Macedo Maria Lígia R, Ribeiro Suzanna F F, Taveira Gabriel B, Gomes Valdirene M, de Barros Karina M C A, Maria-Neto Simone
Laboratório de Purificação de Proteínas e suas Funções Biológicas, Centro de Ciências Biológicas e da Saúde, Universidade Federal de Mato Grosso do Sul, Cidade Universitária S/N, Caixa Postal 549, Campo Grande, MS, 79070-900, Brazil.
Laboratório de Fisiologia e Bioquímica de Microrganismos, Centro de Biociências e Biotecnologia, Universidade Estadual do Norte Fluminense Darcy Ribeiro, Campos dos Goytacazes, RJ, 28013-602, Brazil.
Curr Microbiol. 2016 May;72(5):538-44. doi: 10.1007/s00284-015-0970-z. Epub 2016 Jan 14.
Over the last few years, a growing number of proteinase inhibitors have been isolated from plants and particularly from seeds and have shown antimicrobial activity. A 20,000 Da serine peptidase inhibitor, named ILTI, was isolated from Inga laurina seeds and showed potent inhibitory enzymatic activity against trypsin. The aim of this study was to determine the effects of ILTI on the growth of pathogenic and non-pathogenic microorganisms. We observed that ILTI strongly inhibited in particular the growth of Candida tropicalis and Candida buinensis, inducing cellular agglomeration. However, it was ineffective against human pathogenic bacteria. We also investigated the potential of ILTI to permeabilize the plasma membrane of yeast cells. C. tropicalis and C. buinensis were incubated for 24 h with the ILTI at different concentrations, which showed that this inhibitor induced changes in the membranes of yeast cells, leading to their permeabilization. Interestingly, ILTI induced the production of reactive oxygen species (ROS) in C. tropicalis and C. buinensis cells. Finally, ILTI was coupled with fluorescein isothiocyanate, and subsequent treatment of C. tropicalis and C. buinensis with DAPI revealed the presence of the labeled protein in the intracellular spaces. In conclusion, our results indicated the ability of peptidase inhibitors to induce microbial inhibition; therefore, they might offer templates for the design of new antifungal agents.
在过去几年中，越来越多的蛋白酶抑制剂已从植物中分离出来，尤其是从种子中分离出来，并显示出抗菌活性。一种名为ILTI的20,000 Da丝氨酸蛋白酶抑制剂从劳氏印加树种子中分离出来，对胰蛋白酶显示出强大的抑制酶活性。本研究的目的是确定ILTI对致病性和非致病性微生物生长的影响。我们观察到，ILTI尤其强烈抑制热带假丝酵母和布氏假丝酵母的生长，诱导细胞聚集。然而，它对人类病原菌无效。我们还研究了ILTI使酵母细胞质膜通透化的潜力。将热带假丝酵母和布氏假丝酵母与不同浓度的ILTI孵育24小时，结果表明这种抑制剂诱导酵母细胞膜发生变化，导致其通透化。有趣的是，ILTI在热带假丝酵母和布氏假丝酵母细胞中诱导活性氧（ROS）的产生。最后，将ILTI与异硫氰酸荧光素偶联，随后用DAPI处理热带假丝酵母和布氏假丝酵母，结果显示在细胞内空间存在标记蛋白。总之，我们的结果表明肽酶抑制剂具有诱导微生物抑制的能力；因此，它们可能为新型抗真菌剂的设计提供模板。
