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. 2014 Aug 25;16(2):150–163. doi: 10.1111/mpp.12168

Derivative of plant phenolic compound inhibits the type III secretion system of Dickeya dadantii via HrpX/HrpY two‐component signal transduction and Rsm systems

Yan Li 1, William Hutchins 2, Xiaogang Wu 2, Cuirong Liang 3, Chengfang Zhang 3, Xiaochen Yuan 2, Devanshi Khokhani 2, Xin Chen 3, Yizhou Che 2, Qi Wang 1,✉, Ching‐Hong Yang 2,✉
PMCID: PMC6638520  PMID: 24986378

Summary

The type III secretion system (T3SS) is a major virulence factor in many Gram‐negative bacterial pathogens and represents a particularly appealing target for antimicrobial agents. Previous studies have shown that the plant phenolic compound p‐coumaric acid (PCA) plays a role in the inhibition of T3SS expression of the phytopathogen Dickeya dadantii 3937. This study screened a series of derivatives of plant phenolic compounds and identified that trans‐4‐hydroxycinnamohydroxamic acid (TS103) has an eight‐fold higher inhibitory potency than PCA on the T3SS of D. dadantii. The effect of TS103 on regulatory components of the T3SS was further elucidated. Our results suggest that TS103 inhibits HrpY phosphorylation and leads to reduced levels of hrpS and hrpL transcripts. In addition, through a reduction in the RNA levels of the regulatory small RNA RsmB, TS103 also inhibits hrpL at the post‐transcriptional level via the rsmB‐RsmA regulatory pathway. Finally, TS103 inhibits hrpL transcription and mRNA stability, which leads to reduced expression of HrpL regulon genes, such as hrpA and hrpN. To our knowledge, this is the first inhibitor to affect the T3SS through both the transcriptional and post‐transcriptional pathways in the soft‐rot phytopathogen D. dadantii 3937.

Keywords: plant phenolic compound, Rsm system, T3SS inhibitor, type III secretion system, two‐component signal transduction system

Introduction

Antibiotic treatment is the most commonly used strategy to control pathogenic infections. However, most antibiotics kill bacteria by inhibiting cellular processes essential for survival, which leads to strong selective pressure to develop resistance against antibiotics (Cegelski et al., 2008; Escaich, 2008; Rasko and Sperandio, 2010). In the face of increasing antibiotic resistance, the targeting of bacterial virulence factors rather than bacterial survivability provides a novel alternative approach for the development of new antimicrobials, as virulence‐specific therapeutics would offer a reduced selection pressure for antibiotic‐resistant mutations (Escaich, 2008; Rasko and Sperandio, 2010). The type III secretion system (T3SS) represents a particularly appealing target for antimicrobial agents because it is a major virulence factor in many Gram‐negative plant and animal pathogens (Cornelis, 2006; Tang et al., 2006; Waterman and Holden, 2003; Yang et al., 2002). The T3SS in phytobacteria, also known as the hypersensitive response and pathogenicity (Hrp) system, is a syringe needle‐like structure which is responsible for the secretion and translocation of effector proteins into the host cells, where the effector proteins subvert or inhibit the host cell's defences or facilitate pathogenicity (Alfano and Collmer, 1997; Galán and Collmer, 1999; Ghosh, 2004; Grant et al., 2006; Hueck, 1998; Yang et al., 2005).

Dickeya dadantii 3937 (formerly named Erwinia chrysanthemi), a member of the Enterobacteriaceae family, is a Gram‐negative pathogen which causes soft rot, wilt and blight diseases on a wide range of plant species (Bauer et al., 1994). D. dadantii possesses a T3SS, which is encoded by the hrp gene cluster and thought to be coordinately regulated by various host and environmental factors (Nasser et al., 2005; Yang et al., 2002). Similar to many phytopathogens, the expression of the T3SS of D. dadantii 3937 is repressed in nutrient‐rich media, but induced in the plant apoplast or in nutrient‐deficient inducing medium, which is considered to mimic plant apoplastic conditions (Galán and Collmer, 1999; Tang et al., 2006). The well‐studied T3SS of D. dadantii is regulated by the master regulator HrpL, which is a member of the extracytoplasmic function (ECF) family of alternative sigma factors that up‐regulate many hrp genes downstream of the T3SS regulatory cascade, such as hrpA (encoding a structural protein of the T3SS pilus), dspE (encoding a T3SS effector) and hrpN (encoding a harpin protein) (Fig. 1) (Chatterjee et al., 2002; Wei et al., 1992; Yang et al., 2010; Yap et al., 2005). The expression of hrpL is regulated at both the transcriptional and post‐transcriptional levels. HrpX/HrpY‐HrpS regulates hrpL at the transcriptional level. A two‐component signal transduction system (TCSTS) HrpX/HrpY, encoded by genes in the centre of the hrp gene cluster, positively regulates hrpS, which encodes a σ54 enhancer‐binding protein (Fig. 1) (Tang et al., 2006). HrpS interacts with the σ54 (RpoN)‐containing RNA polymerase holoenzyme and initiates the transcription of hrpL (Fig. 1) (Chatterjee et al., 2002; Yap et al., 2005). The regulator of secondary metabolites (Rsm) RsmA–RsmB pair regulates hrpL at the post‐transcriptional level. RsmA, a small RNA‐binding protein, promotes hrpL mRNA degradation (Fig. 1) (Chatterjee et al., 2002; Cui et al., 1995). RsmB, an untranslated regulatory small RNA, binds to the RsmA protein and neutralizes the activity of RsmA on hrpL mRNA degradation by forming an inactive ribonucleoprotein complex with RsmA (Chatterjee et al., 2002; Liu et al., 1998) (Fig. 1). The global regulatory TCSTS, GacS/GacA, up‐regulates the transcription of the regulatory small RNA RsmB (Tang et al., 2006). In addition to GacS/GacA, other regulators have been identified that control the expression of rsmB in the soft‐rot pathogens Pectobacterium and Dickeya. KdgR, an IcII‐like protein, has been reported to negatively control the transcription of rsmB by binding within the transcribed region of the rsmB gene in P. carotovorum (Miller et al., 2000). Polynucleotide phosphorylase (PNPase) has been reported to decrease the amount of functional rsmB transcripts in D. dadantii (Zeng et al., 2010).

Figure 1.

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Regulatory network controlling the Dickeya dadantii type III secretion system (T3SS). The D. dadantii  T3SS is regulated by the HrpX/HrpY‐HrpS‐HrpL and GacS/GacA‐rsmB‐RsmA‐HrpL regulatory pathways. The two‐component signal transduction system HrpX/HrpY activates hrpS, which encodes a σ54 enhancer. HrpS is required for the expression of the alternative sigma factor, hrpL. HrpL activates the expression of genes encoding the T3SS apparatus and its secreted substrates. RsmA is a small RNA‐binding protein that acts by decreasing the half‐life of hrpL  mRNA. GacS/GacA up‐regulates the expression of rsmB, which increases the mRNA level of hrpL by sequestering RsmA. From this study, we observed that TS103 altered the hrpA promoter activity through both the HrpX/HrpY‐HrpS‐HrpL and rsmB‐RsmA‐HrpL pathways. TS103 inhibits hrpL at the post‐transcriptional level through a decrease in expression of rsmB. In addition, TS103 inhibits hrpS transcription through suppression of phosphorylation of HrpY and also post‐transcriptionally inhibits rpoN. ⊥, negative control; →, positive control. IM: Inner Membrane; OM: Outer Membrane.

