BACKGROUND:The type III secretion system (T3SS) is a key virulence determinant in Dickeya species and is essential for successful host invasion and symptom development. To identify effective antivirulence agents, a series of novel hybrid molecules was designed and synthesized by linking cinnamic acid with hordenine. These compounds were evaluated for their ability to inhibit the T3SS of Dickeya oryzae EC1 without impairing bacterial growth. RESULTS:A total of 22 hybrid derivatives were synthesized. Among them, compound I-3 significantly reduced the promoter activity of the T3SS gene hrpA without inhibiting the growth of D. oryzae EC1. Mechanistic analyses revealed that compound I-3 effectively suppressed the EC1-triggered hypersensitive response in tobacco, down-regulated the expression of multiple T3SS-related genes, and inhibited the secretion of type III effector proteins. In planta assays further confirmed that compound I-3 attenuated the virulence of D. oryzae EC1 on rice and alleviated the disease symptoms caused by Dickeya dadantii 3937 in potato and Dickeya fangzhongdai CL3 in taro, indicating a broad-spectrum antivirulence activity. CONCLUSION:This study identifies compound I-3 as a promising hybrid molecule that suppresses T3SS activity in D. oryzae EC1 and reduces the virulence of multiple Dickeya species across diverse host plants. These findings provide both a theoretical foundation and practical guidance for the development of novel T3SS-targeting antivirulence agents to control soft rot diseases in crops. © 2026 Society of Chemical Industry.

Xanthomonas citri subsp. citri (Xcc), the pathogen responsible for citrus canker, causes major economic damage worldwide. The type III secretion system (T3SS) is essential for Xcc pathogenicity. Here, 39 chiral 1,3,4-oxadiazole sulfide derivatives of mandelic acid were synthesized, with 17 compounds exhibiting significant inhibitory activity (>70% inhibition) against the T3SS of Xcc jx-6 at 40 μg/mL. Among these, the R-configuration WR4 significantly suppressed the symptoms of citrus canker in an in vivo bioassay, whereas neither the S-configuration WS4 nor the racemic mixture WRS4 showed inhibitory effects. Mechanistically, WR4 suppressed T3SS gene transcription and Hpa1 protein secretion, and counteracted pathogen-induced suppression of host immunity, without affecting other virulence factors. From an environmental safety perspective, WR4 exhibited low toxicity against nontarget organisms, including earthworms, silkworms, and zebrafish. These findings reveal the potential of the R-configuration derivatives as sustainable T3SS inhibitors.

Bacterial wilt caused by causes severe crop losses, yet effective agrochemicals remain scarce. Here, we designed a T3SS-targeting antivirulence agent that attenuates the pathogen virulence without inhibiting growth. Two series of mandelic acid and 2-amino-2-phenylacetic acid derivatives containing 1,3,4-oxadiazolyl thioethers were synthesized. High-throughput screening identified W40 (2-(4-chlorophenyl)-2-hydroxy-N-(5-((4-methylbenzyl)thio)-1,3,4-oxadiazol-2-yl)acetamide), which strongly inhibited hrpY expression without affecting bacterial growth. W40 exhibited significant protective activity against infection in tomato and casuarina. qRT-PCR analysis showed that W40 suppressed T3SS-associated regulatory networks by downregulating key regulators, including prhG, phcA, prhA, and prhR. Phenotypic assays revealed that W40 impaired swimming motility but did not affect the extracellular cellulase activity, biofilm formation, or exopolysaccharide production. These findings highlight W40 as a promising lead compound for antivirulence therapy against bacterial wilt.

