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Forward Genetic Approaches in Chlamydia trachomatis
Published on: October 23, 2013
In vitro passage selects for Chlamydia muridarum with enhanced infectivity in cultured cells but attenuated
Chaoqun Chen1, Zhou Zhou1, Turner Conrad2
1Department of Microbiology and Immunology, University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA Departments of Microbiology and Immunology, Institute of Pathobiology, University of South China, Hengyang, Hunan, People's Republic of China.
Abstract:
Although modern Chlamydia muridarum has been passaged for decades, there are no reports on the consequences of serial passage with strong selection pressure on its fitness. In order to explore the potential for Pasteurian selection to induce genomic and phenotypic perturbations to C. muridarum, a starter population was passaged in cultured cells for 28 generations without standard infection assistance. The resultant population, designated CMG28, displays markedly reduced in vitro dependence on centrifugation for infection and low incidence and severity of upper genital tract pathology following intravaginal inoculation into mice compared to the parental C. muridarum population, CMG0. Deep sequencing of CMG0 and CMG28 revealed novel protein variants in the hypothetical genes TC0237 (Q117E) and TC0668 (G322R). In vitro attachment assays of isogenic plaque clone pairs with mutations in either TC0237 and TC0668 or only TC0237 reveal that TC0237(Q117E) is solely responsible for enhanced adherence to host cells. Paradoxically, double mutants, but not TC0237(Q117E) single mutants, display severely attenuated in vivo pathogenicity. These findings implicate TC0237 and TC0668 as novel genetic factors involved in chlamydial attachment and pathogenicity, respectively, and show that serial passage under selection pressure remains an effective tool for studying Chlamydia pathogenicity.
Insights
Serial passage of Chlamydia muridarum under selection pressure created a new strain (CMG28) with enhanced host cell adherence and reduced pathogenicity. Novel variants in TC0237 and TC0668 genes were identified.
Area of Science:
- Microbiology
- Genomics
- Pathogenesis
Background:
- Modern Chlamydia muridarum has undergone decades of passage, but the effects of strong selection pressure on its fitness are unknown.
- Pasteurian selection may induce genomic and phenotypic changes in Chlamydia muridarum.
Purpose of the Study:
- To explore the effects of serial passage under strong selection pressure on Chlamydia muridarum fitness and pathogenicity.
- To identify genomic and phenotypic alterations induced by serial passage.
Main Methods:
- Serial passage of Chlamydia muridarum (CMG0) in cultured cells for 28 generations without standard infection assistance to create CMG28.
- Deep sequencing of CMG0 and CMG28 to identify genetic variations.
- In vitro attachment assays using isogenic plaque clone pairs with mutations in TC0237 and TC0668.
- Intravaginal inoculation of mice to assess in vivo pathogenicity.
Main Results:
- The passaged population (CMG28) showed reduced in vitro dependence on centrifugation for infection.
- CMG28 exhibited lower incidence and severity of upper genital tract pathology in mice compared to CMG0.
- Deep sequencing revealed novel protein variants in hypothetical genes TC0237 (Q117E) and TC0668 (G322R).
- TC0237(Q117E) was solely responsible for enhanced adherence to host cells in vitro.
- Double mutants (TC0237 and TC0668) showed severely attenuated in vivo pathogenicity, unlike TC0237(Q117E) single mutants.
Conclusions:
- TC0237 and TC0668 are novel genetic factors involved in chlamydial attachment and pathogenicity, respectively.
- Serial passage under selection pressure is an effective method for studying Chlamydia pathogenicity.
- Specific genetic variants can decouple host cell adherence from in vivo pathogenicity.
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