In response to microbial attack, plants activate defence responses which lead to the induction of a broad spectrum of antimicrobial defences (Montesano et al., 2005; Van Loon, 2000). These induced defences are regulated by a network of interconnecting signal transduction pathways and eventually lead to the production of defence molecules, such as phenylpropanoids (Dixon and Paiva, 1995; Feys and Parker, 2000; Hahlbrock and Scheel, 1989). Phenylpropanoids are a group of secondary metabolites produced by plants from l‐phenylalanine. Our previous reports have shown that the plant phenolic compound p‐coumaric acid (PCA), an intermediate in phenylpropanoid biosynthesis, plays a role in the inhibition of T3SS expression of D. dadantii 3937 (Li et al., 2009). With the aid of structure–activity relationship (SAR) studies, the para positioning of the hydroxyl group in the phenyl ring and the double bond in PCA have been predicted previously to be essential for its inhibitory activity (Li et al., 2009). As the regulatory mechanism of the T3SS of D. dadantii 3937 is well understood, to develop T3SS inhibitors which are more potent than PCA, a series of derivatives of plant phenolic compounds were screened using D. dadantii 3937 as a model organism. One derivative, TS103, which showed an eight‐fold higher potency in the inhibition of the T3SS vs. PCA, was selected and the regulators responsible for the inhibition of T3SS gene expression by TS103 were further elucidated. Our results showed that TS103 inhibits the T3SS through both the HrpX/HrpY TCSTS and Rsm systems.

Results

Screening for highly potent T3SS inhibitors of D. dadantii

In our previous work, the phenolic acid PCA was found to inhibit T3SS gene expression of D. dadantii 3937 at a concentration of 100 μm (Li et al., 2009). To identify T3SS inhibitors which have higher potency in the inhibition of T3SS expression, 50 derivatives of plant phenolic compounds (Fig. 2) at a concentration of 100 μm were first screened by monitoring the promoter activity of hrpA. The hrpA gene encodes the T3SS pilus, which is required for the translocation of effector proteins into plant cells and is located downstream in the T3SS regulatory pathway (Fig. 1). A reporter plasmid, pPhrpA, which carries a hrpA‐gfp transcriptional fusion, was used to measure the effects of the derivatives of plant phenolic compounds on hrpA expression (Table 1). The wild‐type cells containing pPhrpA were grown in T3SS‐inducing medium (MM) supplemented with each of the compounds at a concentration of 100 μm. Green fluorescent protein (GFP) intensity, which is a measurement of hrpA promoter activity, was assayed by flow cytometry. Among the derivatives of the plant phenolic compounds screened, 13 compounds at a concentration of 100 μm showed strong inhibition of T3SS gene expression of D. dadantii, in which the level of hrpA promoter activity was reduced by more than 50% of the level in MM at both 12 and 24 h of growth after repeated measurements (Table 2).

Figure 2.

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Chemical structures of derivatives of plant phenolic compounds.

Table 1.

Strains and plasmids used in this study

Strains and plasmids Relevant characteristics Reference or source
Strains
Escherichia coli
E. coli S17‐1 λ‐pir λ‐pir lysogen of S17‐1; SpR Sanchez‐Romero et al. (1998)
Dickeya dadantii
3937 Wild‐type, Saintpaulia (African violet) isolate Hugouvieux‐Cotte‐Pattat, N. (Microbiologie Adaptation et Pathogenie CNRS, INSA de Lyon, Universite de Lyon, Lyon F‐69622, France)
ΔhrpY hrpY deletion mutant; KmR This study
ΔhrpYD57A 3937 derivative in which the conserved aspartate residue at position 57 in HrpY was changed by nonconservative substitution to alanine; KmR This study
ΔgacA gacA deletion mutant; KmR This study
ΔgacA::gacA ΔgacA with chromosomal insertion of lacY‐gacA‐cm‐prt; KmR, CmR This study
Δpnp pnp deletion mutant; KmR Zeng et al. (2010)
ΔkdgR kdgR deletion mutant; KmR This study
3937::OpgG‐His6 3937 with a 6 × His epitope sequence tagged to the C‐terminus of OpgG Laboratory stock
Plasmids
pAT pProbe‐AT, promoter‐probe vector; ApR Miller et al. (2000)
pPhrpA pAT derivative with PCR fragment containing hrpA promoter region; ApR Yang et al. (2008a)
pPhrpN pAT derivative with PCR fragment containing hrpN promoter region; ApR Yang et al. (2007)
pPhrpL pAT derivative with PCR fragment containing hrpL promoter region; ApR Yang et al. (2007)
pPhrpS pAT derivative with PCR fragment containing hrpS promoter region; ApR Li et al. (2009)
pPrpoN pAT derivative with PCR fragment containing rpoN promoter region; ApR Yi et al. (2010)
pPrsmB pAT derivative with PCR fragment containing rsmB promoter region; ApR This study
pWM91 Sucrose‐based counter‐selectable plasmid; ApR Metcalf et al. (1996)
pKD4 Template plasmid for kanamycin cassette; KmR Datsenko and Wanner (2000)
pGEM‐T Easy Cloning vector; ApR Promega (Madison, WI, USA)
phrpY pGEM‐T Easy derivative with PCR fragment containing the hrpY ORF and its flanking regions; ApR This study
phrpYD57A phrpY derivative in which the conserved aspartate residue at position 57 in HrpY was changed by nonconservative substitution to alanine; ApR This study
pTCLSCm 6.4‐kb lacY‐cm‐prt region cloned in pGEM‐T Easy; CmR Yap et al. (2008)
pML123 Broad‐host‐range cloning vector; GmR Labes et al. (1990)
pMLkdgR Derivative of pML123 carrying kdgR; GmR This study
pCL1920 Expression vector; SpR Lerner and Inouye (1990)
pCLhrpXY Derivative of pCL1920 carrying hrpXY operon; SpR This study
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ApR, ampicillin resistance; CmR, chloramphenicol resistance; KmR, kanamycin resistance; GmR, gentamicin resistance; ORF, open reading frame; SpR, spectinomycin resistance.

Table 2.

The hrpA expression of D ickeya dadantii 3937 in type III secretion system‐inducing medium (MM) and MM supplemented with different derivatives of plant phenolic compounds