Citrus canker, caused by Xanthomonas citri subsp. citri (Xcc) remains a serious constraint on citrus production. To explore antivirulence strategies, 75 selenium-containing diselenides and monoselenides were synthesized as a bioisosteric extension of previously reported sulfur-based scaffolds and screened using an hpa1 promoter-lux reporter to evaluate T3SS inhibition. Three monoselenides (II6, II19, and II37) markedly inhibited the hpa1 promoter activity. Among them, II19 and II37 suppressed T3SS-dependent phenotypes, including the hypersensitive response in Nicotiana benthamiana and canker lesions on citrus, without affecting bacterial growth in vitro. II19 also improved disease suppression when used together with the DSF-degrading biocontrol strain Burkholderia anthina HN-8. Transcriptome analysis, supported by qRT-PCR and Western blotting, confirmed that II19 downregulates key hrp/hc genes and limits Hpa1 secretion. Toxicity assays in silkworms and earthworms revealed no adverse effects at concentrations exceeding the practical use levels. These findings highlight monoselenides as environmentally acceptable T3SS inhibitors suitable for integrated citrus canker management.

Erwinia amylovora, the causal agent of fire blight, poses a severe and ongoing threat to the global production of Rosaceous crops. We synthesized 54 garlic-derived disulfides and identified compound b as the most potent candidate (EC50 = 2.85 μg/mL), which significantly surpassed allicin and zhongshengmycin (ZSM). In planta assays on Cuiguan pear demonstrated that b provided exceptional curative and protective efficacy, effectively suppressing fire blight infections in flowers, leaves, shoots, and fruits. Mechanistically, b triggered lethal ROS accumulation by inhibiting catalase (CAT) and superoxide dismutase (SOD) activities, leading to compromised cell envelope and ultrastructural damage in E. amylovora. Notably, b attenuated bacterial virulence by suppressing swimming and swarming motility, reducing biofilm formation, and decreasing extracellular cellulase activity, while also inhibiting DNA and protein biosynthesis. Additionally, b exhibited minimal toxicity toward nontarget organisms, such as silkworms (Bombyx mori) and earthworms (Eisenia fetida), highlighting its environmental safety. These findings establish compound b as a highly effective, safe, and eco-friendly candidate for sustainable fire blight management.

Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of rice bacterial leaf blight, relies on a type III secretion system (T3SS) for virulence. Using chemical inhibitors to suppress T3SS without affecting bacterial growth is a novel strategy that minimizes disturbance of the innate leaf microbiome. (Z)-N-(3-(5-(2-nitrophenyl) furan-2-carbonyl) thiazolidin-2-ylidene) cyanamide (II-2) is a chemical inhibitor of T3SS in Xoo and an effective control alternative. This study evaluated whether II-2 treatment and Xoo infection alter the leaf microbiome. Using 16S rRNA gene sequencing, Xoo infection markedly reshaped the leaf microbiome, shifting the dominance from Proteobacteria to Pseudomonadales and Xanthomonadales. II-2 influenced bacterial communities in both healthy and Xoo-infected leaves, with low-dose treatment enriching Gram-negative taxa in diseased leaves, whereas the high dose enriched Sphingomonas in healthy leaves. In healthy plants, higher II-2 doses preserved microbial stability, suggesting minimal ecological disturbance. II-2 exhibited no toxicity in silkworms or earthworms, functioning as a T3SS-targeting, microbiome-compatible antivirulence agent.

This study designed and synthesized 33 novel imidazole ethanol derivatives and evaluated their efficacy against plant bacterial diseases. Most compounds exhibited excellent antibacterial activity. Particularly, compound D11 showed potent in vitro activity against Xanthomonas oryzae pv oryzae (EC50 = 0.51 μg/mL), surpassing bismerthiazol (EC50 = 3.84 μg/mL). In vivo, D11 effectively controlled rice bacterial leaf blight, with protective and curative effects of 82.8% and 73.1% at 200 μg/mL, both exceeding those of bismerthiazol (56.1% and 52.2%). Mechanistic investigations demonstrated that D11 induces elevated levels of reactive oxygen species, suppresses and compromises the cellular integrity of Xoo, and concurrently downregulates the expression of associated virulence factors, thereby implying a potential multitarget mechanism of action. Notably, D11 exhibits favorable selectivity toward nontarget organisms (earthworms and silkworms). These findings highlight D11 as a promising, environmentally friendly, and highly effective candidate for managing rice bacterial leaf blight.

