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Small-molecule inhibitors suppress the expression of both type III secretion and amylovoran biosynthesis genes in Erwinia amylovora.

ABSTRACT: The type III secretion system (T3SS) and exopolysaccharide (EPS) amylovoran are two essential pathogenicity factors in Erwinia amylovora, the causal agent of the serious bacterial disease fire blight. In this study, small molecules that inhibit T3SS gene expression in E. amylovora under hrp (hypersensitive response and pathogenicity)-inducing conditions were identified and characterized using green fluorescent protein (GFP) as a reporter. These compounds belong to salicylidene acylhydrazides and also inhibit amylovoran production. Microarray analysis of E. amylovora treated with compounds 3 and 9 identified a total of 588 significantly differentially expressed genes. Among them, 95 and 78 genes were activated and suppressed by both compounds, respectively, when compared with the dimethylsulphoxide (DMSO) control. The expression of the majority of T3SS genes in E. amylovora, including hrpL and the avrRpt2 effector gene, was suppressed by both compounds. Compound 3 also suppressed the expression of amylovoran precursor and biosynthesis genes. However, both compounds induced significantly the expression of glycogen biosynthesis genes and siderophore biosynthesis, regulatory and transport genes. Furthermore, many membrane, lipoprotein and exported protein-encoding genes were also activated by both compounds. Similar expression patterns were observed for compounds 1, 2 and 4. Using crab apple flower as a model, compound 3 was capable of reducing disease development in pistils. These results suggest a common inhibition mechanism shared by salicylidene acylhydrazides and indicate that small-molecule inhibitors that disable T3SS function could be explored to control fire blight disease.

SUBMITTER: Yang F

PROVIDER: S-EPMC6638656 | biostudies-literature | 2014 Jan

REPOSITORIES: biostudies-literature

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Small-molecule inhibitors suppress the expression of both type III secretion and amylovoran biosynthesis genes in Erwinia amylovora.

Yang Fan F Korban Schuyler S SS Pusey P Lawrence PL Elofsson Michael M Sundin George W GW Zhao Youfu Y

Molecular plant pathology 20130805 1

The type III secretion system (T3SS) and exopolysaccharide (EPS) amylovoran are two essential pathogenicity factors in Erwinia amylovora, the causal agent of the serious bacterial disease fire blight. In this study, small molecules that inhibit T3SS gene expression in E. amylovora under hrp (hypersensitive response and pathogenicity)-inducing conditions were identified and characterized using green fluorescent protein (GFP) as a reporter. These compounds belong to salicylidene acylhydrazides and ...[more]

PMID: 23915008

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Project description:Cyclic di-GMP (c-di-GMP) is a ubiquitous bacterial second messenger molecule that is an important virulence regulator in the plant pathogen Erwinia amylovora Intracellular levels of c-di-GMP are modulated by diguanylate cyclase (DGC) enzymes that synthesize c-di-GMP and by phosphodiesterase (PDE) enzymes that degrade c-di-GMP. The regulatory role of the PDE enzymes in E. amylovora has not been determined. Using a combination of single, double, and triple deletion mutants, we determined the effects of each of the four putative PDE-encoding genes (pdeA, pdeB, pdeC, and edcA) in E. amylovora on cellular processes related to virulence. Our results indicate that pdeA and pdeC are the two phosphodiesterases most active in virulence regulation in E. amylovora Ea1189. The deletion of pdeC resulted in a measurably significant increase in the intracellular pool of c-di-GMP, and the highest intracellular concentrations of c-di-GMP were observed in the Ea1189 ?pdeAC and Ea1189 ?pdeABC mutants. The regulation of virulence traits due to the deletion of the pde genes showed two patterns. A stronger regulatory effect was observed on amylovoran production and biofilm formation, where both Ea1189 ?pdeA and Ea1189 ?pdeC mutants exhibited significant increases in these two phenotypes in vitro In contrast, the deletion of two or more pde genes was required to affect motility and virulence phenotypes. Our results indicate a functional redundancy among the pde genes in E. amylovora for certain traits and indicate that the intracellular degradation of c-di-GMP is mainly regulated by pdeA and pdeC, but they also suggest a role for pdeB in regulating motility and virulence.IMPORTANCE Precise control of the expression of virulence genes is essential for successful infection of apple hosts by the fire blight pathogen, Erwinia amylovora The presence and buildup of a signaling molecule called cyclic di-GMP enables the expression and function of some virulence determinants in E. amylovora, such as amylovoran production and biofilm formation. However, other determinants, such as those for motility and the type III secretion system, are expressed and functional when cyclic di-GMP is absent. Here, we report studies of enzymes called phosphodiesterases, which function in the degradation of cyclic di-GMP. We show the importance of these enzymes in virulence gene regulation and the ability of E. amylovora to cause plant disease.

Project description:BackgroundTwo-component signal transduction systems (TCSTs), consisting of a histidine kinase (HK) and a response regulator (RR), represent a major paradigm for signal transduction in prokaryotes. TCSTs play critical roles in sensing and responding to environmental conditions, and in bacterial pathogenesis. Most TCSTs in Erwinia amylovora have either not been identified or have not yet been studied.ResultsWe used a systems approach to identify TCST and related signal transduction genes in the genome of E. amylovora. Comparative genomic analysis of TCSTs indicated that E. amylovora TCSTs were closely related to those of Erwinia tasmaniensis, a saprophytic enterobacterium isolated from apple flowers, and to other enterobacteria. Forty-six TCST genes in E. amylovora including 17 sensor kinases, three hybrid kinases, 20 DNA- or ligand-binding RRs, four RRs with enzymatic output domain (EAL-GGDEF proteins), and two kinases were characterized in this study. A systematic TCST gene-knockout experiment was conducted, generating a total of 59 single-, double-, and triple-mutants. Virulence assays revealed that five of these mutants were non-pathogenic on immature pear fruits. Results from phenotypic characterization and gene expression experiments indicated that several groups of TCST systems in E. amylovora control amylovoran biosynthesis, one of two major virulence factors in E. amylovora. Both negative and positive regulators of amylovoran biosynthesis were identified, indicating a complex network may control this important feature of pathogenesis. Positive (non-motile, EnvZ/OmpR), negative (hypermotile, GrrS/GrrA), and intermediate regulators for swarming motility in E. amylovora were also identified.ConclusionOur results demonstrated that TCSTs in E. amylovora played major roles in virulence on immature pear fruit and in regulating amylovoran biosynthesis and swarming motility. This suggested presence of regulatory networks governing expression of critical virulence genes in E. amylovora.

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Small-molecule inhibitors suppress the expression of both type III secretion and amylovoran biosynthesis genes in Erwinia amylovora.

Publications

Small-molecule inhibitors suppress the expression of both type III secretion and amylovoran biosynthesis genes in Erwinia amylovora.

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