Forward Genetic Approaches in Chlamydia trachomatis

Summary

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.

Abstract

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.

Introduction

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 inclus....

Protocol

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 d....

Results

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 plaque.......

Discussion

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".......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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