Phenolic compoundb 12 h 24 h
Ave MFI ± SDc %MMd Ave MFI ± SD %MM
MM 35.4 ± 0.8 90.6 ± 11.7
TS100, ethyl trans‐2‐(4‐methoxyphenyl)‐1‐cyclopropanecarboxylate 45.3 ± 3.4a 128.2 119.1 ± 22.1 131.5
TS101, methyl para‐coumarate 17.5 ± 0.7a 49.6 19.9 ± 0.5a 22.0
TS102, trans‐4‐hydroxycinnamide 21.1 ± 1.2a 59.7 60.8 ± 10.6 67.1
TS103, trans‐4‐hydroxycinnamohydroxamic acid 8.7 ± 0.3a 24.7 11.9 ± 0.8a 13.2
TS104, para‐coumaryl alcohol 3.0 ± 0.1a 8.6 12.9 ± 1.8a 14.2
TS105, trans‐2‐(4‐methoxyphenyl)‐1‐cyclopropanecarboxylic acid 22.9 ± 2.6a 64.8 82.4 ± 5.8 90.9
TS106, ethyl trans‐2‐(4‐hydroxyphenyl)‐1‐cyclopropanecarboxylate 10.5 ± 0.8a 29.8 49.4 ± 4.1a 54.5
TS107, trans‐2‐(4‐hydroxyphenyl)‐1‐cyclopropanecarboxylic acid 45.9 ± 2.7a 129.9 122.1 ± 17.9 134.7
TS108, trans‐4‐phenylcinnamic acid 31.6 ± 2.5 89.3 109.0 ± 19.8 120.3
TS109, trans‐4‐chlorocinnamide 35.7 ± 6.3 100.8 81.5 ± 3.8 89.9
TS110, trans‐4‐fluorocinnamic acid 63.4 ± 8.0a 179.4 183.8 ± 19.2a 202.8
TS111, trans‐4‐bromocinnamic acid 23.9 ± 5.2 67.7 54.7 ± 7.2 60.3
TS112, trans‐4‐dimethylaminocinnamic acid 23.8 ± 4.3 67.4 60.7 ± 6.4 67.0
TS113, trans‐4‐trifluoromethylcinnamic acid 24.4 ± 0.9a 69.1 68.5 ± 5.7 75.6
MM 51.4 ± 6.7 77.1 ± 9.1
TS114, diethyl trans‐2‐(4‐hydroxyphenyl)‐vinylphosphonate 55.4 ± 1.0 107.8 76.5 ± 5.6 99.2
TS115, trans‐2‐(4‐hydroxyphenyl)‐vinylphosphonic acid 57.9 ± 1.6 112.7 79.6 ± 3.3 103.2
TS116, N‐(para‐coumaryl)phthalimide 24.3 ± 1.5a 47.2 39.3 ± 3.5a 51.0
TS117, para‐coumarylamine 49.8 ± 1.6 96.9 73.1 ± 2.0 94.9
TS118, N‐(4‐methoxycinnamyl)phthalimide 59.0 ± 0.4 114.7 83.8 ± 1.7 108.8
TS119, trans‐4‐methoxycinnamylamine 47.8 ± 3.2 93.0 68.2 ± 10.4 88.5
TS120, ethyl trans‐2‐(4‐methoxyphenyl)‐ethenylsulphonate 49.9 ± 2.6 97.0 68.6 ± 6.0 89.1
TS121, trans‐2‐(4‐methoxyphenyl)ethenylsulphonic acid tetra(n‐butyl)ammonium salt 57.1 ± 10.0 111.1 72.2 ± 10.6 93.7
TS122, ethyl trans‐2‐(4‐hydroxyphenyl)‐ethenylsulphonate 38.6 ± 1.8 75.1 60.9 ± 0.8 79.0
TS123, trans‐2‐(4‐hydroxyphenyl)ethenylsulphonic acid tetra(n‐butyl)ammonium salt 52.3 ± 2.0 101.6 77.7 ± 6.8 100.8
TS124, trans‐4‐hydroxymethylcinnamic acid 44.9 ± 3.5 87.2 78.1 ± 3.4 101.3
TS125, trans‐4‐methoxycinnamohydroxamic acid 17.4 ± 0.9a 33.8 25.6 ± 2.0a 33.2
TS126, trans‐4‐methoxycinnamyl alcohol 7.8 ± 0.2a 15.2 13.4 ± 0.3a 17.4
TS127, trans‐3‐indoleacrylohydroxamic acid 21.0 ± 3.0a 40.8 35.1 ± 3.0a 45.6
MM 30.1 ± 6.9 74.0 ± 7.9
TS128, trans‐4‐bromocinnamohydroxamic acid 7.5 ± 0.7a 25.0 10.9 ± 4.4a 14.7
TS129, trans‐2‐hydroxycinnamohydroxamic acid 12.0 ± 0.4 39.8 20.2 ± 1.3a 27.3
TS130, trans‐3‐hydroxycinnamohydroxamic acid 28.7 ± 2.5 95.6 41.0 ± 1.1a 55.4
TS131, trans‐3,4‐dihydroxycinnamohydroxamic acid 6.1 ± 0.8a 20.2 16.4 ± 0.6a 22.2
MM 37.7 ± 2.6 56.9 ± 1.4
TS132, trans‐cinnamohydroxamic acid 12.3 ± 0.2a 32.7 44.2 ± 2.9a 77.7
TS133, trans‐3‐(4‐hydroxyphenyl)acrylohydrazide 7.2 ± 0.9a 19.2 17.1 ± 2.5a 30.1
MM 78.7 ± 6.3 92.1 ± 17.1
TS032, cinnamyl alcohol 27.8 ± 5.0a 35.3 53.8 ± 2.9a 58.4
MM 57.9 ± 1.7 165.4 ± 4.5
TS037, N‐(4‐fluorocinnamyl)phthalimide 51.6 ± 3.2 89.1 130.3 ± 1.3a 78.8
TS039, N‐(4‐aminocinnamyl)phthalimide 70.8 ± 2.1a 122.4 138.9 ± 1.0a 84.0
TS040, N‐(4‐dimethylaminocinnamyl)phthalimide 49.6 ± 3.2 85.7 136.1 ± 5.4a 82.3
TS041, N‐(2‐methoxycinnamyl)phthalimide 47.6 ± 1.4a 82.2 120.6 ± 4.8a 72.9
TS042, N‐(3‐methoxycinnamyl)phthalimide 64.4 ± 5.8 111.3 146.8 ± 6.1 88.7
TS138, N‐(2‐hydroxyethyl)‐4‐hydroxycinnamamide 36.4 ± 3.7a 63.0 148.1 ± 10.0 89.6
TS139, 3‐phenylpropionohydroxamic acid 47.9 ± 1.3a 82.8 64.7 ± 2.1a 39.1
TS146, trans‐4‐formylcinnamohydroxamic acid 50.0 ± 2.4a 86.4 93.7 ± 5.7a 56.6
MM 51.5 ± 10.9 149.2 ± 22.2
TS044, trans‐4‐fluorocinnamylamine 43.5 ± 0.9 84.6 118.3 ± 19.1 79.3
TS046, trans‐4‐aminocinnamylamine 45.9 ± 3.9 89.3 109.6 ± 19.8 73.4
TS047, trans‐4‐dimethylaminocinnamylamine 20.9 ± 1.8a 40.5 36.2 ± 6.5a 24.2
TS137, N‐methyl‐4‐hydroxycinnamamide 47.4 ± 2.5 92.1 140.6 ± 6.9 94.2
TS143, 2‐phenoxyacetohydroxamic acid 23.4 ± 1.0 45.4 74.0 ± 5.6a 49.6
TS164, trans‐4‐(benzylcarbonyl)cinnamic acid 101.9 ± 7.9a 198.1 198.9 ± 1.8 133.3
TS165, trans‐2‐(4′‐benzylcarbonyl)phenylcyclopropane‐1‐carboxylic acid 19.4 ± 0.2a 37.6 45.6 ± 1.6a 30.6
p‐Coumaric acid 15.9 ± 1.2a 30.9 22.9 ± 1.8a 15.4
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a

Statistically significant differences in green fluorescent protein (GFP) mean fluorescence intensity (MFI) between bacterial cells grown in MM and MM supplemented with the different compounds (P < 0.01, Student's t‐test).

b

MM was supplemented with 100 μm of the indicated compounds. The compounds were assayed at two different times, with MM supplemented with dimethylsulphoxide (DMSO) as the control treatment (indicated by ‘MM’) for each set of experiments. The compound numbers are as used in Fig. 2.

c

Dickeya dadantii 3937 cells carrying the GFP reporter pPhrpA were used in this study. The promoter activities at 12 and 24 h of bacterial growth were determined. GFP MFI was determined for gated populations of bacterial cells by flow cytometry. Values are representative of two independent experiments, and three replicates were used for each experiment.

d

The relative promoter activity of hrpA in D. dadantii 3937 cells grown in MM supplemented with 100 μm of the indicated compounds compared with that in MM (indicated by ‘%MM’) was calculated by the formula: %MM = 100 × MFI(compound)/MFI(MM).

To identify inhibitors that exhibit higher efficacy of T3SS inhibition, the hrpA expression of D. dadantii cells grown in MM supplemented with inhibitors at a concentration of 10 μm instead of 100 μm was further examined. PCA was used as a reference, in which hrpA expression was inhibited at a concentration of 100 μm, but not at 10 μm (Table 2 and Fig. S1, see Supporting Information) (Li et al., 2009). Our results showed that the level of hrpA expression of D. dadantii 3937 cells grown in MM supplemented with three of the inhibitors (TS103, TS126 and TS131) at a concentration of 10 μm was less than 50% of the level in MM at 12 h of growth (Fig. S1). Among these three compounds, the addition of TS103 at a concentration of 10 μm resulted in the greatest reduction in hrpA expression at 12 h of bacterial growth, in which the level of hrpA expression of D. dadantii cells grown in MM supplemented with TS103 was less than 25% of the level in MM (Fig. S1). The addition of all the selected inhibitors at a concentration of 1 μm did not result in a significant reduction in hrpA promoter activity at 12 h of bacterial growth (data not shown). In addition, growth inhibition was not observed in TS103 at the tested concentrations (Figs S2 and S3, see Supporting Information). These results suggest that TS103 has the highest potency of T3SS inhibition among all the derivatives of plant phenolic compounds screened in this study. To further compare the inhibitory efficacy on the T3SS between TS103 and PCA, we tested the half‐maximal inhibitory concentration (IC50) of these two compounds on T3SS expression. Here, IC50 is defined as the concentration of compound that is required for the inhibition of 50% of the hrpA promoter activity compared with MM. The results showed that the IC50 of TS103 was 2.2 μm, which is one‐eighth of that of PCA (Fig. 3).

