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.
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 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–phenotype associations should be broadly applicable to the large list of medically and environmentally important microorganisms currently intractable to genetic analysis.
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 sequelæ such as pelvic inflammatory disease, ectopic pregnancies, and infertility (1). Chlamydia 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’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 based on chemical mutagenesis, WGS, and a system for DNA exchange within infected cells (14) (Figure 1).
1. Chemical Mutagenesis
Note: We found that the replicative RB form is more amenable to chemical mutagenesis than the EB form. At mid-cycle (between 18 to 20 hpi), RBs are at the greatest numbers prior to RB-EB transition. Because Chlamydia trachomatis is an obligate intracellular pathogen, the effects of the mutagen on the host health can limit bacterial recovery. Vero cells were found to be more resistant to the adverse effects of high levels of EMS than other cell lines tested.
Segregation of mutations by recombination requires selection for antibiotic resistant recombinant progeny. We recommend using drug resistant strains (e.g. rifampin, spectinomycin, or trimethoprim) for generating mutants for the ability to perform recombinant analysis and to isolate isogenic strains. Antibiotic resistant strains were generated by a stepwise selection process (15, 16).
Note: Chlamydia trachomatis (strain L2/434/Bu / ATCC VR902B) is BSL2 pathogen. Refer to institutional standard operating procedures (SOP) for handling such pathogens.
2. Clonal Isolation of Chlamydia trachomatis Mutant Strains by Plaque Purification
3. Whole Genome Sequencing of Selected Chlamydia Mutants
4. Generation of Chlamydia Recombinants
Note: Chlamydia can exchange DNA during infection, which allows for the generation of recombinant strains (12, 13, 20). After co-infection of cells with two unique antibiotic resistant strains, recombinant "progeny" can be selected for dual antibiotic resistance (12, 13). We took advantage of this phenomenon to segregate mutations and to generate co-isogenic strains. Hence, mutant strains were generated in antibiotic resistant background (e.g. rifampin resistance (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).
Table 1: Antibiotic concentrations for selection of recombinants:
Final Concentration | Preparation instructions |
---|---|
200 ng/ml rifampin | Make 25 mg/ml storage stock in dimethyl sulfoxide (DMSO). Make up 200 μg/ml working solution by diluting the storage stock with H2O. Store at -20 °C in the dark. |
200 μg/ml trimethoprim | Make a 100 mg/ml stock in DMSO. Store at -20 °C |
200 μg/ml spectinomycin | Make a 100 mg/ml stock in water in 100 μl aliquots. Store at -20 °C. Avoid repeated freeze thaw. |
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.
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 >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 plaques with altered morphologies, including small plaques (SPQ) (Figure 3). Other morphologies include plaques that appear granular, clumpy, or honeycomb shaped (Figure 3). These phenotypes may reflect defects in processes required for infection and survival within the host environment. Mutants with small plaque (SPQ) morphology generally produce significantly fewer infectious progeny and may take up to 2-3 weeks to amplify. Caution should be exercised when passaging these strains as reversions and suppressor mutations can accumulate at a high frequency.
The doses of EMS used in these studies can lead to between 3 to 30 mutations per Chlamydia genome, as assessed experimentally by WGS (Figure 4). Although gradient purified EBs are largely free of host cell material, host DNA is also detected and consists about 10-15% of genomic preps.
Co-infection of two strains can generate progeny with genetic contributions from both strains and occurs at frequencies of 10-4 to 10-3, approximately 104 times more frequently than spontaneous resistance (13) . Recombination break points typically occur between ~100 kb to 800 kb (12). An analysis of such recombinant strains can reveal mutations that are genetically linked to the phenotype under study.
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 "fitter" mutants can quickly lead to mutant strains being outcompeted. Similarly, mapping mutations by whole genome sequencing of non-clonal samples can lead to ambiguous results. Isolating mutants from heavily plaqued wells or plugging plaques that are too close to each other should be avoided. In addition, for slow growing mutants, revertants can emerge at relatively high frequencies. We recommend preserving original or low passage stocks. Replaque mutants if cross contamination or revertants are suspected.
The concentration of EMS can be lowered to decrease the frequency of mutations. The mutation rate, as assessed by the generation of rifampin resistant variants (due to point mutations in the gene encoding RNA polymerase, rpoB) was optimal in our hands at 20 mg/ml EMS. The induction of rifampin resistance can be assessed by the frequency of plaques formed in the presence of rifampin and should correlate to the frequency of chemically induced mutations.
EMS can be replaced with other mutagens to widen the spectrum of mutations that can be obtained. For instance, DNA intercalating agents like psoralen and its derivatives can be used to induce deletions and insertions (21).
