title: Cyclic dimeric GMP signaling regulates intracellular aggregation, sessility, and growth of Ehrlichia chaffeensis.
abstract: Cyclic dimeric GMP (c-di-GMP), a bacterial second messenger, is known to regulate bacterial biofilm and sessility. Replication of an obligatory intracellular pathogen, Ehrlichia chaffeensis, is characterized by formation of bacterial aggregates called morulae inside membrane-bound inclusions. When E. chaffeensis matures into an infectious form, morulae become loose to allow bacteria to exit from host cells to infect adjacent cells. E. chaffeensis expresses a sensor kinase, PleC, and a cognate response regulator, PleD, which can produce c-di-GMP. A hydrophobic c-di-GMP antagonist, 2'-O-di(tert-butyldimethysilyl)-c-di-GMP (CDGA) inhibits E. chaffeensis internalization into host cells by facilitating degradation of some bacterial surface proteins via endogenous serine proteases. In the present study, we found that PleC and PleD were upregulated synchronously during exponential growth of bacteria, concomitant with increased morula size. While CDGA did not affect host cells, when infected cells were treated with CDGA, bacterial proliferation was inhibited, morulae became less compact, and the intracellular movement of bacteria was enhanced. Concurrently, CDGA treatment facilitated the extracellular release of bacteria with lower infectivity than those spontaneously released from sham-treated cells. Addition of CDGA to isolated inclusions induced dispersion of the morulae, degradation of an inclusion matrix protein TRP120, and bacterial intrainclusion movement, all of which were blocked by a serine protease inhibitor. These results suggest that c-di-GMP signaling regulates aggregation and sessility of E. chaffeensis within the inclusion through stabilization of matrix proteins by preventing the serine protease activity, which is associated with bacterial intracellular proliferation and maturation.
authors.affiliation: Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH 43210-1093, USA.
authors.forename: Yumi
authors.initials: Y
authors.lastname: Kumagai
authors.forename: Junji
authors.initials: J
authors.lastname: Matsuo
authors.forename: Zhihui
authors.initials: Z
authors.lastname: Cheng
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Hayakawa
authors.forename: Yasuko
authors.initials: Y
authors.lastname: Rikihisa
mesh_terms: D001422:Bacterial Adhesion / Q000187:drug effects*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D018441:Biofilms; D002460:Cell Line; D006152:Cyclic GMP / Q000378:metabolism / Q000494:pharmacology*; D019281:Dimerization; D018469:Ehrlichia chaffeensis / Q000254:growth & development* / Q000378:metabolism / Q000472:pathogenicity* / Q000502:physiology; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D000071677:Histidine Kinase; D054884:Host-Pathogen Interactions; D006801:Humans; D002479:Inclusion Bodies / Q000382:microbiology*; D009000:Monocytes / Q000382:microbiology* / Q000648:ultrastructure*; D019761:Phosphorus-Oxygen Lyases / Q000235:genetics / Q000378:metabolism; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism; D015290:Second Messenger Systems; D015398:Signal Transduction; D015854:Up-Regulation
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D011494:Protein Kinases; D000071677:Histidine Kinase; D019761:Phosphorus-Oxygen Lyases; C060720:diguanylate cyclase; D006152:Cyclic GMP