CsrD regulates amylovoran biosynthesis and virulence in Erwinia amylovora in a novel cyclic-di-GMP dependent manner.
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ABSTRACT:
SUBMITTER: Kharadi RR
PROVIDER: S-EPMC9276943 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The second messenger cyclic di-GMP (c-di-GMP) is a nearly ubiquitous intracellular signal molecule known to regulate various cellular processes, including biofilm formation, motility, and virulence. The intracellular concentration of c-di-GMP is inversely governed by diguanylate cyclase (DGC) enzymes and phosphodiesterase (PDE) enzymes, which synthesize and degrade c-di-GMP, respectively. The role of c-di-GMP in the plant pathogen and causal agent of fire blight disease Erwinia amylovora has not been studied previously. Here we demonstrate that three of the five predicted DGC genes in E. amylovora (edc genes, for Erwinia diguanylate cyclase), edcA, edcC, and edcE, are active diguanylate cyclases. We show that c-di-GMP positively regulates the secretion of the main exopolysaccharide in E. amylovora, amylovoran, leading to increased biofilm formation, and negatively regulates flagellar swimming motility. Although amylovoran secretion and biofilm formation are important for the colonization of plant xylem tissues and the development of systemic infections, deletion of the two biofilm-promoting DGCs increased tissue necrosis in an immature-pear infection assay and an apple shoot infection model, suggesting that c-di-GMP negatively regulates virulence. In addition, c-di-GMP inhibited the expression of hrpA, a gene encoding the major structural component of the type III secretion pilus. Our results are the first to describe a role for c-di-GMP in E. amylovora and suggest that downregulation of motility and type III secretion by c-di-GMP during infection plays a key role in the coordination of pathogenesis.
| S-EPMC3650540 | biostudies-literature
Project description:The second messenger cyclic-di-GMP (c-di-GMP) is a critical regulator of biofilm formation in the plant pathogen Erwinia amylovora. Phosphodiesterase (PDE) enzymes are responsible for the degradation of intracellular c-di-GMP. Previously, we found that the deletion of one or more of the three PDE enzyme encoding genes (pdeA, pdeB, and pdeC) in E. amylovora Ea1189 led to an increase in biofilm formation. However, in mutants Ea1189ΔpdeAC and Ea1189ΔpdeABC, biofilm formation was reduced compared to the other single and double deletion mutants. Here, we attribute this to an autoaggregation phenotype observed in these two mutants. Examination of Ea1189ΔpdeABC cellular aggregates using scanning electron microscopy indicated that a subset of cells were impaired in cell separation post cell division. Concomitant with this phenotype, Ea1189ΔpdeABC also exhibited increased transcription of the cell-division inhibitor gene sulA and reduced transcription of ftsZ. Ea1189ΔpdeABC showed a significant reduction in biofilm formation, and biofilms formed by Ea1189ΔpdeABC exhibited a distinctive morphology of sparsely scattered aggregates rather than an evenly distributed biofilm as observed in WT Ea1189. Our results suggest that highly elevated levels of c-di-GMP lead to increased cell-cell interactions that contribute to autoaggregation and impair cell-surface interaction, negatively affecting biofilm formation.
| S-EPMC6423407 | biostudies-literature
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.
| S-EPMC6293108 | biostudies-literature
Project description:Erwinia amylovora is a plant-pathogenic bacterium that causes fire blight disease in many economically important plants, including apples and pears. This bacterium produces three exopolysaccharides (EPSs), amylovoran, levan, and cellulose, and forms biofilms in host plant vascular tissues, which are crucial for pathogenesis. Here, we demonstrate that ProQ, a conserved bacterial RNA chaperone, was required for the virulence of E. amylovora in apple shoots and for biofilm formation in planta. In vitro experiments revealed that the deletion of proQ increased the production of amylovoran and cellulose. Prc is a putative periplasmic protease, and the prc gene is located adjacent to proQ. We found that Prc and the associated lipoprotein NlpI negatively affected amylovoran production, whereas Spr, a peptidoglycan hydrolase degraded by Prc, positively regulated amylovoran. Since the prc promoter is likely located within proQ, our data showed that proQ deletion significantly reduced the prc mRNA levels. We used a genome-wide transposon mutagenesis experiment to uncover the involvement of the bacterial second messenger c-di-GMP in ProQ-mediated cellulose production. The deletion of proQ resulted in elevated intracellular c-di-GMP levels and cellulose production, which were restored to wild-type levels by deleting genes encoding c-di-GMP biosynthesis enzymes. Moreover, ProQ positively affected the mRNA levels of genes encoding c-di-GMP-degrading phosphodiesterase enzymes via a mechanism independent of mRNA decay. In summary, our study revealed a detailed function of E. amylovora ProQ in coordinating cellulose biosynthesis and, for the first time, linked ProQ with c-di-GMP metabolism and also uncovered a role of Prc in the regulation of amylovoran production. IMPORTANCE Fire blight, caused by the bacterium Erwinia amylovora, is an important disease affecting many rosaceous plants, including apple and pear, that can lead to devastating economic losses worldwide. Similar to many xylem-invading pathogens, E. amylovora forms biofilms that rely on the production of exopolysaccharides (EPSs). In this paper, we identified the RNA-binding protein ProQ as an important virulence regulator. ProQ played a central role in controlling the production of EPSs and participated in the regulation of several conserved bacterial signal transduction pathways, including the second messenger c-di-GMP and the periplasmic protease Prc-mediated systems. Since ProQ has recently been recognized as a global posttranscriptional regulator in many bacteria, these findings provide new insights into multitiered regulatory mechanisms for the precise control of virulence factor production in bacterial pathogens.