Figure 3.

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Effectiveness of TS103 and p‐coumaric acid (PCA) to inhibit hrpA promoter activity. hrpA promoter activity of Dickeya dadantii was determined in the presence of TS103 or PCA at the respective concentrations. The IC 50 of these compounds represents the inhibition of 50% of the promoter activity of hrpA compared with the dimethylsulphoxide (DMSO) control. The data are representative of two independent experiments. Three replicates were used in each experiment.

TS103 inhibits the transcription and production of T3SS structural‐ and harpin‐encoding genes

To confirm the inhibitory effect of TS103 on the T3SS of D. dadantii, the promoter activities and mRNA levels of two representative hrp genes, hrpA and hrpN, were examined in the presence and absence of TS103. Similar to the results obtained from the screening above (Table 2), a lower hrpA promoter activity was observed in MM supplemented with TS103 compared with that in MM alone (Table 3). Considerably lower promoter activity of hrpN was observed in the cells grown in MM supplemented with TS103 in comparison with that in MM (Table 3). Quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR) analysis revealed a significant decrease in hrpA (relative expression ratio 0.221, P = 0.049) and hrpN (relative expression ratio 0.276, P = 0.049) mRNA levels in the cells grown in MM supplemented with TS103 compared with that in MM (Fig. 4). HrpN protein production was further analysed by Western blot in the presence of TS103. Compared with that in MM, less HrpN was detected in protein extracts from D. dadantii 3937 grown in MM supplemented with TS103 (Fig. 5). These results demonstrate that TS103 inhibits hrpA and hrpN expression and HrpN protein production.

Table 3.

The expression of type III secretion system (T3SS) genes hrpA, hrpN, hrpS, hrpL and rpoN of D ickeya dadantii 3937 (3937) in T3SS‐inducing medium (MM) and MM supplemented with TS103 (MM103)

Strain Average MFI ± SD for growth in the indicated mediumb
12 h 24 h
MM MM103 %MMd MM MM103 %MMd
3937 (pPhrpA) 58.7 ± 6.1 8.9 ± 0.7a 15.2 66.5 ± 5.4 8.9 ± 0.2a 13.4
3937 (pPhrpN) 46.8 ± 2.9 5.8 ± 0.7a 12.4 49.9 ± 2.1 5.4 ± 0.3a 10.8
3937 (pPhrpS) 73.2 ± 0.6 27.7 ± 1.5a 37.8 90.9 ± 1.2 26.4 ± 0.6a 29.0
3937 (pPhrpL) 20.2 ± 1.8 7.6 ± 0.2a 37.6 21.5 ± 0.5 7.7 ± 0.2a 35.8
3937 (pPrpoN) 215.3 ± 26.1 175.8 ± 15.5 —c —
3937 (pAT) 4.2 ± 0.5 3.0 ± 0.6 5.7 ± 1.8 4.3 ± 0.6
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a

Statistically significant differences in green fluorescent protein (GFP) intensity between bacterial cells grown in MM (MM) and MM supplemented with 100 μm TS103 (MM103) (P < 0.01, Student's t‐test).

b

The promoter activities were compared in MM and MM supplemented with 100 μm TS103 at 12 and 24 h of bacterial growth. GFP mean fluorescence intensity (MFI) was determined for gated populations of bacterial cells by flow cytometry. Values are representative of two independent experiments, and three replicates were used for each experiment.

c

—, not determined.

d

The relative promoter activity of hrp genes in D. dadantii 3937 cells grown in MM supplemented with 100 μm TS103 compared with that in MM (indicated by ‘%MM’) was calculated by the formula: %MM = 100 × MFI(MM103)/MFI(MM).

Figure 4.

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Relative mRNA levels of hrpA, hrpN, hrpS, hrpL and rpoN as determined by quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR). Relative mRNA levels of hrpA, hrpN, hrpS, hrpL and rpoN genes of Dickeya dadantii 3937 in type III secretion system‐inducing medium (MM) supplemented with 100 μm TS103 (TS103) compared with that in MM (MM). Asterisks indicate statistically significant differences in mRNA level of cells grown in MM supplemented with 100 μm TS103 compared with that in MM. rplU was used as an endogenous control for data analysis (Pfaffl et al., 2002). The data are representative of two independent experiments. Three replicates were used in each experiment.

Figure 5.

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HrpN (A) and OpgG (B) protein expression of Dickeya dadantii 3937 in type III secretion system‐inducing medium (MM) and MM supplemented with TS103. Lane 1, D. dadantii 3937 grown in MM; lane 2, D. dadantii 3937 grown in MM supplemented with 100 μm TS103.

TS103 inhibits hrpL transcription through both HrpS and RpoN

In D. dadantii, HrpL is a master regulator that controls the expression of genes encoding T3SS ‐associated filamentous structure and harpin proteins (Chatterjee et al., 2002; Wei et al., 1992; Yang et al., 2010; Yap et al., 2005). We hypothesize that TS103 lowers the level of hrpL transcription, which further leads to a decrease in the expression of hrpA and hrpN. To test this, the promoter activity of hrpL was investigated in cells in the presence and absence of TS103. About a three‐fold decrease in hrpL promoter activity was observed in the cells grown in MM supplemented with TS103 compared with that in MM (Table 3), suggesting that TS103 inhibits hrpL at the transcriptional level. Previous reports have shown that HrpS, a σ54 enhancer‐binding protein, interacts with the σ54 (RpoN)‐containing RNA polymerase holoenzyme and initiates the transcription of hrpL (Chatterjee et al., 2002; Yap et al., 2005). As TS103 inhibits hrpL promoter activity, expression levels of hrpS and rpoN were examined in cells grown in MM supplemented with TS103 and in MM. Interestingly, a reduction in hrpS promoter activity was observed in the cells grown in MM supplemented with TS103 compared with that in MM, whereas rpoN transcriptional levels were similar (Table 3). qRT‐PCR analysis revealed a significant decrease in hrpS (relative expression ratio 0.178, P = 0.049) and rpoN (relative expression ratio 0.265, P = 0.046) mRNA levels in D. dadantii 3937 grown in MM amended with TS103 in comparison with that in MM (Fig. 4). These results suggest that TS103 inhibits hrpL transcription through both hrpS and rpoN.

TS103 inhibits hrpL at the post‐transcriptional level through rsmB

In addition to the regulation at the transcriptional level through HrpS and RpoN, hrpL is also regulated post‐transcriptionally by the RsmA–RsmB pair (Chatterjee et al., 2002; Liu et al., 1998). To determine whether TS103 inhibits the expression of rsmA and rsmB, a Northern blot was performed to examine the RNA levels of rsmA and rsmB in D. dadantii 3937 grown in MM with and without TS103. Similar levels of rsmA mRNA were observed in cells grown in MM with and without TS103 (Fig. 6A). However, significantly lower rsmB RNA levels were observed in cells grown in MM supplemented with TS103 in comparison with that in MM (Fig. 6B). These results indicate that TS103 inhibits hrpL at the post‐transcriptional level through rsmB.