Bacterial wilt caused by Ralstonia solanacearum causes severe crop losses, yet effective agrochemicals remain scarce. Here, we designed a T3SS-targeting antivirulence agent that attenuates the pathogen virulence without inhibiting growth. Two series of mandelic acid and 2-amino-2-phenylacetic acid derivatives containing 1,3,4-oxadiazolyl thioethers were synthesized. High-throughput screening identified W40 (2-(4-chlorophenyl)-2-hydroxy-N-(5-((4-methylbenzyl)thio)-1,3,4-oxadiazol-2-yl)acetamide), which strongly inhibited hrpY expression without affecting bacterial growth. W40 exhibited significant protective activity against R. solanacearum infection in tomato and casuarina. qRT-PCR analysis showed that W40 suppressed T3SS-associated regulatory networks by downregulating key regulators, including prhG, phcA, prhA, and prhR. Phenotypic assays revealed that W40 impaired swimming motility but did not affect the extracellular cellulase activity, biofilm formation, or exopolysaccharide production. These findings highlight W40 as a promising lead compound for antivirulence therapy against bacterial wilt.

In this study, we evaluated the inhibitory effects of cinnamic acid and its 18 commercially available derivatives on phosphodiesterase 4 (PDE4) to investigate their therapeutic potential for chronic obstructive pulmonary disease (COPD). Among the tested compounds, p-coumaric acid and trans-4-methoxycinnamic acid exhibited potent PDE4B inhibitory activity (IC50 = 2.2 μM and 8.2 μM, respectively) and notable selectivity over PDE4D, with IC50 values against TNF-α release in human mononuclear cells of 21.5 μM and 30.8 μM, respectively, comparable to rolipram. Structure-activity relationship (SAR) analysis indicated that para-substituted derivatives generally showed higher activity than their meta- or ortho-substituted counterparts. A validated CoMSIA model (q 2 = 0.514, r 2 = 0.971) highlighted the importance of electrostatic properties, revealing that electron-donating groups at the para position enhance inhibitory activity. Molecular docking illustrated that active derivatives bind in the PDE4B active site, forming key interactions with Gln443 and His234, which was refined by molecular dynamics simulations and free energy calculations. For p-coumaric acid, binding is primarily driven by a strong hydrogen bond with His234, whereas for trans-4-methoxycinnamic acid, enhanced hydrophobic interactions within the M pocket compensate for the lack of this hydrogen bond, revealing a dual mechanism for high-affinity binding. In vivo studies further confirmed significant anti-inflammatory effects, where p-coumaric acid inhibited TNF-α release by 41.1% and LPS-induced neutrophilia by 32.5%. Additionally, in silico ADMET profiling predicted favorable drug-like properties including high oral bioavailability and low CNS penetration, while identifying CYP2C8 inhibition as an optimizable liability. These integrated results underscore monosubstituted cinnamic acids, especially para-hydroxy and para-methoxy derivatives, as privileged scaffolds for developing novel PDE4B-targeted therapeutics for COPD.

The rice foot rot disease caused by Dickeya oryzae is an important bacterial disease that could cause tremendous economic losses. The virulence factor modulating cluster (Vfm) quorum sensing (QS) system, a major virulence regulatory mechanism conserved in the Dickeya genus, controls the production of zeamines and various extracellular cell wall degradation enzymes in D. oryzae. In this study, we targeted the Vfm QS system of D. oryzae strain EC1 to screen for chemical compounds capable of attenuating its virulence. Using a high-throughput vfmE-lux reporter assay, we screened over 2000 compounds and identified a series of long aliphatic chain compounds - quaternary ammonium cationic surfactants (QACs) that substantially reduced the vfmE promoter activity. Further analysis revealed that QACs blocked strain EC1 response to Vfm signals. Quantitative PCR results showed that the transcriptional expression of the vfm gene cluster and its downstream virulence genes, including those encoding production of phytotoxin zeamines and CWDEs were significantly downregulated. Consistent with their role in blocking Vfm signaling, QACs markedly inhibited the production of virulence determinant zeamines and suppressed the secretion of cellulase and pectinase in strain EC1. Infection assays demonstrated that these compounds substantially reduced the virulence of D. oryzae EC1 toward rice seeds and alleviated the soft rot symptoms in potato tubers. This is the first report that QACs are potent inhibitors of the D. oryzae Vfm QS system, illustrating a promising QS-targeted approach for the control of bacterial rice foot rot disease.

