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.

Figure 1

Effect of small‐molecule inhibitors on the promoter activities of hrpN, dspE, hrpL and hrpA genes in Erwinia amylovora strain Ea273. Bacterial strains were grown in hrp (hypersensitive response and pathogenicity)‐inducing minimal medium (HMM) in the presence of 100 μm of the corresponding compounds at 18 °C for 18 h. The equivalent volume of dimethylsulphoxide (DMSO) was added as a control. Error bars represent the standard deviation. One‐way analysis of variance (ANOVA) and Turkey's W‐test (P = 0.05) were carried out to determine the difference in the means using the SAS program.

Figure 2

Venn diagrams showing the number of differentially expressed genes in compound 3‐ and 9‐treated samples relative to dimethylsulphoxide (DMSO) control. Differentially expressed genes were those that were activated (>twofold) or suppressed (<twofold) by compounds 3 (50 μm) and 9 (50 μm), respectively. Erwinia amylovora strain Ea273 was grown in hrp (hypersensitive response and pathogenicity)‐inducing minimal medium (HMM) at 18 °C for 18 h.

Figure 3

Verification of microarray data by quantitative real‐time polymerase chain reaction (qRT‐PCR). The relative fold change of each gene was derived from the comparison of the compound‐treated versus dimethylsulphoxide (DMSO)‐treated Erwinia amylovora strain Ea273 in hrp (hypersensitive response and pathogenicity)‐inducing minimal medium (HMM) for compounds 3 and 9 using qRT‐PCR. The 16S rDNA (rrsA) gene was used as a control. The values of the relative fold change were means of three replicates. The experiments were repeated three times with similar results. Error bars indicate the standard deviation.

Figure 4

Effect of salicylidene acylhydrazide inhibitors on virulence gene expression. Relative fold changes were determined by quantitative real‐time polymerase chain reaction (qRT‐PCR) for hrpA, dspE, hrpL, hrpN, amsG, EAM_359 and hmuS genes. Erwinia amylovora  Ea273 was grown in the presence of compounds 1–4 and 9 (50 μm) in hrp (hypersensitive response and pathogenicity)‐inducing minimal medium (HMM) at 18 °C for 18 h. The 16S rDNA (rrsA) gene was used as a control. The values of the relative fold change were means of three replicates. The experiments were repeated three times with similar results. Error bars indicate the standard deviation.

Figure 5

Effect of small‐molecule inhibitors on amylovoran production. Erwinia amylovora strain Ea273 was grown in MBMA medium with 1% sorbitol at 28 °C for 24 h with shaking. Chemical compounds and an equal volume of dimethylsulphoxide (DMSO) were added to the cell culture at a final concentration of 25 μm. Amylovoran concentrations were measured by the cetylpyrimidinium chloride (CPC) method and normalized to a cell density of unity. Data points represent the means of three replicates ± standard deviations. Each experiment was performed at least three times with similar results.

Figure 6

Virulence assay on crab apple (Malus mandshurica) flowers. Symptoms of crab apple flowers at 6 days post‐inoculation with Erwinia amylovora strain Ea273, dspE mutant, Ea273 treated with dimethylsulphoxide (DMSO) or compound 3 at 50 μm. Photographs were taken at 6 days post‐inoculation. The experiments were repeated three times with similar results. Arrow indicates representative inoculation site (stigma) and symptoms below on style and ovary.