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.
<...

<ol>
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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 border="0" cellspacing="0" cols="4">
	<colgroup width="291">
	</colgroup>
	<colgroup width="123">
	</colgroup>
	<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

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JoVE Journal

Immunology and Infection

12.7K visualizações.  Duke University Medical Center.  Descreve-se um método para realizar a análise genética baseada em Chlamydia mutagénese química e sequenciação do genoma inteiro. Além disso, um sistema de intercâmbio de ADN no interior das células infectadas está descrito que pode ser usado para o mapeamento genético. Este método pode ser largamente aplicável a sistemas microbianos sem sistemas de transformação e técnicas de genética molecular. 