Expression of Chlamydia muridarum plasmid genes and immunogenicity of pGP3 and pGP4 in different mouse strains.

Chlamydia muridarum carries a cryptic plasmid (pMoPn) of 7.5kb, which encodes seven genes. Our aims were to describe the transcriptional pattern of the pMoPn genes in C. muridarum-infected mice and to evaluate the host immune responses against pGP3 and pGP4 proteins. BALB/c and C57BL/6N female mice were inoculated intranasally with C. muridarum and sacrificed at different time points, and the total RNA was extracted from the lung suspensions to determine the levels of expression of the different plasmid genes by RT qPCR. The supernatants of the lungs were subjected to the quantitation of recoverable C. muridarum. TCA04 and TCA05, which encode pGP3 and pGP4, respectively, were amplified by PCR and cloned into the pET vector. The proteins were overexpressed in E. coli HB101 and purified. Selected groups of BALB/c and C57BL/6N mice were infected with C. muridarum 1-3 times. The humoral immune responses in the sera of the mice to the proteins encoded by TCA04 and TCA05 were tested by Western blotting, and the cellular immune responses were assessed in lymphocyte proliferation assays. The proteins recognized by the mouse sera were further analysed by a LC/MSMS technique. The kinetics of C. muridarum growth were similar in the mouse strains used, but the pathogen burden was higher in the BALB/c mice in the late phase of infection. All the plasmid genes in the BALB/c mice showed an increased level of expression on day 7, whereas the expression of the same genes did not change on day 7 in the C57BL/6N mice. The levels of expression of the plasmid genes were higher in the C57BL/6N mice at later time points. In Western blot assays, the sera of the singly infected C57BL/6N mice reacted with the monomeric form of pGP3, whereas the sera of the singly infected BALB/c mice reacted with the trimeric form of pGP3. The sera of the multiply infected C57BL/6N mice also recognized pGP4. Similarly to the humoral immune response, cellular immune responses to pGP3 and pGP4 were detected in the C. muridarum-infected C57BL/6N mice, but the spleen cells of BALB/c mice responded with proliferation only to the pGP3 protein. These results suggest that the proteins encoded by pMoPn genes may modulate the host immune response during C. muridarum infection, and that the evolved immune response against plasmid proteins, similarly to that against other chlamydial proteins, depends on the genetic background of the host.