Cyclic Di-GMP modulates the disease progression of Erwinia amylovora

Abstract

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.

Fig 1

The five putative DGC enzymes present in the genome of Erwinia amylovora Ea1189. (A) The EAL and GGDEF domains of these proteins are shown with the protein lengths (in amino acids) and gene locus tags. Protein domains were predicted using Pfam, version 25.0, and are drawn to scale. Membrane-spanning domains were predicted by TMHMM, version 2.0, and are shown as vertical filled bars. (B) The GGDEF domain proteins from E. amylovora were aligned with HmsT, an active DGC from Yersinia pestis. Protein sequences were aligned using ClustalW on the MEGA 5.0 platform. Conserved amino acids (>80%) are highlighted in black. Residues required for enzymatic activity from this domain are indicated by black arrows above the amino acid alignment. It should be noted that the third amino acid of the GGDEF sequence of active DGCs can be either an aspartate or a glutamate residue.

Fig 2

c-di-GMP inhibits flagellar motility in E. amylovora. Motility was examined in strain Ea1189 overexpressing DGC genes (A) or in DGC mutant strains (B). Values are normalized to the value for pEVS141, the vector control (Vector) (similarly labeled in all subsequent figures), or to the value for wild-type Ea1189. The complemented gene was expressed from a plasmid (indicated by a lowercase “p”). Data represent three biological replicates, and error bars indicate the standard errors of the means. Different letters above bars indicate statistically significant differences (P < 0.05 by Student's t test).

Fig 3

c-di-GMP increases the production of amylovoran in E. amylovora. (A) Overexpression strains were grown in 3 parts MBMA medium with 1% sorbitol and 1 part LB medium supplemented with Km and 0.1 mM IPTG. (B) The WT, deletion mutants, and corresponding complemented strains were grown in MBMA medium amended with 1% sorbitol for 48 h at 28°C. The complemented gene was expressed from a plasmid (indicated by a lowercase “p”). Amylovoran production was quantified using the turbidometric CPC-binding assay, and the values were normalized to the cell density. The Ea1189Δams mutant is deficient in amylovoran production and was used as a negative control. Data represent three biological replicates, and error bars indicate the standard errors of the means. Different letters above bars indicate statistically significant differences of the means (P < 0.05 by Student's t test).

Fig 4

c-di-GMP induces biofilm formation of E. amylovora grown under static conditions. Biofilm formation by DGC overexpression strains (A) and DGC mutants (B) was determined by quantifying crystal violet (CV) binding as described in Materials and Methods. For the quantification of biofilm formation, E. amylovora Ea1189, deletion mutants, and complemented strains were grown in 0.5× LB medium for 48 h at 28°C. The complemented gene was expressed from a plasmid (indicated by a lowercase “p”). Biofilm formation was quantified as the absorbance after CV staining. Values are means for 12 replicates from one representative experiment. This assay was repeated three times with similar results. Error bars indicate the standard errors of the means, and different letters above the bars indicate statistically significant differences of the means (P < 0.05 by Student's t test).

Fig 5

Virulence of E. amylovora DGC mutant strains in an immature-pear infection model. (A) Sizes of lesions on immature pears inoculated with 2 ml of the indicated strains at ≈1 × 10⁴ CFU ml⁻¹. Lesion size was measured using calipers at 4 dpi. The experiment was repeated three times with similar results. The ΔedcCE mutant was complemented with edcC and edcE expressed from a plasmid (indicated by a lowercase “p”). Values are means for 10 replicates from one representative experiment, and error bars represent the standard errors. Different letters above the bars indicate statistically significant differences (P < 0.05 by Student's t test). (B) Representative pears illustrate symptom development at 4 dpi for Ea1189 and the mutant strains.

Fig 6

c-di-GMP inhibits acute virulence and migration of E. amylovora in an apple shoot infection model. Symptom development on apple shoots at 4 dpi is shown. The youngest leaves of central shoots were clip-inoculated with scissors previously dipped in a bacterial suspension of the indicated strains at a concentration of ≈5 × 10⁸ CFU ml⁻¹. The mutations were complemented with genes expressed from a plasmid (indicated by a lowercase “p”). This assay was repeated three times with similar results.

Fig 7

c-di-GMP controls the transcription of genes involved in amylovoran production and type III secretion. The expression of an amsG-lux transcriptional fusion (A) and of hrpA and hrpS transcriptional fusions to gfp (B) was examined upon expression of the indicated DGCs. Luciferase expression is shown as relative light units (RLU), determined by dividing luminescence by the OD₆₀₀ of the culture. Similarly, gfp is expressed in normalized units (fluorescence units/OD₆₀₀). Error bars indicate the standard errors. Symbols above the bars indicate statistically significant differences from the vector controls (WT) by Student's t test (*, P < 0.05).