[Objective]To screen highly effective fungicides against rice leaf blight caused by the new pathogen Pantoea ananatis.[Method]Total 16 fungicides were screened via in vitro antibacterial experiments.The effects of three commercial agents on P.ananatis were systematically evaluated by apoptosis,reactive oxygen species(ROS)accumulation and in vivo efficacy.[Result]Both 3 g/L tetramycin AS and 80%(ω)ethylicin EC had good antibacterial effects against P.ananatis,with EC50 of 0.76 and 3.70 μg/mL,respectively.Further mechanism research found that 3 g/L tetramycin AS obviousily induced apoptosis of P.ananatis,increasing the early apoptosis rate to 55.89%,and markedly boosted the level of ROS,causing a marked rise in fluorescence intensity.In the efficacy test of rice bacterial leaf blight caused by P.ananatis,the protective activity of 80%ethylicin EC reached 73.18%,significantly outperforming 3 g/L tetramycin AS(60.71%).Conversely,3 g/L tetramycin AS showed remarkable therapeutic activity at 69.32%.[Conclusion]Tetracymycin can be used to treat the disease effectively by inducing apoptosis and ROS accumulation after the occurrence of the disease,while the outstanding protective activity of ethylicin is more suitable for early prevention.

Pseudomonas aeruginosa is a widely encountered bacterium linked to the deterioration of food products and represents a notable concern for public health safety. Disulfides serve as significant pharmacologically active scaffolds exhibiting antibacterial, antiviral, and anticancer properties; however, reports on their activity as quorum sensing inhibitors (QSIs) against P. aeruginosa are limited. In our work, asymmetrical disulfides were designed and synthesized, utilizing natural products, such as allicin, ajoene, diallyl disulfide (DADS), hordenine, and cinnamic acid, as lead compounds. By screening for lasB, rhlA, and pqsA promoter activity, two highly effective QSIs were identified. Compounds 7d and 4c show effectiveness in reducing the synthesis of different virulence factors, the creation of biofilms, and movement capabilities. Subsequent validation using the Galleria mellonella larvae model confirmed their robust in vivo efficacy. Moreover, their combination with antibiotics markedly augmented the antibacterial activity. Mechanism studies employed by transcriptome analysis, quantitative reverse transcription-PCR (qRT-PCR), surface plasmon resonance, and molecular docking demonstrate that compound 7d disrupts the quorum sensing system by interacting with PqsR. These findings suggest that our disulfide derivatives hold promise for treating P. aeruginosa infections.

Cancer remains a leading cause of mortality worldwide, and new chemical leads are essential for developing potent anticancer therapies. Evidence suggests that Pseudomonas aeruginosa (Pa) may suppress tumorigenesis, although the underlying mechanisms remain largely unclear. This study characterized a novel small molecule quinolone, JH62 (E-2-(tridec-4-en-1-yl)-quinolin-4(1H)-one, C22H31NO), from Pa. JH62 exhibited broad-spectrum anticancer activity, inhibiting the proliferation of A549 lung cancer cells in a time- and dose-dependent manner with an IC50 of 15 μM, while showed low cytotoxicity toward normal cells. In xenograft mice model, treatment with JH62 (10 mg/kg) reduced tumor weight and volume by 73% and 79%, respectively. Mechanistically, treatment with JH62 induced structural and functional disruption of mitochondria in cancer cells, triggered autophagic cell death, and did not cause DNA damage. Genetic analysis confirmed that JH62 biosynthesis depends on the pqsABCDE gene cluster and that JH62 positively regulates its own production. ADMET profiling further indicated promising drug-like properties for future development. These findings establish JH62 as a promising anticancer lead compound derived from microbial metabolism.

