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Evaluation of cytotoxicity features of antimicrobial peptides with potential to control bacterial diseases of citrus.

ABSTRACT: Antimicrobial peptides (AMPs) can be found in various organisms, and could be considered an alternative for pesticides used to control plant pathogens, including those affecting citrus. Brazil is the largest producer and exporter of frozen concentrated orange juice in the world. However, the citrus industry has been affected by several diseases such as citrus canker and huanglongbing (HLB), caused by the bacteria Xanthomonas citri subsp. citri (X.citri) and Candidatus Liberibacter asiaticus (CaLas), respectively. In order to control these pathogens, putative AMPs were prospected in databases containing citrus sequences. Furthermore, AMPs already reported in the literature were also used for in vitro and in vivo assays against X.citri. Since CaLas cannot be cultivated in vitro, surrogates as Sinorhizobium meliloti and Agrobacterium tumefaciens were used. This study reports the evaluation of six AMPs obtained from different sources, two of them from Citrus spp. (citrus-amp1 and citrus-amp2), three from amphibians (Hylin-a1, K0-W6-Hy-a1 and Ocellatin 4-analogue) and one from porcine (Tritrpticin). Peptides K0-W6-Hy-a1, Ocellatin 4-analogue, and citrus-amp1 showed bactericidal activity against X.citri and S. meliloti and bacteriostatic effect on A. tumefaciens. These results were confirmed for X.citri in planta. In addition cytotoxicity evaluations of these molecules were performed. The AMPs that showed the lowest hemolytic activities were Triptrpticin, citrus-amp1 and citrus-amp2. Citrus-amp1 and citrus-amp2 not presented toxicity in experiments using in vivo model, G. mellonella and U87 MG cells. To verify the interaction of these AMPs with bacteria and erythrocyte cell membranes, vesicles mimicking these cells were built. Citrus-amp1 and Tritrpticin exhibited higher affinity to bacterial membranes, while Ocellatin 4-analogue and Hylin-a1 showed higher affinity to erythrocyte membranes; exclude their use in citrus. This work demonstrates an essential alternative, trough AMPs obtained from Citrus spp., which can be feasibly used to control bacterial pathogens.

SUBMITTER: Inui Kishi RN

PROVIDER: S-EPMC6128562 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Evaluation of cytotoxicity features of antimicrobial peptides with potential to control bacterial diseases of citrus.

Inui Kishi Rosangela Naomi RN Stach-Machado Dagmar D Singulani Junya de Lacorte JL Dos Santos Claudia Tavares CT Fusco-Almeida Ana Marisa AM Cilli Eduardo Maffud EM Freitas-Astúa Juliana J Picchi Simone Cristina SC Machado Marcos Antonio MA

PloS one 20180907 9

Antimicrobial peptides (AMPs) can be found in various organisms, and could be considered an alternative for pesticides used to control plant pathogens, including those affecting citrus. Brazil is the largest producer and exporter of frozen concentrated orange juice in the world. However, the citrus industry has been affected by several diseases such as citrus canker and huanglongbing (HLB), caused by the bacteria Xanthomonas citri subsp. citri (X.citri) and Candidatus Liberibacter asiaticus (CaL ...[more]

PMID: 30192822

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Project description:Although orthodox medications are available for skin diseases, expensive dermatological services have necessitated the use of medicinal plants as a cheaper alternative. This study evaluated the pharmacological and phytochemical profiles of four medicinal plants (Drimia sanguinea, Elephantorrhiza elephantina, Helichrysum paronychioides, and Senecio longiflorus) used for treating skin diseases. Petroleum ether and 50% methanol extracts of the plants were screened for antimicrobial activity against six microbes: Bacillus cereus, Shigella flexneri, Candida glabrata, Candida krusei, Trichophyton rubrum and Trichophyton tonsurans using the micro-dilution technique. Antioxidant activity was conducted using 2,2-diphenyl-1-picryhydrazyl (DPPH) free radical scavenging and β-carotene linoleic acid models. Cytotoxicity was determined against African green monkey Vero kidney cells based on the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay. Spectrophotometric and Gas Chromatography-Mass Spectrometry (GC-MS) methods were used to evaluate the phytochemical constituents. All the extracts demonstrated varying degrees of antimicrobial potencies. Shigella flexneri, Candida glabrata, Trichophyton rubrum and Trichophyton tonsurans were most susceptible at 0.10 mg/mL. In the DPPH test, EC50 values ranged from approximately 6-93 µg/mL and 65%-85% antioxidant activity in the β-carotene linoleic acid antioxidant activity model. The phenolic and flavonoid contents ranged from 3.5-64 mg GAE/g and 1.25-28 mg CE/g DW, respectively. The LC50 values of the cytotoxicity assay ranged from 0.015-5622 µg/mL. GC-MS analysis revealed a rich pool (94-198) of bioactive compounds including dotriacontane, benzothiazole, heptacosane, bumetrizole, phthalic acid, stigmasterol, hexanoic acid and eicosanoic acid, which were common to the four plants. The current findings provide some degree of scientific evidence supporting the use of these four plants in folk medicine. However, the plants with high cytotoxicity need to be used with caution.

Project description:Antimicrobial activity of many AMPs can be improved by lysine-to-arginine substitution due to a more favourable interaction of arginine guanidinium moiety with bacterial membranes. In a previous work, the structural and functional characterization of an amphipathic antimicrobial peptide named RiLK1, including lysine and arginine as the positively charged amino acids in its sequence, was reported. Specifically, RiLK1 retained its β-sheet structure under a wide range of environmental conditions (temperature, pH, and ionic strength), and exhibited bactericidal activity against Gram-positive and Gram-negative bacteria and fungal pathogens with no evidence of toxicity on mammalian cells. To further elucidate the influence of a lysine-to-arginine replacement on RiLK1 conformational properties, antimicrobial activity and peptide-liposome interaction, a new RiLK1-derivative, named RiLK3, in which the lysine is replaced with an arginine residue, was projected and characterised in comparison with its parental compound. The results evidenced that lysine-to-arginine mutation not only did not assure an improvement in the antimicrobial potency of RiLK1 in terms of bactericidal, virucidal and fungicidal activities, but rather it was completely abolished against the hepatitis A virus. Therefore, RiLK1 exhibited a wide range of antimicrobial activity like other cationic peptides, although the exact mechanisms of action are not completely understood. Moreover, tryptophan fluorescence measurements confirmed that RiLK3 bound to negatively charged lipid vesicles with an affinity lower than that of RiLK1, although no substantial differences from the structural and self-assembled point of view were evidenced. Therefore, our findings imply that antimicrobial efficacy and selectivity are affected by several complex and interrelated factors related to substitution of lysine with arginine, such as their relative proportion and position. In this context, this study could provide a better rationalisation for the optimization of antimicrobial peptide sequences, paving the way for the development of novel AMPs with broad applications.

Dataset Information

Evaluation of cytotoxicity features of antimicrobial peptides with potential to control bacterial diseases of citrus.

Publications

Evaluation of cytotoxicity features of antimicrobial peptides with potential to control bacterial diseases of citrus.

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