RESUMO
"Candidatus Liberibacter asiaticus" (Ca. L. asiaticus) is the causal agent of Huanglongbing disease of citrus and current study focuses on the discovery of novel small-molecule inhibitors against SecA protein of Ca. L. asiaticus. In this study, homologous modeling was used to construct the three-dimensional structure of SecA. Then, molecular docking-based virtual screening and two rounds of in vitro bacteriostatic experiments were utilized to identify novel small-molecule inhibitors of SecA. Encouragingly, 93 compounds were obtained and two of them (P684-2850, P684-3808) showed strong antimicrobial activities against Liberibacter crescens BT-1 in bacteriostatic experiments. Finally, molecular dynamics simulations were employed to explore the binding modes of the receptor-ligand complexes. Results in MD simulations showed that compound P684-3808 was relatively stable during simulation, while compound P684-2850 left the binding pocket. Compound P684-3808 might be suitable as a lead compound for further development of antimicrobial compounds against SecA of Ca. L. asiaticus.
Assuntos
Proteínas de Bactérias/antagonistas & inibidores , Liberibacter/metabolismo , Proteínas SecA/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Avaliação Pré-Clínica de Medicamentos , Concentração Inibidora 50 , Liberibacter/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Proteínas SecA/metabolismo , Bibliotecas de Moléculas Pequenas/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
Citrus Huanglongbing (HLB), caused by a vector-transmitted phloem-limited bacterium Candidatus Liberibacter asiaticus (CLas), is the most devastating citrus disease worldwide. Currently, there are no effective strategies to prevent infection or to cure HLB-positive trees. Here, using comparative analysis between HLB-sensitive citrus cultivars and HLB-tolerant citrus hybrids and relatives, we identified a novel class of stable antimicrobial peptides (SAMPs). The SAMP from Microcitrusaustraliasica can rapidly kill Liberibacter crescens (Lcr), a culturable Liberibacter strain, and inhibit infections of CLas and CL. solanacearum in plants. In controlled greenhouse trials, SAMP not only effectively reduced CLas titer and disease symptoms in HLB-positive trees but also induced innate immunity to prevent and inhibit infections. Importantly, unlike antibiotics, SAMP is heat stable, making it better suited for field applications. Spray-applied SAMP was taken up by citrus leaves, stayed stable inside the plants for at least a week, and moved systemically through the vascular system where CLas is located. We further demonstrate that SAMP is most effective on α-proteobacteria and causes rapid cytosol leakage and cell lysis. The α-helix-2 domain of SAMP is sufficient to kill Lcr Future field trials will help determine the efficacy of SAMP in controlling HLB and the ideal mode of application.
Assuntos
Citrus/efeitos dos fármacos , Doenças das Plantas/prevenção & controle , Proteínas Citotóxicas Formadoras de Poros/farmacologia , Rutaceae/química , Citrus/microbiologia , Resistência à Doença/genética , Liberibacter/efeitos dos fármacos , Liberibacter/patogenicidade , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Folhas de Planta/microbiologia , Proteínas Citotóxicas Formadoras de Poros/química , Proteínas Citotóxicas Formadoras de Poros/genéticaRESUMO
Pierce's disease of grapevine and citrus huanglongbing are caused by the bacterial pathogens Xylella fastidiosa and Candidatus Liberibacter asiaticus (CLas), respectively. Both pathogens reside within the plant vascular system, occluding water and nutrient transport, leading to a decrease in productivity and fruit marketability and ultimately death of their hosts. Field observations of apparently healthy plants in disease-affected vineyards and groves led to the hypothesis that natural products from endophytes may inhibit these bacterial pathogens. Previously, we showed that the natural product radicinin from Cochliobolus sp. inhibits X. fastidiosa. Herein we describe a chemical synthesis of deoxyradicinin and establish it as an inhibitor of both X. fastidiosa and Liberibacter crescens, a culturable surrogate for CLas. The key to this three-step route is a zinc-mediated enolate C-acylation, which allows for direct introduction of the propenyl side chain without extraneous redox manipulations.