Fire blight, caused by Erwinia amylovora, poses a significant threat to rosaceous plants, such as pears, apples, and hawthorns. The type III secretion system (T3SS) is a critical pathogenicity factor in the pathogenesis of E. amylovora. Disulfide compounds, including those derived from garlic extract, exhibit good bioactivity against both bacteria and fungi. In this study, we synthesized 39 disulfide compounds based on garlic extract and developed a high-throughput screening system incorporating the bacterial luciferase lux reporter gene. Compound 5c was identified as the most effective inhibitor, significantly suppressing the promoter expression of T3SS-related genes, such as hrpA and hrpL, in E. amylovora CFBP1430. Furthermore, compound 5c inhibited the hypersensitive response (HR) triggered by E. amylovora CFBP1430 in tobacco without affecting bacterial growth. Compound 5c also reduced the level of secretion of the pathogenic protein HrpN and diminished the pathogenicity of E. amylovora CFBP1430 in pear infection assays. These findings offer a theoretical foundation for the development of novel T3SS inhibitors aimed at the prevention and control of fire blight disease.

BACKGROUND:The Gram-negative bacterium Erwinia amylovora induces fire blight disease in Rosaceae hosts, severely damaging pome fruit trees including pear, apple, and hawthorn cultivars. Traditional control methods, such as antibiotics and copper-based agents, are associated with plant damage and the emergence of resistant bacterial strains, and present significant challenges. This study aims to design and synthesize a novel eco-friendly bactericide. RESULTS:In this study, we designed and synthesized 30 small-molecule compounds by combining 1,3,4-thiadiazole, a key moiety of zinc thiazole, with 5-phenyl-2-furan. Structural characterization of these compounds was performed using 1H and 13C NMR spectroscopy, complemented by elemental composition analysis. Compound II-10 was identified as the most promising compound in initial screenings, with a median effective concentration (EC50) of 9.04 μg/mL. Inoculation experiments demonstrated that at a concentration of 200 μg/mL, II-10 effectively inhibited E. amylovora and controlled pear fire blight, with its protective activity exceeding its curative effects. Furthermore, fluorescence microscopy, flow cytometry and scanning electron microscopy analysis revealed a potential mechanism of antimicrobial action involving reactive oxygen species generation, apoptosis and changes in bacterial morphology. Toxicity assessment showed that II-10 was non-toxic to zebrafish, earthworm and silkworm. CONCLUSION:This study established that II-10 was a lead scaffold for the development to control pear fire blight, providing theoretical support for optimizing small-molecule compounds. © 2025 Society of Chemical Industry.

Rice bacterial leaf blight, caused by Xanthomonas oryzae pv. oryzae (Xoo), is a severe bacterial disease that can lead to fatal damage to rice plants when infection is severe. In this study, an active splicing strategy was employed to design and synthesize 36 novel acetoguanidine derivatives. The structures of the target compounds were confirmed by 1H NMR and 13C NMR spectroscopy, and their antibacterial activity against Xoo was evaluated. Among them, compounds E5, E6, and E9 exhibited significant inhibitory effects on Xoo, with EC50 values of 17.27, 18.15, and 19.35 mu g/mL, respectively. Further investigation into the preliminary mechanism of action of E5 revealed that treatment with this compound notably influenced reactive oxygen species (ROS) production. Additionally, safety assessments conducted on earthworms and silkworms indicated that E5 possesses favorable biosafety. These findings suggest that E5 is a promising candidate for antibacterial agents, laying a theoretical foundation for the development of small-molecule inhibitors.

