Identification of proteins differentially expressed by Chlamydia trachomatis treated with chlamydiaphage capsid protein VP1 during intracellular growth

Abstract

Chlamydia trachomatis infection is one of the most prevalent sexually transmitted diseases. Our research pertains to the inhibitory effect and molecular mechanism of the chlamydiaphage capsid protein VP1 on the growth of Chlamydia trachomatis. In this research, the capsid protein VP1 of the guinea-pig conjunctivitis chlamydiaphage phiCPG1 was expressed, purified and identified, and then, it was applied to the cultivation of different serovars of Chlamydia trachomatis and Chlamydia psittaci. The inhibitory effect was observed in each serovar of Chlamydia trachomatis (D, E, F, G, H, I, K, and L2) and Chlamydia psittaci inoculated with VP1 protein. The inhibition affection of VP1 on the growth of Chlamydia trachomatis was caused by the changes of expressions of some related proteins including 36 proteins up-regulated and 81 proteins down-regulated in the development cycle of Ct through the label-free test, and the transcription levels of these proteins, including Hc1, pmpD, and MOMP, were confirmed by RT-PCR. It provides information that is essential for understanding the mechanism of chlamydiaphage capsid protein VP1 on chlamydia and a new direction for further clinical treatment of chlamydial infection.

Keywords: Chlamydia trachomatis; Chlamydiaphages; Inhibition; VP1.

Fig. 1

Inhibition effect of VP1 on the growth of different Ct strains and GPIC. (1) Different Ct strains (D, E, F, G, H, I, K, L2) or GPIC strain was preincubated with PBS (normal group), BSA in PBS (control group), or purified VP1 in PBS (VP1 group), and then, these mixtures were inoculated into the HeLa cells with PBS (D1, E1, F1, G1, H1, I1, K1, L1, 1), BSA in PBS (D2, E2, F2, G2, H2, I2, K2, L2, 2), or purified VP1 in PBS (D3, E3, F3, G3, H3, I3, K3, L3, 3). After 48 h, HeLa cells with the different Ct strains were stained with iodine and those with GPIC were used for double immunofluorescence labeling (1–3) for chlamydial organisms (green) and DNA (blue). Note that VP1 has an obviously inhibitive effect on the growth of Chlamydia trachomatis and GPIC. (2) Bar graph shows the obvious inhibition effects of VP1 on different Ct strains from D to L2 and GPIC (color figure online)

Fig. 1

Inhibition effect of VP1 on the growth of different Ct strains and GPIC. (1) Different Ct strains (D, E, F, G, H, I, K, L2) or GPIC strain was preincubated with PBS (normal group), BSA in PBS (control group), or purified VP1 in PBS (VP1 group), and then, these mixtures were inoculated into the HeLa cells with PBS (D1, E1, F1, G1, H1, I1, K1, L1, 1), BSA in PBS (D2, E2, F2, G2, H2, I2, K2, L2, 2), or purified VP1 in PBS (D3, E3, F3, G3, H3, I3, K3, L3, 3). After 48 h, HeLa cells with the different Ct strains were stained with iodine and those with GPIC were used for double immunofluorescence labeling (1–3) for chlamydial organisms (green) and DNA (blue). Note that VP1 has an obviously inhibitive effect on the growth of Chlamydia trachomatis and GPIC. (2) Bar graph shows the obvious inhibition effects of VP1 on different Ct strains from D to L2 and GPIC (color figure online)

Fig. 2

Inhibition effect of VP1 with different concentrations on Chlamydia trachomatis serovar E. The Ct serovar E was treated with VP1 from 50 ug/mL (a, g), 40 ug/mL (b, h), 30 ug/mL (c, i), 20 ug/mL (d, j), 10 ug/mL (e, k) to 0 ug/mL (f, l) following the method described in the Fig. 1; after culturing for 48 h, the iodine staining (a–f) and immunofluorescent staining (g–l) were used to observe the result. With the reduction of VP1 concentration, the number of inclusions increased significantly

Fig. 3

Real-time polymerase chain reaction analysis of transcription level of investigated genes (Hc1, MOMP, and pmpD). Transcription levels were measured by real-time PCR during the developmental cycle of C. trachomatis treated with or without 10 ug/mL VP1 at 0, 12, 24, 36, and 48 h after infection. Transcription level of the tufA gene encoding EF-Tu was used for comparison. The 2^−ΔΔCt of the transcription level of pmpD > 1, indicating that the transcription of pmpD was increased at different timepoints after VP1 treatment, whereas 2^−ΔΔCt of the transcription level of MOMP or Hc1 < 1, indicating that transcription of these two genes was decreased. The changes of the transcription level of pmpD, MOMP, or Hc1 were consistent with the changes of the protein level detected by label-free quantitative test