Chlamydiaphage φCPG1 Capsid Protein Vp1 Inhibits Chlamydia trachomatis Growth via the Mitogen-Activated Protein Kinase Pathway

Abstract

Chlamydia trachomatis is the most common cause of curable bacterial sexually transmitted infections worldwide. Although the pathogen is well established, the pathogenic mechanisms remain unclear. Given the current challenges of antibiotic resistance and blocked processes of vaccine development, the use of a specific chlamydiaphage may be a new treatment solution. φCPG1 is a lytic phage specific for Chlamydia caviae, and shows over 90% nucleotide sequence identity with other chlamydiaphages. Vp1 is the major capsid protein of φCPG1. Purified Vp1 was previously confirmed to inhibit Chlamydia trachomatis growth. We here report the first attempt at exploring the relationship between Vp1-treated C. trachomatis and the protein and gene levels of the mitogen-activated/extracellular regulated protein kinase (MAPK/ERK) pathway by Western blotting and real-time PCR, respectively. Moreover, we evaluated the levels of pro-inflammatory cytokines interleukin (IL)-8 and IL-1 by enzyme-linked immunosorbent assay after Vp1 treatment. After 48 h of incubation, the p-ERK level of the Vp1-treated group decreased compared with that of the Chlamydia infection group. Accordingly, ERK1 and ERK2 mRNA expression levels of the Vp1-treated group also decreased compared with the Chlamydia infection group. IL-8 and IL-1 levels were also decreased after Vp1 treatment compared with the untreated group. Our results demonstrate that the inhibition effect of the chlamydiaphage φCPG1 capsid protein Vp1 on C. trachomatis is associated with the MAPK pathway, and inhibits production of the pro-inflammatory cytokines IL-8 and IL-1. The bacteriophages may provide insight into a new signaling transduction mechanism to influence their hosts, in addition to bacteriolysis.

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

“…In our study Chlamydia trachomatis was pretreated and sufficiently combined with VP1 protein before inoculation, which was similar to the infection process of chlamydiaphages, and was then treated with the VP1 protein throughout the development cycle (Liu et al 2012 ). VP1 protein may play a similar but inferior role with chlamydiaphages by inhibiting the development of RBs, so that they would not become mature EBs (Guo et al 2016 ). The VP1 protein has obvious inhibition to the Ct at 3 × 10 5 IFU/ml treated with VP1 of the concentration 50 ug/ml.…”

“…The VP1 protein was expressed, purified, and quantified as described previously (Guo et al 2016 ). The quantified VP1 protein was aliquoted and stored in freezer for the following experiment.…”

“…After 48 h in an incubator at 37 °C, the liquid was discarded and the cells were allowed to dry naturally, after which they were fixed with methanol for 10 min at room temperature and iodine-stained. We counted the numbers of inclusions of every Ct, including D, E, F, G, H, I, K, and L2, obtaining an average of 30 fields of microscope’s view (Guo et al 2016 ). We also observed the inclusions of GPIC by fluorescent microscopy using polyclonal antibodies (anti-EB serum 1:1000) and secondary antibody of goat anti-rabbit-FITC (1:80) (Solarbio, China).…”

Capsid protein Vp1 from chlamydiaphage φCPG1 effectively alleviates cytotoxicity induced by Chlamydia�trachomatis

“…HeLa cells were seeded in a 24-well plate and after 24 h, the cells were pre-treated with Diethylaminoethyl-dextran (Merck KGaA) for 30 min to increase their susceptibility to C. trachomatis infection. C. trachomatis strain E (ATCC, Manassas, VA, USA) was subjected to two freeze-thaw cycles, followed by vortex at 3, 200 rpm for 1 min, room temperature.. Next, C. trachomatis cells were pre-incubated with 60 µg/ml purified Vp1 in PBS or 60 µg/ml bovine serum albumin (BSA; Beijing Solarbio Science and Technology Co., Ltd.) in PBS for 1 h. According to our previous study, 60 µg/ml was selected as the optimal concentration of Vp1 and BSA ( 15 ). Following Vp1 pretreatment, C. trachomatis cells were used to infect HeLa cells at a multiplicity of infection (MOI) of 1.…”

“…Vp1 capsid proteins of φCPG1 have been demonstrated to inhibit C. trachomatis growth and cause a decrease in the number of C. trachomatis inclusion bodies during infection ( 15 – 17 ). Vp1 has also been indicated to exert inhibitory effects on the proliferation of C. trachomatis in the mouse genital tract ( 16 ).…”