Visualization of IFN-β mRNA in Chlamydia-infected and adjacent uninfected cells and the contribution of CX43 in intercellular transfer of cGAMP

Abstract

Background IFNβ has been implicated a key effector of oviduct pathology after genital chlamydial infection in mice. We reported that the host DNA sensor cGAS (cGAMP synthase) is essential for IFNβ expression during Chlamydia trachomatis infection. Sensing during infection leads to generation of the second messenger, 2′3-cGAMP, which can migrate to uninfected adjacent cells via gap-junction mediated transfer. cGAMP binding to central adaptor molecule, STING drives IFNβ induction. The goals of this study were to visualize the mechanism of c-GAMP transfer and to identify the DNA source triggering this response. Methods cGAMP production was detected by mass spectrometry. The contribution of gap junction proteins, connexins CX43 and CX45 in intercellular transfer of cGAMP was assayed by siRNA depletion and subsequent co-culture experiments. view RNATM methodology was used to detect IFNβ mRNA transcripts in Hela cells. Mitochondrial DNA as IFNβ inducer was assessed in mtDNA depleted cell lines. Results Mass spectroscopy confirmed cGAMP in infected host cytosol. Connexin depletion studies revealed that CX43 but not CX45 was essential for optimal IFNβ expression during infection. IFNβ mRNA transcripts were visualized in infected and adjacent, uninfected cells. In contrast, distal uninfected cells lacking cell-cell contact were negative. IFNβ induction after chlamydia infection was unchanged in mtDNA depleted cells. Conclusion Overall, our results confirm the production of cGAMP in chlamydia-infected cells and its transfer via CX43 to adjacent cells resulting in IFNβ production. Our studies exclude mtDNA as a potential source initiating this response. We are currently investigating the role of Chlamydial-DNA as an initiator.