Identification Of Cell Invasion-related Virulence Genes In Chlamydia Plasmid In Vitro

Objective: Culture, amplification, purification of Chlamydia trachomatis(Ct) mouse pneumonitis(Mopn) strains in vitro. To compare the infectivity of several transformed(Mopn) strains to host cells, and to identify cell invasion-related virulence genes in Chlamydial plasmids, Compare the influence of different experimental conditions on the adhesion ability of invasion-related virulence genes, To identify which genes play a role in chlamydial cell to cell spreading and glycogen accumulation in inclusion in vitro.Methods: He La cells was choosed as the host cells, Strains Ct strains, including wild-type Ct Mopn strain(WT strain), plasmid-free Ct strain(CMUT3 strain), Ct Mopn strain transformed with the shuttle vector carry p GFP and the complete C.muridarum(CM) plasmid(p GFP::CM strain), Ct Mopn strains transformed with shuttle vectors carrying p GFP and mutant CM plasmids with single in-frame deletions of Pgp3, 4, 5 or 7(p GFP::CM△Pgp3, 4, 5, 7 strains), were cultured, amplified, collected,centrifuge, purify the Ct elementary bodies by discontinuous renograffin gradient. The existence and concentration of Chlamydia trachomatis(Ct) was identified and calculated by indirect immunofluorescence. After the concentrations of Ct were determined, each of these strains was divided into four groups to be inoculated at a same amount(1.5×104 inclusion forming units, IFU) followed by four different treatments respectively: centrifugalization+DEAE-D group treated with centrifugalizati- on followed by ion-exchange chromatography on diethylaminoethyl(DEAE-D)-cellulose columns, centrifugalization group treated with centrifugalization only, DEAE-D group treated with chromatography on DEAE-D-cellulose columns only, control group receiving no treatment. After additional culture for 20 — 24 hours, indirect immunofluorescence assay was performed to count the number of chlamydial inclusions, Cells were fixed at different times(20h,40 h,60h) separately after infection and processed by IFA staining, comparing the forming speed and size of plague from the first generations to the third generations in different transformed strains. Observe glycogen accumulation in inclusion by Logus′s iodine dying.Results: A higr titer Chlamydia trachomatis EB(elementary body) stock was obstained. The inclusion number in the centrifugalization + DEAE-D group, centrifugalization group, DEAE-D group and control group was 10.20 ± 1.30, 6.80 ± 0.44, 3.00 ± 1.22 and 0.80 ± 0.45 respectively for the p GFP::CM△Pgp4 strain, 6.40 ± 0.89, 3.80 ± 0.83, 1.60 ± 0.89 and 0.60 ± 0.54 respectively for the CMUT3 strain. Under same experiment conditions, the p GFP::CM△Pgp4 strain and CMUT3 strain showed similar infectivity, and formed less inclusions compared with the other Ct strains(all P < 0.01). The number of inclusions formed by the same Ct strains were significantly different among the 4 groups(F = 845.310, P < 0.01), and were highest in the centrifugalization + DEAE-D group for all the strains. The p GFP::CM△Pgp4 strain showed significantly lower growth rate and area of plaques with an abnormality in glycogen accumulation compared with the other strains at 20, 40 and 60 hours after infection.Conclusions: This method can remove the influence of cellular constituent to the result of the experiment,And can ensure the reliability of the results compared with previous routine methods.The plasmid-encoding gene Pgp4 may be a cell invasion-associated virulence gene in chlamydial plasmids, which decrease glycogen accumulation and has an effect on Chlamydia between host cells and cells spread and growth ability in vitro.Centrifugalization and DEAE-D can affect the ability to adhesive and invasive host cells of the virulence genes in vitro.