title: Cyclic di-GMP signaling regulates invasion by Ehrlichia chaffeensis of human monocytes.
abstract: Cyclic di-GMP (c-di-GMP) is a bacterial second messenger produced by GGDEF domain-containing proteins. The genome of Ehrlichia chaffeensis, an obligatory intracellular bacterium that causes human monocytic ehrlichiosis, encodes a single protein that contains a GGDEF domain, called PleD. In this study, we investigated the effects of c-di-GMP signaling on E. chaffeensis infection of the human monocytic cell line THP-1. Recombinant E. chaffeensis PleD showed diguanylate cyclase activity as it generated c-di-GMP in vitro. Because c-di-GMP is not cell permeable, the c-di-GMP hydrophobic analog 2'-O-di(tert-butyldimethylsilyl)-c-di-GMP (CDGA) was used to examine intracellular c-di-GMP signaling. CDGA activity was first tested with Salmonella enterica serovar Typhimurium. CDGA inhibited well-defined c-di-GMP-regulated phenomena, including cellulose synthesis, clumping, and upregulation of csgD and adrA mRNA, indicating that CDGA acts as an antagonist in c-di-GMP signaling. [(32)P]c-di-GMP bound several E. chaffeensis native proteins and two E. chaffeensis recombinant I-site proteins, and this binding was blocked by CDGA. Although pretreatment of E. chaffeensis with CDGA did not reduce bacterial binding to THP-1 cells, bacterial internalization was reduced. CDGA facilitated protease-dependent degradation of particular, but not all, bacterial surface-exposed proteins, including TRP120, which is associated with bacterial internalization. Indeed, the serine protease HtrA was detected on the surface of E. chaffeensis, and TRP120 was degraded by treatment of E. chaffeensis with recombinant E. chaffeensis HtrA. Furthermore, anti-HtrA inhibited CDGA-induced TRP120 degradation. Our results suggest that E. chaffeensis invasion is regulated by c-di-GMP signaling, which stabilizes some bacterial surface-exposed proteins against proteases.
authors.affiliation: Department of Veterinary Biosciences, The Ohio State University, Columbus, OH 43210, USA.
authors.forename: Yumi
authors.initials: Y
authors.lastname: Kumagai
authors.forename: Junji
authors.initials: J
authors.lastname: Matsuo
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Hayakawa
authors.forename: Yasuko
authors.initials: Y
authors.lastname: Rikihisa
mesh_terms: D001422:Bacterial Adhesion; D001426:Bacterial Proteins / Q000378:metabolism; D002460:Cell Line; D002482:Cellulose / Q000378:metabolism; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000378:metabolism; D018469:Ehrlichia chaffeensis / Q000472:pathogenicity*; D029968:Escherichia coli Proteins; D020869:Gene Expression Profiling; D015964:Gene Expression Regulation, Bacterial; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D009000:Monocytes / Q000382:microbiology*; D019761:Phosphorus-Oxygen Lyases / Q000378:metabolism; D012329:RNA, Bacterial / Q000096:biosynthesis; D012333:RNA, Messenger / Q000096:biosynthesis; D015398:Signal Transduction; D015534:Trans-Activators / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D012329:RNA, Bacterial; D012333:RNA, Messenger; D015534:Trans-Activators; C062025:bis(3',5')-cyclic diguanylic acid; D002482:Cellulose; D019761:Phosphorus-Oxygen Lyases; C060720:diguanylate cyclase; D006152:Cyclic GMP