Figure 6.

figure

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The relative RNA levels of rsmA and rsmB as determined by Northern blot. Cells were cultured in type III secretion system‐inducing medium (MM) or MM supplemented with 100 μm TS103 for 12 h before RNA isolation. rRNA was used as an internal control. (A) Lane 1, 3937 grown in MM; lane 2, 3937 grown in MM supplemented with TS103. (B) Lane 1, 3937 grown in MM; lane 2, 3937 grown in MM supplemented with TS103. (C) Lane 1, Δpnp grown in MM; lane 2, Δpnp grown in MM supplemented with TS103. (D) Lane 1, ΔcsrD grown in MM; lane 2, ΔcsrD grown in MM supplemented with TS103; lane 3, 3937 grown in MM. (E) Lane 1, ΔkdgR (pMLkdgR) grown in MM; lane 2, 3937 (pML123) grown in MM; lane 3, ΔkdgR (pML123) grown in MM; lane 4, ΔkdgR (pML123) grown in MM supplemented with TS103. Numbers below the Northern blots indicate the relative intensity of rsmA/rsmB  RNA provided by ImageJ.

Given that hrpS, rpoN and rsmB RNA levels were reduced in cells grown in MM supplemented with TS103, the hrpL mRNA level was further examined in cells grown in MM supplemented with TS103. qRT‐PCR analysis was performed and our data revealed that a significant reduction in the hrpL mRNA level (relative expression ratio 0.254, P < 0.05) was observed in cells grown in MM supplemented with TS103 compared with that in MM (Fig. 4). Together, these results suggest that TS103 inhibits hrpL through both the HrpS‐RpoN and rsmB‐RsmA pathways, and consequently lowers the expression of HrpL regulon genes, such as hrpA and hrpN.

TS103 inhibits hrpS transcription through phosphorylation of HrpY

The phosphorylation of HrpY is mandatory for the transcriptional activation of hrpS in D. dadantii and other phytopathogens (Nizan‐Koren et al., 2003; Yap et al., 2008). The aspartate residue at position 57 (D57) in HrpY has been proven to be the phosphorylation site needed for its activity in Pantoea stewartii ssp. stewartii and Erwinia herbicola pv. gypsophilae (Merighi et al., 2006; Nizan‐Koren et al., 2003). We hypothesize that the inhibition of HrpY phosphorylation by TS103 leads to low levels of hrpS transcript of D. dadantii 3937. To test this, ΔhrpYD57A was constructed by site‐directed mutagenesis, in which the conserved D57 in HrpY was changed by nonconservative substitution to alanine (D57A), and hrpS promoter activity was assayed in both the wild‐type and ΔhrpYD57A strains. A lower level of hrpS promoter activity was observed in ΔhrpYD57A in comparison with that in the wild‐type strain (Fig. 7). Moreover, similar levels of hrpS promoter activity were observed in ΔhrpYD57A and ΔhrpY (Fig. 7). As HrpX/HrpY is the TCSTS that activates the expression of hrpS (Tang et al., 2006), hrpS promoter activity was also determined in ΔhrpX. As expected, in ΔhrpX, the transcription of hrpS was reduced in comparison with that in the wild‐type strain (Fig. 7). Furthermore, the promoter activity of hrpS was restored to the wild‐type level when ΔhrpYD57A, ΔhrpY and ΔhrpX were complemented with pCLhrpXY in trans (Fig. 7). These results indicate that D57 of HrpY is the phosphorylation site required for the activity of HrpY in D. dadantii 3937. To further investigate whether TS103 inhibits hrpS transcription through its influence on the phosphorylation of HrpY, the promoter activity of hrpS was measured in ΔhrpYD57A grown in MM and MM supplemented with TS103. Similar levels of hrpS promoter activity were observed in ΔhrpYD57A grown in MM and MM supplemented with 100 μm TS103 (Table 4). Furthermore, similar levels of hrpS promoter activity were observed among ΔhrpYD57A, ΔhrpX and the wild‐type strain when they were grown in MM supplemented with 100 μm TS103 (Table 4). These results indicate that TS103 inhibits hrpS expression through its influence on the phosphorylation of HrpY.

Figure 7.

figure

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The hrpS promoter activity of Dickeya dadantii derivatives in type III secretion system‐inducing medium (MM) at 12 h of growth. Strains carrying the green fluorescent protein (GFP) reporter pPhrpS were used in this study. The promoter activities at 12 h of bacterial growth were determined. GFP mean fluorescence intensity (MFI) was determined for gated populations of bacterial cells by flow cytometry. Values are representative of two experiments, and three replicates were used for each experiment. Asterisks indicate statistically significant differences in GFP MFI between the wild‐type strain and mutants or complementary strains (P < 0.01, Student's t‐test).

Table 4.

The hrpS promoter activity of D ickeya dadantii 3937 (3937) and its derivatives in type III secretion system‐inducing medium (MM) and MM supplemented with TS103 (MM103)

Strain Average MFI ± SD for growth in the indicated mediumb
12 h 24 h
MM MM103 MM MM103
3937 92.0 ± 6.6 37.5 ± 0.9a 63.2 ± 6.4 28.5 ± 1.1a
ΔhrpYD57A 40.7 ± 1.1 35.2 ± 0.7 32.6 ± 1.8 26.4 ± 0.5
ΔhrpX 33.2 ± 2.0 34.2 ± 0.2 25.5 ± 0.6 25.4 ± 0.6
ΔgacA 94.2 ± 2.5 35.9 ± 1.6a 57.7 ± 3.0 24.5 ± 0.4a
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a

Statistically significant differences in green fluorescent protein (GFP) intensity between bacterial cells grown in MM (MM) and MM supplemented with 100 μm TS103 (MM103) (P < 0.01, Student's t‐test).

b

The promoter activities were compared in MM and MM supplemented with 100 μm TS103 at 12 and 24 h of bacterial growth. GFP mean fluorescence intensity (MFI) was determined for gated populations of bacterial cells by flow cytometry. Values are representative of two independent experiments, and three replicates were used for each experiment.

GacS/GacA is another TCSTS that regulates T3SS through the Rsm system (Tang et al., 2006), but has no regulatory effect on HrpS as no significant difference in hrpS promoter activity was observed between ΔgacA and its parental strain when they were grown in MM (Table 4). To demonstrate that TS103‐inhibited hrpS expression is HrpY specific, we compared the hrpS promoter activities in ΔgacA and the wild‐type D. dadantii 3937 when they were grown in MM supplemented with TS103. The addition of TS103 led to a similar reduction in hrpS promoter activity in the ΔgacA and wild‐type strains (Table 4), suggesting that the inhibition of hrpS by TS103 is HrpY specific and is not via GacS/GacA. Together, these results suggest that specific inhibition of HrpY phosphorylation by TS103 leads to low levels of hrpS transcript of D. dadantii 3937.

TS103 inhibits rsmB through unknown regulators

As a reduced level of rsmB RNA was observed when D. dadantii 3937 was grown in MM supplemented with TS103 compared with that in MM (Fig. 6B), the regulators of rsmB by TS103 were further investigated. The TCSTS GacS/GacA has been reported to be the transcriptional regulator of rsmB in several bacteria (Liu et al., 1999; Mukherjee et al., 2000; Tang et al., 2006). To confirm that rsmB is a GacA regulon gene in D. dadantii 3937, the promoter activity of rsmB in ΔgacA was examined. A dramatic reduction in rsmB promoter activity was observed in ΔgacA compared with the wild‐type strain (Table 5). Complementation of ΔgacA with a gacA gene coupled with its native promoter into the chromosome of the mutant strain at an intergenic region (ΔgacA::gacA) restored the transcription of rsmB to near wild‐type levels (Table 5). These results demonstrate that GacA is a transcriptional regulator of rsmB in D. dadantii 3937. To investigate whether TS103 inhibits the activity of GacS/GacA, and leads to a lower level of rsmB RNA, the promoter activity of rsmB was examined in the wild‐type and ΔgacA strains grown in MM and MM supplemented with TS103. Similar levels of rsmB promoter activity were observed in the cells grown in MM and MM supplemented with TS103 (Table 5). These results suggest that GacS/GacA positively regulates rsmB transcription in D. dadantii 3937 and the inhibition of TS103 on rsmB does not occur through the transcriptional regulator GacS/GacA.

Table 5.

