Identification of Benzyloxy Carbonimidoyl Dicyanide Derivatives as Novel Type III Secretion System Inhibitors via High-Throughput Screening.

Yi-Nan Ma,Wen-Jun Jiang,Li-Qun Zhang,Nai-Guo Si,Liang Chen,Jun-Li Liu,Zhi-Gang Zhou

doi:10.3389/fpls.2019.01059

Abstract

The type III secretion system (T3SS) in many Gram-negative bacterial pathogens is regarded as the most critical virulence determinant and an attractive target for novel anti-virulence drugs. In this study, we constructed a T3SS secretion reporter containing the β-lactamase gene fused with a signal peptide sequence of the T3SS effector gene, and established a high-throughput screening system for T3SS inhibitors in the plant pathogenic bacterium Acidovorax citrulli. From a library of 12,000 chemical compounds, we identified a series of benzyloxy carbonimidoyl dicyanide (BCD) derivatives that effectively blocked T3SS-dependent β-lactamase secretion. Substitution of halogens or nitro groups at the para-position on the benzene ring contributed to an increased inhibitory activity. One representative compound, BCD03 (3,4-dichloro-benzyloxy carbonimidoyl dicyanide), dramatically reduced pathogenicity of A. citrulli on melon seedlings, and attenuated hypersensitive responses in the non-host Nicotiana tabacum caused by pathogenic bacteria A. citrulli, Xanthomonas oryzae pv. oryzae and Pseudomonas syringae pv. tomato at sub-MIC concentrations. Western blotting assay further confirmed that BCD03 inhibited effector secretion from the above bacteria via T3SS in the liquid medium. Taken together, our data suggest that BCD derivatives act as novel inhibitors of T3SS in multiple plant bacterial pathogens.

Highlights

Antibiotic resistance is becoming a major concern in agricultural industries
We constructed a T3SS effectors (T3Es) secretion reporter plasmid containing a T3E signal peptide sequence fused with the β-lactamase gene in A. citrulli MH21 to screen for T3SS inhibitors
We developed a high-throughput system based on a T3E secretion reporter to screen bacterial T3SS inhibitors, which is distinguished from most previous studies using transcriptional reporters of T3SS structure genes, regulatory genes or T3E genes (Kauppi et al, 2003; Gauthier et al, 2005; Li et al, 2005; Nordfelth et al, 2005; Pan et al, 2007; Felise et al, 2008; Pan et al, 2009; Aiello et al, 2010; Enquist et al, 2012; Fan et al, 2017)

Summary

Introduction

Traditional antibiotics, which target growth and survival, induce high selective pressure on pathogenic bacteria. One promising way to reduce bacterial resistance is to target virulence factors, rather than growth (Rasko and Sperandio, 2010). The type III secretion system (T3SS) is highly conserved among Gramnegative bacteria and is directly related to pathogenicity during host cell invasion. T3SS is not necessary for bacterial survival, making it an attractive target for novel antibacterial drugs (Büttner, 2012). A number of chemical compounds acting as T3SS inhibitors have been identified in a wide range of important plant and animal bacterial pathogens, such as Erwinia, Pseudomonas, Xanthomonas, Escherichia, Yersinia and Salmonella species (Felise et al, 2008; Wang et al, 2011; Jessen et al, 2013; Yang et al, 2014; Anantharajah et al, 2016; Fan et al, 2017)

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Results

Conclusion