| S-EPMC9088287 | biostudies-literature
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.
| S-EPMC2698875 | biostudies-literature
Project description:Erwinia amylovora is the causal agent of fire blight, an economically impactful disease that affects apple and pear production worldwide. E. amylovora pathogenesis is comprised of distinct type III secretion-dependent and biofilm-dependent stages. Alterations in the intracellular levels of cyclic-di-GMP (c-di-GMP) regulate the transition between the different stages of infection in E. amylovora. We previously reported that hyper-elevation of c-di-GMP levels in E. amylovora Ea1189, resulting from the deletion of all three c-di-GMP specific phosphodiesterase genes (Ea1189ΔpdeABC), resulted in an autoaggregation phenotype. The two major exopolysaccharides, amylovoran and cellulose, were also shown to partially contribute to autoaggregation. In this study, we aimed to identify the c-di-GMP dependent factor(s) that contributes to autoaggregation. We conducted a transposon mutant screen in Ea1189ΔpdeABC and selected for loss of autoaggregation. Our search identified a peptidoglycan hydrolase, specifically, a D, D-endopeptidase of the metallopeptidase class, EagA (Erwinia aggregation factor A), that was found to physiologically contribute to autoaggregation in a c-di-GMP dependent manner. The production of amylovoran was also positively affected by EagA levels. An eagA deletion mutant (Ea1189ΔeagA) was significantly reduced in virulence compared to the wild type E. amylovora Ea1189. eagA is part of the znuABC zinc uptake gene cluster and is located within an operon downstream of znuA. The znuAeagA/znuCB gene cluster was transcriptionally regulated by elevated levels of c-di-GMP as well as by the zinc-dependent transcriptional repressor Zur. We also observed that with an influx of Zn2+ in the environment, the transcription of the znuAeagA/znuBC gene cluster is regulated by both Zur and a yet to be characterized c-di-GMP dependent pathway.
| S-EPMC7705223 | biostudies-literature
Project description: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.
| S-EPMC6638656 | biostudies-literature
Project description:In this study, we attempted to understand the role of an orphan gene amyR in Erwinia amylovora, a functionally conserved ortholog of ybjN in Escherichia coli, which has recently been characterized. Amylovoran, a high molecular weight acidic heteropolymer exopolysaccharide, is a virulent factor of E. amylovora. As reported earlier, amylovoran production in an amyR knockout mutant was about eight-fold higher than that in the wild type (WT) strain of E. amylovora. When a multicopy plasmid containing the amyR gene was introduced into the amyR mutant or WT strains, amylovoran production was strongly inhibited. Furthermore, amylovoran production was also suppressed in various amylovoran-over-producing mutants, such as grrSA containing multicopies of the amyR gene. Consistent with amylovoran production, an inverse correlation was observed between in vitro expression of amyR and that of amylovoran biosynthetic genes. However, both the amyR knockout mutant and over-expression strains showed reduced levan production, another exopolysaccharide produced by E. amylovora. Virulence assays demonstrated that while the amyR mutant was capable of inducing slightly greater disease severity than that of the WT strain, strains over-expressing the amyR gene did not incite disease on apple shoots or leaves, and only caused reduced disease on immature pear fruits. Microarray studies revealed that amylovoran biosynthesis and related membrane protein-encoding genes were highly expressed in the amyR mutant, but down-regulated in the amyR over-expression strains in vitro. Down-regulation of amylovoran biosynthesis genes in the amyR over-expression strain partially explained why over-expression of amyR led to non-pathogenic or reduced virulence in vivo. These results suggest that AmyR plays an important role in regulating exopolysaccharide production, and thus virulence in E. amylovora.
| S-EPMC3445560 | biostudies-literature
Project description:We compared the transcriptomes of S. flexneri strains expressing the diguanylate cyclase VCA0956 (derived from V. cholerae)
2021-09-01 | GSE173759 | GEO
Project description:Microarray analysis of E. amylovora treated with compounds no. 3 and no. 9 identified a total of 588 significantly differentially expressed genes. Among them, 95 and 78 genes were, respectively, induced and suppressed by both compounds as compared to DMSO control. Majority of T3SS genes in E. amylovora including hrpL and avrRpt2 effector gene were suppressed by both compounds. Compound no. 3 also suppressed the transcription of amylovoran precursor and biosynthesis genes. On the other hand, both compounds significantly induced 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.
2013-12-24 | GSE45602 | GEO