BACKGROUND:Rice, a staple food crop for half of the global population, faces severe threats from bacterial leaf blight caused by the pathogen Pantoea ananatis. The conventional chemical control strategy poses significant environmental risks and contributes to resistance development, thus necessitating the discovery of eco-friendly antimicrobial agents with enhanced cost-effectiveness. Although disulfides derived from garlic exhibit broad-spectrum antibacterial activity, their molecular instability limits practical applications. This study aimed to design and synthesize disulfide derivatives with improved stability and evaluate their inhibitory potential against P. ananatis. RESULTS:In this study, 41 disulfides were successfully synthesized. Among them, compound D7 demonstrated the strongest inhibitory activity against P. ananatis with a half-maximal effective concentration (EC50) value of 0.87 μg/mL, markedly surpassing the control agent Zhongshengmycin, which exhibited an EC50 value of 14.73 μg/mL. Mechanistic studies revealed that D7 disrupts the bacterial oxidative defense system by inducing reactive oxygen species accumulation and inhibiting superoxide dismutase and catalase activities, thereby triggering apoptosis. Electron microscopy observations confirmed that D7 causes membrane disintegration and cytoplasmic leakage. In vivo experiments demonstrated that D7 (200 μg/mL) achieved 77.50% curative and 79.49% protective efficacy against rice bacterial leaf blight, with low acute toxicity observed in non-target organisms. CONCLUSION:This study confirms that compound D7 efficiently suppresses P. ananatis and treatment bacterial leaf blight via dual oxidative stress and membrane damage mechanisms, while also posing low environmental risks. © 2025 Society of Chemical Industry.

Plant bacterial diseases have inflicted substantial economic losses in global crop, fruit, and vegetable production. The conventional methods for managing these diseases typically rely on the application of antibiotics. However, these antibiotics often target the growth factors of the pathogenic bacteria, leading to the accumulation and emergence of drug-resistant strains, which exacerbates antibiotic resistance. Innovative methods are urgently needed to treat and prevent the toxicity caused by these pathogenic bacteria. Targeting virulence mechanisms in pathogens is a globally recognized and effective strategy for mitigating bacterial resistance. Type III secretion system (T3SS) serves as a crucial virulence determinant in Gram-negative pathogens, and its non-essentials for pathogen growth renders it an ideal target. Targeting the T3SS holds significant potential to alleviate selective pressure for resistance mutations in pathogens. Therefore, targeting T3SS in pathogenic bacteria, while preserving their growth, has emerged as a novel avenue for the development of antimicrobial drugs. In recent years, a multitude of small molecular inhibitors targeting T3SS have been identified. This article offers a comprehensive review of T3SS inhibitors in plant pathogens, while also presenting the latest research advancements in this research direction.

With the increasing reports of antibiotic resistance in this species, Pseudomonas aeruginosa is a common human pathogen with important implications for public health. Bacterial quorum sensing (QS) systems are potentially broad and versatile targets for developing new antimicrobial compounds. While previous reports have demonstrated that certain amide compounds can inhibit bacterial growth, there are few reports on the specific inhibitory effects of these compounds on bacterial quorum sensing systems. In this study, thirty-one amide derivatives were synthesized. The results of the biological activity assessment indicated that A9 and B6 could significantly inhibit the expression of lasB, rhlA, and pqsA, effectively reducing several virulence factors regulated by the QS systems of PAO1. Additionally, compound A9 attenuated the pathogenicity of PAO1 to Galleria mellonella larvae. Meanwhile, RT-qPCR, SPR, and molecular docking studies were conducted to explore the mechanism of these compounds, which suggests that compound A9 inhibited the QS systems by binding with LasR and PqsR, especially PqsR. In conclusion, amide derivatives A9 and B6 exhibit promising potential for further development as novel QS inhibitors in P. aeruginosa.

Although RNA structures play important roles in regulating gene expression, the mechanism and function of mRNA folding in plant bacterial pathogens remain elusive. Therefore, we perform dimethyl sulfate sequencing (DMS-seq) on the Pseudomonas syringae under nutrition-rich and -deficient conditions, revealing that the mRNA structure changes substantially in the minimal medium (MM) that tunes global translation efficiency (TE), thereby inducing virulence. This process is led by the increased expression of hfq, which is directly activated by transcription regulators RpoS and CysB. The co-occurrence of Hfq and RpoS in diverse bacteria and the deep conservation of Hfq Y25 is critical for RNA-mediated regulation and implicates the wider biological importance of mRNA structure and feedback loops in the control of global gene expression.