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Identification of Plasmid-Free Chlamydia muridarum Organisms Using a Pgp3 Detection-Based Immunofluorescence Assay
Chaoqun Chen1,2, Guangming Zhong1,2, Lin Ren1
1Departments of Medical Microbiology and Immunology, Hunan Province Key Laboratory for Special Pathogens Prevention and Control, University of South China School of Medicine, Hengyang, Hunan 421001, P.R. China.
Abstract:
Chlamydia possesses a conserved 7.5 kb plasmid that is known to play an important role in chlamydial pathogenesis, since some chlamydial organisms lacking the plasmid are attenuated. The chlamydial transformation system developed recently required the use of plasmid-free organisms. Thus, the generation and identification of plasmid-free organisms represent a key step in understanding chlamydial pathogenic mechanisms. A tricolor immunofluorescence assay for simultaneously detecting the plasmid-encoded Pgp3 and whole organisms plus DNA staining was used to screen C. muridarum organisms selected with novobiocin. PCR was used to detect the plasmid genes. Next-generation sequencing was then used to sequence the genomes of plasmid-free C. muridarum candidates and the parental C. muridarum Nigg strain. We generated five independent clones of plasmid-free C. muridarum organisms by using a combination of novobiocin treatment and screening plaque-purified clones with anti-Pgp3 antibody. The clones were confirmed to lack plasmid genes by PCR analysis. No GlgA protein or glycogen accumulation was detected in cells infected with the plasmid-free clones. More importantly, whole-genome sequencing characterization of the plasmid-free C. muridarum organism and the parental C. muridarum Nigg strain revealed no additional mutations other than loss of the plasmid in the plasmid-free C. muridarum organism. Thus, the Pgp3-based immunofluorescence assay has allowed us to identify authentic plasmid-free organisms that are useful for further investigating chlamydial pathogenic mechanisms.
Insights
Researchers developed a method to create and identify plasmid-free Chlamydia muridarum. This advancement is crucial for understanding chlamydial pathogenesis and developing new Chlamydia treatments.
Area of Science:
- Microbiology
- Pathogenesis Research
- Bacterial Genetics
Background:
- Chlamydia possesses a conserved 7.5 kb plasmid essential for pathogenesis; plasmid-free strains are attenuated.
- A functional chlamydial transformation system requires plasmid-free organisms.
- Generating plasmid-free strains is vital for studying chlamydial pathogenic mechanisms.
Purpose of the Study:
- To generate and identify authentic plasmid-free Chlamydia muridarum.
- To establish a reliable method for screening plasmid-free Chlamydia strains.
- To provide essential tools for investigating chlamydial pathogenesis.
Main Methods:
- Novobiocin treatment combined with plaque purification to select for potential plasmid-free strains.
- Tricolor immunofluorescence assay using anti-Pgp3 antibody to detect plasmid-encoded proteins.
- Polymerase Chain Reaction (PCR) to confirm the absence of plasmid genes.
- Whole-genome sequencing to ensure no additional mutations in plasmid-free strains.
Main Results:
- Five independent clones of plasmid-free C. muridarum were successfully generated.
- PCR analysis confirmed the absence of plasmid genes in the generated clones.
- Absence of GlgA protein and glycogen accumulation observed in plasmid-free C. muridarum infections.
- Whole-genome sequencing verified that plasmid-free strains only lacked the plasmid, with no other genomic alterations.
Conclusions:
- A Pgp3-based immunofluorescence assay is effective for identifying authentic plasmid-free Chlamydia organisms.
- The generated plasmid-free C. muridarum strains are suitable for further research into chlamydial pathogenesis.
- This methodology facilitates the study of plasmid's role in Chlamydia infection and disease.