The rsmB promoter activity of D ickeya dadantii 3937 (3937) and its derivatives in type III secretion system‐inducing medium (MM) and MM supplemented with 100 μm TS103 (MM103)

Strain Average MFI ± SD for growth in the indicated mediuma
6 h 12 h
MM MM103 MM MM103
3937 1525.3 ± 20.2 1603.4 ± 28.0 2531.4 ± 150.9 2628.6 ± 86.9
ΔgacA 74.6 ± 2.0 53.5 ± 1.4 86.0 ± 6.7 63.1 ± 4.0
ΔgacA::gacA 1956.2 ± 140.8 2052.3 ± 60.8 2779.6 ± 48.1 2905.1 ± 104.4
3937 (pML123) —b — 1724.8 ± 9.1 —
ΔkdgR (pML123) — — 1700.4 ± 12.0 —
ΔkdgR (pMLkdgR) — — 1582.2 ± 42.0 —
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a

The promoter activities were compared in MM and MM supplemented with 100 μM TS103 at 12 and 24 h of bacterial growth. Green fluorescent protein (GFP) mean fluorescence intensity (MFI) was determined for gated populations of bacterial cells by flow cytometry. Values are representative of two independent experiments, and three replicates were used for each experiment.

b

—, not determined.

PNPase plays an important role in reducing rpoN mRNA stability and rsmB RNA turnover, which, in turn, down‐regulates hrpL and HrpL regulon genes, such as hrpA and hrpN in D. dadantii 3937 (Zeng et al., 2010). As a significant reduction in rpoN mRNA and rsmB RNA levels was observed in cells grown in MM supplemented with TS103 compared with that in MM (Figs 4 and 6B), we speculate that TS103 may inhibit T3SS through PNPase. If this is the case, a similar rsmB RNA level should be observed in the pnp mutant (Δpnp) grown in MM and MM supplemented with TS103. To test this, Northern blot analysis was performed to compare the rsmB RNA level in Δpnp grown in MM with and without TS103. Unexpectedly, significantly smaller amounts of rsmB RNA were observed in the Δpnp cells grown in TS103 compared with that in MM (Fig. 6C), suggesting that the inhibition of TS103 on rsmB is not through PNPase.

CsrD, a regulator of RNA turnover, was found to be essential for the decay of the small RNAs CsrB (homologue of RsmB) and CsrC in Escherichia coli (Suzuki et al., 2006). Inactivation of csrD resulted in an increase in the csrB RNA level (Suzuki et al., 2006). In D. dadantii, an increased rsmB RNA level was also observed in ΔcsrD compared with the wild‐type strain (Fig. 6D), suggesting that CsrD also negatively regulates the rsmB RNA level in D. dadantii. To determine whether TS103 inhibits rsmB RNA through CsrD, Northern blot analysis was performed to compare the rsmB RNA levels in ΔcsrD grown in MM with and without TS103. Smaller amounts of rsmB RNA were observed in the ΔcsrD cells grown in MM supplemented with TS103 compared with that in MM (Fig. 6D). These results suggest that csrD negatively regulates rsmB RNA in D. dadantii 3937 and the inhibition of TS103 on rsmB is not through CsrD.

Our recent work has demonstrated that OpgG, a component of Osmo‐regulated periplasmic glucans, positively regulates rsmB at the post‐transcriptional level in D. dadantii (X. Wu et al., in press). To test whether the inhibition of TS103 on rsmB is through OpgG, the original promoter and the whole open reading frame (ORF) of opgG were fused in frame with the His × 6 tag, and the OpgG protein level of the wild‐type cells grown in MM and MM supplemented with TS103 was examined by Western blot. Similar levels of the OpgG protein were observed in the cells grown in MM and MM supplemented with TS103 (Fig. 5B), suggesting that the inhibition of TS103 on rsmB is not through OpgG.

The IcIR‐like regulator KdgR has been reported to be a regulator of rsmB in Pectobacterium (Liu et al., 1999). To study whether KdgR is a regulator of rsmB in D. dadantii 3937, and whether TS103 inhibits rsmB through KdgR, the promoter activity and RNA level of rsmB in ΔkdgR and the wild‐type strains were examined. Similar levels of rsmB promoter and RNA were observed in ΔkdgR compared with that in the wild‐type strain grown in MM (Table 5 and Fig. 6E). Furthermore, significantly smaller amounts of rsmB RNA were observed in ΔkdgR cells grown in MM supplemented with TS103 compared with that in MM (Fig. 6E). These results suggest that KdgR is not a regulator of rsmB in D. dadantii 3937 and the inhibition of TS103 on rsmB is not through KdgR.

Discussion

The T3SS is an essential virulence factor of many Gram‐negative bacterial pathogens. This secretion system has emerged as an attractive target for small‐molecule anti‐virulence therapeutics (Cegelski et al., 2008; Duncan et al., 2012; Escaich, 2008). Recently, a number of T3SS inhibitors have been identified in multiple bacterial species, including Yersinia spp., Chlamydia spp., Salmonella spp., Pseudomonas aeruginosa, Erwinia amylovora and D. dadantii, using screening‐based approaches under interdisciplinary efforts between chemists and microbiologists (Hudson et al., 2007; Khokhani et al., 2013; Li et al., 2009; Muschiol et al., 2006; Pan et al., 2007; Yamazaki et al., 2012). The T3SS regulatory pathways and regulatory components of D. dadantii are well understood (Lebeau et al., 2008; Li et al., 2010; Yang et al., 2008b, 2010; Yi et al., 2010; Zeng et al., 2010). In this study, D. dadantii 3937 was used to screen high‐potency T3SS inhibitors and to identify inhibitor targets in the T3SS pathways. The compound TS103 was found to be a highly potent inhibitor of the T3SS master regulator HrpL via HrpX‐HrpY and the rsmB‐RsmA regulatory pathway. Although no growth inhibition was observed in TS103 at low concentrations (1 or 10 μm), a further study showed that a slight promotion of bacterial growth was observed in TS103 at higher concentrations (100 μm) (Figs S2 and S3). This indicates that D. dadantii 3937 may possibly use TS103 as a carbon or energy source for growth. Nevertheless, TS103 dramatically inhibits the T3SS at all three concentrations used in this study. It is worth mentioning that TS103 also plays a role in inhibiting the T3SS of other hrp Group I phytobacteria, such as E. amylovora (Khokhani et al., 2013). However, the effect of TS103 on the T3SS of hrp Group II phytobacteria remains to be determined.

Bacteria use a wide variety of mechanisms to sense and respond to environmental changes, with TCSTSs being the dominant methods (Calva and Oropeza, 2006). The signal transduction lies in the recognition and interpretation of environmental signals that are related to host infection, and conversion of these signals into specific protein–protein interactions and transcriptional activation (Calva and Oropeza, 2006; Hoch, 2000). HrpX/Y is a TCSTS encoded within the hrp gene cluster of D. dadantii. HrpX, a transmembrane sensor histidine kinase, senses environmental stimuli and activates its cognate response regulator HrpY by phosphorylation. The phosphorylated HrpY then binds the hrpS promoter and promotes the transcription of hrpS (Yap et al., 2008). In P. stewartii ssp. stewartii and E. herbicola pv. gypsophilae, the aspartate residue at position 57 has been proven to be the phosphorylation site of HrpY (Merighi et al., 2006; Nizan‐Koren et al., 2003). Although there is no direct evidence on the phosphorylation site of HrpY in D. dadantii, the HrpY protein without acetyl phosphate treatment was unable to bind the hrpS promoter, suggesting that in vitro phosphorylation is required for HrpY activity in D. dadantii (Yap et al., 2008). In this study, conservative and structurally neutral amino acid substitutions of aspartate to alanine at position 57 reduced the expression of hrpS (Table 4 and Fig. 7). In agreement with the observations in P. stewartii ssp. stewartii and E. herbicola pv. gypsophilae, our data suggest that D57 is needed for the activity of HrpY in D. dadantii 3937 and that D57 is the phosphorylation site of HrpY. Moreover, a similar level of hrpS promoter activity was observed in ΔhrpYD57A grown in MM and MM supplemented with 100 μm TS103 (Table 4), indicating that TS103 inhibits hrpS transcription through the phosphorylation of HrpY. This is the first report of a phenolic compound targeting the specific phosphorylated response regulator HrpY to inhibit the T3SS.