Mutations that map close to each other in the genome (<100 kb apart) are difficult to unlink through recombination. When high mutagenesis rates are achieved, it may be difficult to unambiguously identify a causal mutation. However, since transformation of C. trachomatis is now possible with shuttle plasmids (22), albeit inefficiently, it may be possible to address these linkage problems by complementing mutations in trans with a wild type copy of the mutated gene on a plasmid.
Figure 1. Strategy for forward genetic analysis and recombination-based mapping in Chlamydia. Rifampin resistant (RifR) C. trachomatis was mutagenized during its replicative stage and used to infect Vero cell monolayers until visible plaques formed. Individual clones of mutants were collected and assayed for specific phenotypes, such as altered plaque morphotypes. The genomes of mutants sharing a common phenotype were sequenced to identify common genetic lesions. To establish linkage between these gene lesions and specific plaque morphologies, Vero cells were co-infected with mutants generated in aRifR background and wild-type SpcR Chlamydia strains, and recombinant RifR SpcR strains among the resulting infectious progeny were selected in the presence of rifampin and spectinomycin ("crosses"). The segregation of individual mutations present in the parental RifR mutant strain among the recombinant bacteria displaying altered plaque morphology was determined by targeted DNA sequencing. (Reproduced with permission from PNAS (14)) Click here to view larger image.
Figure 2. Schematic representation of EMS mutagenesis protocol. Chlamydia trachomatis LGV-L2-infected cells were exposed to EMS during the RB stage of the infectious cycle, and the infection was allowed to proceed for 72 h to allow for the generation of infectious elementary bodies (EB). Mutagenized EB pools were harvested and titered for inclusion forming units (IFU) and plaque-forming units on Vero cells. N, nucleus. (Reproduced with permission from PNAS (14)) Click here to view larger image.
Figure 3. Examples of common plaque morphologies among EMS-mutagenized C. trachomatis. Mutagenized C. trachomatis LGV-L2 allowed to form plaques on Vero cell monolayers for 14 day. Plaques varied in size (A) and morphology (B), which can be isolated, amplified in Vero cells, and used in reinfections of Vero monolayers to confirm the stability of the plaque morphotypes. Examples of common phenotypes are shown including honeycomb (Hcm), clumped (Clmp), small plaque (Spq), and granular (Grn). Arrows indicate large granular deposits within a Grn plaque. (Reproduced with permission from PNAS (14)) Click here to view larger image.
Figure 4. Identification of EMS-induced gene lesions. Chromosomal location of nucleotide variants in mutants that display the Grn plaque morphotype. Whole-genome sequencing of three Grn mutants identified mutations that lead to amino acid changes in glgB, coding for a glycogen-branching enzyme. (Reproduced with permission from PNAS (14)) Click here to view larger image.
The authors have nothing to disclose.
Dulbecco’s Modified Eagle Medium (DMEM) | Life Technologies | 11995-073 | |
Fetal Bovine Serum (FBS) | Cellgro | 35-010-CV | |
Ethyl methanesulfonate (EMS) | Sigma | M0880 | |
Cyclohexamide | Sigma | C4859-1ML | |
Gentamicin | Life Technologies | 15750-060 | |
Phosphate buffered saline (PBS) | Life Technologies | 14190-144 | |
Phosphate buffered saline (PBS) solution with 0.493 mM MgCl2 and 0.901 mM CaCl2 (PBS+MgCl2/CaCl2) | Life Technologies | 14040-133 | |
1 M NaOH | |||
5x SPG buffer (1.25 M sucrose, 50 mM sodium phosphate, 25 mM glutamic acid) | |||
SPG buffer (0.25 M sucrose, 10 mM sodium phosphate, 5 mM glutamic acid) | |||
Water (sterile, tissue culture grade) | |||
2x DMEM (prepared from powder, buffered with 7.4 g/L Sodium Bicarbonate | Sigma | D7777 | |
Nonessential amino acids (NEAA) | Life Technologies | 11140-050 | |
1.2% GTG Agarose , autoclaved | Lonzo | 50070 | |
Genomic DNA purification kits | Qiagen | 69504 | |
DNAzol | Life Technologies | 10503-027 | |
Ethanol (molecular biology grade) | |||
8 mM NaOH | |||
0.1 M HEPES ( 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer | |||
25 cm2 tissue culture flasks (T25 flasks) | |||
6-well tissue culture plates | |||
12-well tissue culture plates | |||
96-well tissue culture plates | |||
Chemical safety hood | |||
Biological safety hood | |||
>Centrifuge and adaptors for spinning tissue plates | |||
>Dissection microscope | |||
Fluorometer (Qubit) | Invitrogen | Q32866 | |
Adaptive Focused Acoustics S220 instrument | Covaris |