Interrogating Genes That Mediate Chlamydia trachomatis Survival in Cell Culture Using Conditional Mutants and Recombination

Abstract

Intracellular bacterial pathogens in the family Chlamydiaceae are causes of human blindness, sexually transmitted disease, and pneumonia. Genetic dissection of the mechanisms of chlamydial pathogenicity has been hindered by multiple limitations, including the inability to inactivate genes that would prevent the production of elementary bodies. Many genes are also Chlamydia-specific genes, and chlamydial genomes have undergone extensive reductive evolution, so functions often cannot be inferred from homologs in other organisms. Conditional mutants have been used to study essential genes of many microorganisms, so we screened a library of 4,184 ethyl methanesulfonate-mutagenized Chlamydia trachomatis isolates for temperature-sensitive (TS) mutants that developed normally at physiological temperature (37°C) but not at nonphysiological temperatures. Heat-sensitive TS mutants were identified at a high frequency, while cold-sensitive mutants were less common. Twelve TS mutants were mapped using a novel markerless recombination approach, PCR, and genome sequencing. TS alleles of genes that play essential roles in other bacteria and chlamydia-specific open reading frames (ORFs) of unknown function were identified. Temperatureshift assays determined that phenotypes of the mutants manifested at distinct points in the developmental cycle. Genome sequencing of a larger population of TS mutants also revealed that the screen had not reached saturation. In summary, we describe the first approach for studying essential chlamydial genes and broadly applicable strategies for genetic mapping in Chlamydia spp. and mutants that both define checkpoints and provide insights into the biology of the chlamydial developmental cycle. IMPORTANCEStudy of the pathogenesis of Chlamydia spp. has historically been hampered by a lack of genetic tools. Although there has been recent progress in chlamydial genetics, the existing approaches have limitations for the study of the genes that mediate growth of these organisms in cell culture. We used a genetic screen to identify conditional Chlamydia mutants and then mapped these alleles using a broadly applicable recombination strategy. Phenotypes of the mutants provide fundamental insights into unexplored areas of chlamydial pathogenesis and intracellular biology. Finally, the reagents and approaches we describe are powerful resources for the investigation of these organisms. Chlamydiae are obligate intracellular pathogens that share a characteristic biphasic developmental cycle where these organisms alternate between extracellular, infectious elementary body (EB) and intracellular, replicative reticulate body (RB) forms (1). Multiple species of the family Chlamydiaceae, including Chlamydia trachomatis, C. pneumoniae, and C. psittaci, are common or incidental pathogens of humans (2). Despite their medical importance, a lack of tools for genetic manipulation of these organisms was a major impediment in chlamydial research until recently (3, 4).Only ϳ60% of Chlamydiaceae genes share significant ...

Beyond Tryptophan Synthase: Identification of Genes That Contribute to Chlamydia trachomatis Survival during Gamma Interferon-Induced Persistence and Reactivation

“…C. trachomatis L2-GFP (L2-green fluorescent protein) was created by transforming C. trachomatis L2 434/Bu (C. trachomatis) with pGFP::SW2 (a kind gift from Ian Clarke, University of Southampton, United Kingdom) (40). Mutant screens were performed using a library of plaque-cloned ethane methylsulfonate (EMS)-mutagenized L2-GFP isolates (41).…”

“…This technique produces markerless recombinants that have swapped the detrimental mutant allele for a wild-type copy (41). HeLa cells were coinfected with different pairs of the Sip mutants, and then coinfections were repeatedly passaged in HeLa cells cultivated under the persistence screen conditions.…”

Chlamydia trachomatis Is Resistant to Inclusion Ubiquitination and Associated Host Defense in Gamma Interferon-Primed Human Epithelial Cells

“…To understand how different Chlamydia species escape from inclusion ubiquitination in their respective hosts, we will need to identify the chlamydial factors that enable evasion of inclusion ubiquitination. Forward genetic screens using recently developed Chlamydia mutant libraries (41–43) provide one possible avenue to achieve this goal.…”

Engineering of obligate intracellular bacteria: progress, challenges and paradigms

“…To date, only Chlamydia abortus (formerly C. psittaci var. ovis ) and C. trachomatis have been successfully mutagenized using DNA-alkylating agents to generate libraries 8,45–47 . Chemical mutagenesis does not require bacterial transformation, and obligate intracellular bacteria do not need to be purified from the host cell before application of the mutagen.…”

“…Repeated rounds of co-infection, phenotypic screening and genotyping are carried out until a single mutation can be linked to a phenotype. Other strategies involve using temperature-sensitive mutants to carry out genetic mapping, which does not require the generation of antibiotic- resistant strains of C. trachomatis 47 . Libraries of mutant C. trachomatis strains obtained through ethyl methanesulfonate mutagenesis (EMS mutagenesis) have been screened using various approaches, including temperature, forward genetics and reverse genetics 46–48 .…”