Generation And Characterization Of Plasmid-free Chlamydia Muridarum And The Effect Of In Vitro Passage On Biological Characteristics And Pathogenicity Of C. Muridarum

Chlamydiae are obligately intracellular prokaryotic parasites that cause a spectrum of diseases in humans and animals. They are characterized by a unique biphasic developmental cycle consisting of an infectious, extracellular metabolically inactive elementary body（EB）, and an intracellular replicative, non-infectious reticulate body（RB）. Human pathogens include Chlamydia trachomatis, C. pneumonia and C. psittaci. Chlamydial infection is a public health concern worldwide, which has a great influence on health and economy.To effectively meet the challenges of chlamydial infections to public health, strengthening basic research on chlamydiae, in-depth study of the pathogenic mechanisms of chlamydia-induced diseases, developing chlamydial vaccine is an important issue. Chlamydia muridarum, a mouse pathogen, formerly known as C. trachomatis mouse pneumonitis biovar（designated Mo Pn）, causes no known human diseases, has been widely used to study the pathogenesis and immunity of chlamydial disease. Genital tract infection of mice with C. muridarum can cause hydrosalpinx that closely mimics the tubal pathology induced by C. trachomatis in humans.Part 1 Generation and characterization of plasmid-free Chlamydia muridarumBackground and Objective: 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 highly attenuated. The chlamydial plasmid-mediated transformation system developed recently required the use of plasmid-free organisms. Thus, generation and identification of plasmid-free organisms represent a key step in understanding chlamydial pathogenic mechanisms.Methods: A tricolor immunofluorescence assay for simultaneously detecting the plasmid encoded Pgp3（red） and whole organisms（green） plus DNA staining（blue） was used to screen C. muridarum organisms selected with novobiocin. PCR was used to detect the plasmid genes. He La cells infected with C. muridarum organisms were used for iodine staining of glycogen accumulation. Female C3H/He J mice were intravaginally infected with plasmid-competen or plasmid-free C. muridarum organisms to evaluate the mouse genital tract pathology.Results: In a single screening assay carried out in a 96-well microplate format, we identified 5 plasmid-free C. muridarum clones designated as CMUT1-3, 9 & 15. These clones were confirmed to lack plasmid genes by PCR analysis. No Glg A protein or glycogen accumulation was detected in cells infected with the plasmid-free clones. More importantly, a representative clone CMUT3 failed to induce hydrosalpinx in mice. Pgp3 is a conserved and abundant plasmid protein and the mouse anti-Pgp3 antiserum raised with C. muridarum Pgp3 recognized Pgp3 expressed by all strains/serovars of other chlamydial species evaluated.Conclusion: 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 approach.Part 2 The effect of in vitro passage on biological characteristics and pathogenicity of C. muridarumBackground and Objective: Although modern C. muridarum has been passaged for decades, there are no reports on the consequences of serial passage with strong selection pressure on its fitness. Recently, we passaged the wild type and plasmid-free C. muridarum organisms for multiple generations under the unassisted-infection conditions and assisted-infection conditions using DEAE-dextran pre-treatment plus centrifugation treatment, then we evaluated the infectivity of different generation organisms in cell culture. We found that with the passage increases, it showed that the passaged wild type CMG28 and plasmid-free C. muridaum CMUT3G40 organisms became less dependent on centrifugation for infecting He La cells. We developed C3H/He J mice intravaginally infection wild type C. muridarum（CMG0） and CMG28, the mice intravaginally infection CMG28 induced less pathology in the oviduct compared to CMG0. Thus, there is a big discrepancy between in vitro invasiveness and in vivo pathogenicity.Methods: In vitro serially passage plasmid-free and wild type C. muridarum for multiple generations, then extract the genomic DNA of passaged C. muridarum and parental C. muridarum. Next-generation sequencing technology was then used to sequence the genomes of plasmid-free C. muridarum CMUT3, passaged CMUT3, passaged C. muridarum and the parental C. muridarum Nigg strain, the resulting genomic data were analyzed comparatively. Female C3H/He J mice were intravaginally infected with parental and passaged C. muridarum organisms to evaluate the mouse genital tract pathology.Results: Deep sequencing of CMG0 and CMG28 genomes revealed that mutations accumulated in three open reading frames（ORFs） during passaging: a Q117 E substitution in TC0237（0% in CMG0 versus 100% in CMG28 genomes）, a G216* nonsense（0% versus 6.4%） and a G322 R substitution（0% versus 26%） in TC0668, and multiple preexisting lesions in TC0412 resulting in nonsense and frameshift polymorphisms. Despite the enhanced attachment of CMG28 to the cultured cells, C3H/He J mice infected with CMG28 developed significantly reduced levels of hydrosalpinx. However, CMG0 and CMG28 both developed similar levels of live organism shedding from mouse lower genital tract and ascending infection. Nevertheless, a significant reduction in inflammatory cytokines was detected in the oviduct tissue of mice infected with CMG28, which correlated with a significant decrease in oviduct inflammatory cell infiltration. Since TC0237（Q117E） is the only mutation shared by CMG28 and CMUT3G40 organisms, this mutation is likely responsible for the in vitro attachment enhancement phenotype. These paradoxical observations have demonstrated that the mutations in TC0237, TC₀668, and TC₀412, although resulting in the enhancement of attachment to cultured cells, can significantly attenuate pathogenicity in the mouse upper genital tract by reducing inflammatory stimulation.Conclusion: The mutation in TC0237 can be attributed solely to an in vitro attachment enhancement phenotype, while TC0237 and TC0668 double mutants display severely attenuated in vivo pathogenicity.