title: Identification of Plasmid-Free Chlamydia muridarum Organisms Using a Pgp3 Detection-Based Immunofluorescence Assay.
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.
authors.affiliation: Departments 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.|Department of Microbiology and Immunology, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA.
authors.forename: Chaoqun
authors.initials: C
authors.lastname: Chen
authors.affiliation: Departments 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.|Department of Microbiology and Immunology, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA.
authors.forename: Guangming
authors.initials: G
authors.lastname: Zhong
authors.affiliation: Departments 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.
authors.forename: Lin
authors.initials: L
authors.lastname: Ren
authors.affiliation: Departments 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.
authors.forename: Chunxue
authors.initials: C
authors.lastname: Lu
authors.affiliation: Departments 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.
authors.forename: Zhonggyu
authors.initials: Z
authors.lastname: Li
authors.affiliation: Departments 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.
authors.forename: Yimou
authors.initials: Y
authors.lastname: Wu
mesh_terms: D000942:Antigens, Bacterial / Q000032:analysis*; D001426:Bacterial Proteins / Q000032:analysis*; D023502:Chlamydia muridarum / Q000235:genetics*; D005455:Fluorescent Antibody Technique / Q000379:methods*; D005820:Genetic Testing / Q000379:methods*; D059014:High-Throughput Nucleotide Sequencing; D010957:Plasmids / Q000032:analysis*; D016133:Polymerase Chain Reaction; D017384:Sequence Deletion
chemical_list: D000942:Antigens, Bacterial; D001426:Bacterial Proteins; C082169:pgp3 protein, Chlamydia
keywords: Chlamydia muridarum; Immunofluorescence; Pgp3 detection; plasmid depletion