Rsm, also known as Csr in E. coli, is one of the most studied post‐transcriptional regulators in bacteria (Romeo et al., 2012; Timmermans and Van Melderen, 2010). The Rsm/Csr system is present in many plant‐ and animal‐associated pathogenic bacteria (Babitzke and Romeo, 2007; Bejerano‐Sagie and Xavier, 2007; Liu et al., 1998; Suzuki et al., 2002; Toledo‐Arana et al., 2007). It is composed of two regulatory components: the RNA‐binding protein (RsmA in soft rot phytopathogens, RsmA and RsmE in Pseudomonas, CsrA in E. coli) and non‐coding regulatory small RNAs (RsmB in soft rot phytopathogens, RsmY and RsmZ in Pseudomonas, CsrB in E. coli) (Gudapaty et al., 2001; Romeo, 1998). The central component of the Rsm/Csr systems is a homodimeric RNA‐binding protein (CsrA or RsmA), which either represses or activates the expression of target mRNAs post‐transcriptionally (Babitzke and Romeo, 2007; Mercante et al., 2009). A common feature of Rsm/Csr systems is that a TCSTS is responsible for the activation of the transcription of each small RNA in response to an unknown signal(s) (Babitzke and Romeo, 2007). In D. dadantii, the conserved TCSTS GacA/GacS regulates the small RNA RsmB at the transcriptional level (Table 5). In this TCS, GacS, a tripartite sensor histidine kinase, senses environmental stimuli and activates its cognate response regulator, GacA, by phosphorylation, which, in turn, induces the expression of the regulatory small RNA RsmB (Blumer et al., 1999; Cui et al., 2001; Heeb and Haas, 2001). rsmB transcripts then bind to and sequester RsmA, which eventually affects the expression of downstream genes (Chatterjee et al., 2002; Liu et al., 1998). In P. aeruginosa, the authors deduced that TS103 affects the transcripts of the small RNAs rsmY and rsmZ via the activation of GacA through GacS and/or other two‐component sensor proteins that cross‐talk to GacS/GacA (Yamazaki et al., 2012). Although we speculate that the strong negative effect of TS103 on rsmB of D. dadantii might involve the interference of GacS/GacA, our observations do not support such a hypothesis, because TS103 has no inhibitory effect on rsmB promoter activity in the wild‐type strain, whereas rsmB promoter activity was dramatically reduced in ΔgacA compared with that in the wild‐type strain (Table 5).

RNA turnover is an important process in the regulation of gene expression and is tightly regulated (Mata et al., 2005). In E. coli, RNA decay is often initiated by an endoribonuclease, RNase E, which preferentially binds to the 5′ monophosphorylated terminus of transcripts, with cleavage occurring in A/U‐rich regions adjacent to stem‐loop structures. The resulting cleavage products are then rapidly degraded by the processive 3′ to 5′ exoribonucleases PNPase and RNase II (Carpousis, 2007; Romeo et al., 2012). Recently, the turnover of the small RNAs CsrB and CsrC in E. coli was reported to require a novel regulator, CsrD, in addition to RNase E and PNPase (Romeo et al., 2012; Suzuki et al., 2006). In D. dadantii, PNPase also plays an important role in rsmB RNA turnover (Zeng et al., 2010). Consistent with our previous work (X. Wu et al., in press), an increased rsmB RNA level was observed in ΔcsrD compared with that in the wild‐type strain (Fig. 6E), suggesting that CsrD negatively regulates the rsmB RNA level in D. dadantii. However, smaller amounts of rsmB RNA were observed in both Δpnp and ΔcsrD cells grown in MM supplemented with TS103 compared with that in MM (Fig. 6C,D), suggesting that the inhibition of TS103 on rsmB RNA is not through CsrD and PNPase. In addition, our recent work found that CsrD regulates the rsmB RNA level through OpgG in D. dadantii (X. Wu et al., in press). In this study, similar levels of OpgG protein were observed in the cells grown in MM and MM supplemented with TS103 (Fig. 5B), suggesting that the inhibition of TS103 on rsmB is not through CsrD. Overall, the knowledge of the post‐transcriptional regulators of the small RNA RsmB is limited. Moreover, the regulation of RsmB by TS103 remains to be determined.

In summary, this study screened a series of derivatives of plant phenolic compounds and identified that TS103 has the highest inhibitory potency on T3SS of D. dadantii. The effect of TS103 on the regulatory components of the T3SS was further elucidated and revealed that the inhibition goes through both the HrpX/Y‐HrpS‐HrpL and rsmB‐RsmA‐HrpL regulatory pathways. To our knowledge, this is the first inhibitor which affects the T3SS through both transcriptional and post‐transcriptional pathways in the soft‐rot pathogen D. dadantii 3937.

Experimental Procedures

Bacterial strains, plasmids, primers and growth conditions

The bacterial strains and plasmids used in this study are listed in Table 1. Escherichia coli and D. dadantii were grown in Luria–Bertani (LB), MM or mannitol–glutamic acid (MG) medium at 37 °C and 28 °C, respectively (Yang et al., 2007, 2008b; Zeng et al., 2010; Zou et al., 2012). Medium was supplemented with chloramphenicol (20 μg/mL), ampicillin (100 μg/mL), kanamycin (50 μg/mL), spectinomycin (50 μg/mL) and gentamicin (15 μg/mL) when required. The primers used for PCR in this work are listed in Table S1 (see Supporting Information).

Sources of the screened compounds

Compounds TS32 and TS108–TS113 were purchased from commercial sources (Sigma‐Aldrich, Louis, MO, USA; Alfa Aesar, Ward Hill, MA, USA or TCI, Cambridge, MA, USA). TS37, TS39–TS42, TS44, TS46 and TS47 were synthesized by the methods described in Methods S1. The remaining compounds were synthesized via the routes described in our recent publications (Khokhani et al., 2013; Yamazaki et al., 2012). DMSO stock solutions were prepared and stored at −20 °C. The compounds were added at a final concentration of 100 μm, except when indicated otherwise. DMSO was used as a control (indicated by ‘MM’).

Flow cytometry analysis

Promoter activities of hrpA, hrpN, hrpS, hrpL, rsmB and rpoN were measured by flow cytometry as described previously (Peng et al., 2006). Briefly, the bacterial cells carrying the promoter–GFP transcriptional fusion plasmid were cultured in LB broth at 28 °C overnight and subcultured 1:100 in MM and MM supplemented with compounds in 20‐mL glass culture tubes. Samples were diluted to the appropriate concentration with 1 × phosphate‐buffered saline (PBS) at 12 and 24 h after inoculation. The promoter activities were analysed by measuring the GFP intensity using flow cytometry (BD Biosciences, San Jose, CA, USA). Meanwhile, the optical density of the samples was also measured at 600 nm (OD600) when required.

Construction of plasmids and mutants

The ΔgacA, ΔhrpY and ΔkdgR deletion mutants were constructed by marker exchange mutagenesis (Yang et al., 2002). Briefly, two fragments flanking each target gene were obtained. A kanamycin cassette, amplified from pKD4 (Table 1), was ligated with these two fragments and cloned into the BamHI and XhoI sites in pWM91. This construct was transferred into D. dadantii 3937 by conjugation using E. coli S17‐1λpir. To select strains with chromosomal deletions, transconjugants with kanamycin and ampicillin resistance were plated onto MG containing 5% sucrose and kanamycin. Colonies having sucrose and kanamycin resistance and ampicillin sensitivity were isolated and confirmed by PCR using outside primers.

