or the bacterial protein may not physically bind host proteins, depriving the researcher of crucial information about the precise host pathway being manipulated. A modified yeast toxicity screen coupled with a suppressor screen has been adapted to identify host pathways impacted by bacterial effector proteins. The toxicity screen relies on a toxic effect in yeast caused by the effector protein interfering with the host biological pathways, which often manifests as a growth defect. Expression of a yeast genomic library is used to identify host factors that suppress the toxicity of the bacterial effector protein and thus identify proteins in the pathway that the effector protein targets. This protocol contains detailed instructions for both the toxicity and suppressor screens. These techniques can be performed in any lab capable of molecular cloning and cultivation of yeast and Escherichia coli.

Bacterial pathogens secrete proteins into the host that target crucial biological processes. Identifying the host pathways targeted by bacterial effector proteins is key to addressing molecular pathogenesis. Here, a method using a modified yeast suppressor and toxicity screen to elucidate host pathways targeted by toxic bacterial effector proteins is described.

Intracellular bacteria secrete virulence factors called effector proteins into the host cytosol that act to subvert host proteins and/or their associated ...

Genetics

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).

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 ...

Application of Biolayer Interferometry (BLI) for Studying Protein-Protein Interactions in Transcription

A transcription factor&#160;(TF) is a protein that regulates gene expression by interacting with the RNA polymerase, another TF, and/or template DNA. GrgA is a novel transcription activator found specifically in the obligate intracellular bacterial pathogen Chlamydia. Protein pulldown assays using affinity beads have revealed that GrgA binds two &#963; factors, namely &#963;66 and &#963;28, which recognize different sets of promoters for genes whose products are differentially required at developmental stages. We have used BLI to confirm and further characterize the interactions. BLI demonstrates several advantages over pulldown: 1) It reveals real-time association and dissociation between binding partners, 2) It generates quantitative kinetic parameters, and 3) It can detect bindings that pulldown assays often fail to detect. These characteristics have enabled us to deduce the physiological roles of GrgA in gene expression regulation in Chlamydia, and possible detailed interaction mechanisms. We envision that this relatively affordable technology can be extremely useful for studying transcription and other biological processes.

Application of Biolayer Interferometry BLI for Studying Protein-Protein Interactions in Transcription

Interactions of transcription factors (TFs) with the RNA polymerase are usually studied using pulldown assays. We apply a Biolayer Interferometry (BLI) technology to characterize the interaction of GrgA with the chlamydial RNA polymerase. Compared to pulldown assays, BLI detects real-time association and dissociation, offers higher sensitivity, and is highly quantitative.

A transcription factor&#160;(TF) is a protein that regulates gene expression by interacting with the RNA polymerase, another TF, and/or template DNA. GrgA ...

Biochemistry

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 ...

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.

Chlamydia trachomatis is an obligate intracellular pathogen that has been historically difficult to genetically manipulate. Definitive progress in ...

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.

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.

The intracellular bacterial pathogen Chlamydia trachomatis undergoes a developmental cycle consisting of two morphologically discrete developmental forms. ...

Cell-Free Scaled Production and Adjuvant Addition to a Recombinant Major Outer Membrane Protein from Chlamydia muridarum for Vaccine Development

Subunit vaccines offer advantages over more traditional inactivated or attenuated whole-cell-derived vaccines in safety, stability, and standard manufacturing. To achieve an effective protein-based subunit vaccine, the protein antigen often needs to adopt a native-like conformation. This is particularly important for pathogen-surface antigens that are membrane-bound proteins. Cell-free methods have been successfully used to produce correctly folded functional membrane protein through the co-translation of nanolipoprotein particles (NLPs), commonly known as nanodiscs. 
This strategy can be used to produce subunit vaccines consisting of membrane proteins in a lipid-bound environment. However, cell-free protein production is often limited to small scale (&lt;1 mL). The amount of protein produced in small-scale production runs is usually sufficient for biochemical and biophysical studies. However, the cell-free process needs to be scaled up, optimized, and carefully tested to obtain enough protein for vaccine studies in animal models. Other processes involved in vaccine production, such as purification, adjuvant addition, and lyophilization, need to be optimized in parallel. This paper reports the development of a scaled-up protocol to express, purify, and formulate a membrane-bound protein subunit vaccine. 
Scaled-up cell-free reactions require optimization of plasmid concentrations and ratios when using multiple plasmid expression vectors, lipid selection, and adjuvant addition for high-level production of formulated nanolipoprotein particles. The method is demonstrated here with the expression of a chlamydial major outer membrane protein (MOMP) but may be widely applied to other membrane protein antigens. Antigen effectiveness can be evaluated in vivo through immunization studies to measure antibody production, as demonstrated here.

Cell-Free Scaled Production and Adjuvant Addition to a Recombinant Major Outer Membrane Protein from Chlamydia muridarum for Vaccine Development

This protocol describes using commercial, cell-free protein expression kits to produce membrane proteins supported in nanodisc that can be used as antigens in subunit vaccines.

Forward Genetic Approaches in Chlamydia trachomatis

Summary

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Chapters in this video

A Human Fallopian Tube Model for Investigation of C. trachomatis Infections

Identification of Host Pathways Targeted by Bacterial Effector Proteins using Yeast Toxicity and Suppressor Screens

Genetic Manipulation in Δku80 Strains for Functional Genomic Analysis of Toxoplasma gondii

Application of Biolayer Interferometry (BLI) for Studying Protein-Protein Interactions in Transcription

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

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

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

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

Live-Cell Forward Genetic Approach to Identify and Isolate Developmental Mutants in Chlamydia trachomatis

Cell-Free Scaled Production and Adjuvant Addition to a Recombinant Major Outer Membrane Protein from Chlamydia muridarum for Vaccine Development