| Bacterial pathogenesis generally depends on the ability to effectively coordinate the synthesis and expression of pathogenicity and virulence factors in order to successfully colonize a host. In some cases, this involves a need to rapidly reprogram cellular behaviors in response to host defenses or based on spatial location in the host. This genetic regulation often involves small signaling molecules such as cyclic di-GMP (c-di-GMP), a ubiquitous secondary messenger synthesized intracellularly and degraded by diguanylate cyclases (DGC) and phosphodiesterases (PDE), respectively. This nucleotide signaling molecule is involved in the regulation of many cellular processes in numerous bacterial species, including the transition from motile to sessile lifestyle, virulence, biosynthesis of exopolysaccharides and adhesion structures, and cell differentiation. Signal transduction and phenotypic modulation is determined by binding of c-di-GMP to specific downstream receptor molecules.;Erwinia amylovora uses motility and the hypertensive response and pathogenicity- (Hrp-) type III secretion system (T3SS) during initial phases of plant infection. Once inside the xylem, this pathogen aggregates due to the production of exopolysaccharides (EPS) and attachment structures forming a biofilm that ultimately plugs the xylem vessels. Although c-di-GMP has been shown to be an important intracellular signal in several plant pathogenic bacteria, the importance of this molecule in Erwinia amylovora has not been previously investigated. This doctoral research explores the role of c-di-GMP in the genetic regulation of key cellular processes associated with pathogenesis in E. amylovora. Five active DGC enzymes (EdcA, EdcB, EdcC, EdcD and EdcE) were identified in this bacterial pathogen. Phenotypic analyses demonstrated that c-di-GMP positively regulates the biosynthesis of both cellulose and amylovoran, positively regulates biofilm formation, and represses motility. Disease assays demonstrated that c-di-GMP negatively regulates virulence in these infection models.;The exopolysaccharides amylovoran and levan and attachment structures such as fimbriae, type IV pili and curli, have been demonstrated to be critical for the formation of a mature biofilm in E. amylovora. The results presented in this work demonstrated that c-di-GMP binds to the receptor protein BcsA, the cellulose synthase subunit, and activates the biosynthesis of cellulose in E. amylovora at a post-translational level. In addition, using SEM and confocal microscopy, it was demonstrated that cellulose is a main component of the biofilms formed by E. amylovora in vitro and in the host. Gene overexpression and site-directed mutagenesis analyses, suggest that BcsZ, an endoglucanase, is also required for cyclic di-GMP activation of cellulose biosynthesis and biofilm formation.;Further investigation into the mechanisms of c-di-GMP dependent regulation of virulence, with a special emphasis on the regulation of hrp-T3SS gene expression, revealed that high intracellular levels of c-di-GMP via DGC overexpression lead to a significant reduction in gene expression for hrpL, the hrp alternative sigma factor, and dspE. C-di-GMP binding assays suggest that HrpS, the sigma54 dependent transcriptional regulator of hrpL, is a candidate receptor of c-di-GMP. Moreover, the results presented in this work indicate that HrpS can bind to other guanosine-containing signaling molecules including the c-di-GMP degradation product pGpG. This work provides an overview of some of the molecular mechanisms for c-di-GMP-dependent regulation on the main pathogenicity determinants in E. amylovora, in order to orchestrate pathogenesis and successfully cause disease. |