ΔhrpYD57A, in which the conserved aspartate residue at position 57 in HrpY was changed by nonconservative substitution to alanine, was constructed in a similar manner as above using plasmid phrpYD57A as the PCR template to obtain the flanking fragments, and a kanamycin cassette was placed at the 3′ end of the hrpY ORF for selection. The plasmid phrpYD57A was constructed as follows. A 1830‐bp fragment containing the hrpY ORF and its flanking region was amplified using primers hrpYH1 and hrpYH2 and 3937 genome DNA as the template. The PCR fragment was ligated into pGEM‐T Easy vector to generate plasmid phrpY. phrpYD57A, bearing a D57A change in hrpY primary amino acid sequence, was generated by a QuikChange Lightning Site‐Directed Mutagenesis Kit (Agilent Technologies, La Jolla, CA, USA) according to the manual, using the primers hrpYD57A‐Frd and hrpYD57A‐RC and phrpY as the template.

To construct plasmids for complementation, the ORF and promoter region of target genes were amplified and cloned into low‐copy‐number plasmids pML123 or pCL1920 (Table 1). gacA was inserted into the chromosome using allelic exchange mutagenesis for complementation. To construct plasmids to complement the gacA mutation, the promoter and coding regions of gacA were PCR amplified and cloned into the chromosomal integration vector pTCLSCm (Jahn et al., 2008; Yap et al., 2008). The resulting plasmid was electrotransformed into ΔgacA and allelic exchange was used to insert gacA into the chromosome to generate the complementation strain ΔgacA::gacA (Jahn et al., 2008). All of the constructs and mutants described above were verified by PCR and DNA sequencing.

Northern blot analysis

About 10 μg of RNA of D. dadantii in MM and MM supplemented with TS103 were determined using Northern blot as described previously (Li et al., 2009). In brief, D. dadantii was grown in MM and MM supplemented with TS103 for 12 h, total RNA was isolated using TRI reagent and residual DNA was removed with the TURBO DNA‐free kit (Ambion, Austin, TX, USA). RNA samples were analysed using biotin‐labelled probe and a biotin detection system (BrightStar Psoralen–Biotin and Bright Star BioDetect, Ambion). rRNA was used as an internal control.

qRT‐PCR analysis

The mRNA levels were measured by qRT‐PCR. Dickeya dadantii 3937 was cultured in MM or MM supplemented with 100 μm TS103 for 12 h. Cells were harvested and total RNA was isolated as described previously (Li et al., 2009; Yang et al., 2008a). The cDNA level of target genes in different samples was quantified by real‐time PCR using a Real Master Mix (Eppendorf, Westbury, NY, USA), as described previously (Peng et al., 2006). Data were analysed using a Relative Expression Software Tool (Pfaffl et al., 2002). The expression level of rplU was used as an endogenous control for data analysis (Mah et al., 2003).

Western blot analysis

Proteins were separated by 12.5% sodium dodecylsulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and transferred to an Immobilion poly(vinylidene difluoride) (PVDF) transfer membrane (Millipore, Bedford, MA, USA) using a Trans‐Blot SD Semi‐Dry Transfer Cell (Bio‐Rad, Hercules, CA, USA). The blot (immunoblot) was then probed with an anti‐HrpN peptide antibody (1:5000) or anti‐His polyclonal antibody (1:2000; Southern Biotech, Birmingham, AL, USA). The blot was incubated for 2 h with the primary antibody and then washed in PBS containing 0.1% Tween 20. Antigen–antibody complexes were visualized by incubation of the blots in a 1:5000 dilution of horseradish peroxidase‐conjugated goat anti‐rabbit immunoglobulin G secondary antibody (Abcam, Cambridge, MA, USA) using an ECL detection system (GE Healthcare UK Ltd, England, UK).

Supporting information

Fig. S1 The relative promoter activity of hrpA in Dickeya dadantii 3937 cells grown in type III secretion system‐inducing medium (MM) supplemented with 10 μm of the indicated compounds compared with that in MM (indicated by ‘%MM’). Green fluorescent protein (GFP) mean fluorescence intensity (MFI) was determined for gated populations of bacterial cells by flow cytometry. %MM was calculated by the formula: %MM = 100 × MFI(compound)/MFI(MM). Asterisks indicate statistically significant difference in hrpA promoter activity of D. dadantii 3937 cells grown in MM supplemented with 10 μm of the indicated compounds compared with that in MM. Two independent experiments were performed and three replicates were used for each experiment.

Fig. S2 The growth of Dickeya dadantii 3937 in type III secretion system‐inducing medium (MM) and MM supplemented with TS103 at different concentrations at 12 and 24 h. To study the effect of TS103 on bacterial growth, 50 μL of bacterial suspension [optical density at 600 nm (OD600) = 1.0] was used as the initial inoculum and added to 5 mL of MM and MM supplemented with TS103. The growth of D. dadantii 3937 was recorded. Results from one representative experiment are shown. Three replicates were used in this experiment, and the experiment was repeated twice.

Fig. S3 The growth curve of Dickeya dadantii 3937 in type III secretion system‐inducing medium (MM) supplemented with TS103 at different concentrations. To detect the growth curve of D. dadantii 3937 in MM supplemented with TS103 at concentrations of 0, 1, 10 and 100 μm, overnight cultured bacteria were added to MM and MM supplemented with TS103 [initial inoculum optical density at 600 nm (OD600) = 0.01]. The OD600 of D. dadantii 3937 was recorded at 2‐h intervals. Results from one representative experiment are shown. Three replicates were used in this experiment, and the experiment was repeated twice.

Table S1 Primers used in this study.

Methods S1 General procedure for the preparation of cinnamyl amine derivatives TS37, TS39–TS42, TS44, TS46 and TS47.

Click here for additional data file. (207.5KB, doc)

Acknowledgements

This work is dedicated to Noel T. Keen. This project was supported by grants from the National Science Foundation (award no. EF‐0332163), the Research Growth Initiative of the University of Wisconsin‐Milwaukee, the Catalyst Grant in advanced Automation of UWM Research Foundation for Ching‐Hong Yang, the National Science Foundation of China (grant No. 21272029) for Xin Chen, the 948 Project of the Ministry of Agriculture, China (grant No. 2011‐S8) for Yan Li, and the 111 project B13006, China.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Fig. S1 The relative promoter activity of hrpA in Dickeya dadantii 3937 cells grown in type III secretion system‐inducing medium (MM) supplemented with 10 μm of the indicated compounds compared with that in MM (indicated by ‘%MM’). Green fluorescent protein (GFP) mean fluorescence intensity (MFI) was determined for gated populations of bacterial cells by flow cytometry. %MM was calculated by the formula: %MM = 100 × MFI(compound)/MFI(MM). Asterisks indicate statistically significant difference in hrpA promoter activity of D. dadantii 3937 cells grown in MM supplemented with 10 μm of the indicated compounds compared with that in MM. Two independent experiments were performed and three replicates were used for each experiment.

Fig. S2 The growth of Dickeya dadantii 3937 in type III secretion system‐inducing medium (MM) and MM supplemented with TS103 at different concentrations at 12 and 24 h. To study the effect of TS103 on bacterial growth, 50 μL of bacterial suspension [optical density at 600 nm (OD600) = 1.0] was used as the initial inoculum and added to 5 mL of MM and MM supplemented with TS103. The growth of D. dadantii 3937 was recorded. Results from one representative experiment are shown. Three replicates were used in this experiment, and the experiment was repeated twice.

Fig. S3 The growth curve of Dickeya dadantii 3937 in type III secretion system‐inducing medium (MM) supplemented with TS103 at different concentrations. To detect the growth curve of D. dadantii 3937 in MM supplemented with TS103 at concentrations of 0, 1, 10 and 100 μm, overnight cultured bacteria were added to MM and MM supplemented with TS103 [initial inoculum optical density at 600 nm (OD600) = 0.01]. The OD600 of D. dadantii 3937 was recorded at 2‐h intervals. Results from one representative experiment are shown. Three replicates were used in this experiment, and the experiment was repeated twice.

Table S1 Primers used in this study.

Methods S1 General procedure for the preparation of cinnamyl amine derivatives TS37, TS39–TS42, TS44, TS46 and TS47.

Click here for additional data file. (207.5KB, doc)

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