Biofilm development and computational screening for new putative inhibitors of a homolog of the regulatory protein BrpA in Streptococcus dysgalactiae subsp. dysgalactiae
Biofilm development and computational screening for new putative inhibitors of a homolog of the regulatory protein BrpA in Streptococcus dysgalactiae subsp. dysgalactiae
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DOI：
10.1016/j.ijmm.2019.02.001
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发表时间：
2019-05-01
影响因子：
4.1
通讯作者：
Santos-Sanches, Ilda
中科院分区：
文献类型：
--
作者：
Alves-Barroco, Cinthia;Roma-Rodrigues, Catarina;Santos-Sanches, Ilda
乳酸链球菌亚种。乳房发育不良(SDSD)是一种兰斯菲尔德C组链球菌(GCS)，是引起奶牛乳房炎的常见原因。这种高度流行的疾病是乳制品行业中代价最高的。黏附和生物被膜的产生是链球菌致病的重要因素。我们之前已经描述了SDSD分离株在人类细胞中的黏附和内化，现在我们描述了这种细菌的生物被膜生产能力。在这项工作中，我们结合微生物学、成像和计算方法来评估SDSD分离株的生物被膜能力；评估生物被膜调节蛋白BrPA同源物在生物被膜生产者中的存在；并预测BrPA样蛋白的结构模型及其与假定的抑制剂的结合。结果表明，SDSD分离株在玻璃(亲水)和聚苯乙烯(疏水)等非生物表面形成生物膜，其中玻璃表面形成的生物膜最强。这种能力主要与蛋白质类细胞外基质有关，在蛋白酶K和胰酶处理后生物膜的分散证实了这一点。共聚焦激光扫描显微镜(CLSM)和扫描电子显微镜(SEM)也证实了SDSD分离株的生物被膜形成。扫描电子显微镜观察，VSD16菌株在生物膜生长过程中形成细胞聚集体，而VSD9和VSD10菌株在生物膜生长过程中形成光滑的薄膜。我们发现BrPA类基因在SDSD生物膜产生菌中存在并表达，其表达水平与生物膜的生产能力相关，与生物膜生长相比，BrPA样基因在浮游生长指数后期的表达更多。已知的生物膜抑制剂和BrPA结合分子Fisetin在测试浓度下显著抑制SDSD分离物的生物膜形成，但不影响浮游生长。同源模建用于预测类BrPA蛋白的三维结构。通过高通量的虚拟筛选和分子对接，我们筛选出5个与蛋白质的疏水裂隙具有较强结合亲和力的配体分子，使它们成为SDSD BrPA样蛋白的潜在抑制剂候选。这些结果为开发SDSD抗生物被膜治疗的新策略提供了进一步的研究。
Streptococcus dysgalactiae subsp. dysgalactiae (SDSD), a Lancefield group C streptococci (GCS), is a frequent cause of bovine mastitis. This highly prevalent disease is the costliest in dairy industry. Adherence and biofilm production are important factors in streptoccocal pathogenesis. We have previously described the adhesion and internalization of SDSD isolates in human cells and now we describe the biofilm production capability of this bacterium. In this work we integrated microbiology, imaging and computational methods to evaluate the biofilm production capability of SDSD isolates; to assess the presence of biofilm regulatory protein BrpA homolog in the biofilm producers; and to predict a structural model of BrpA-like protein and its binding to putative inhibitors. Our results show that SDSD isolates form biofilms on abiotic surface such as glass (hydrophilic) and polystyrene (hydrophobic), with the strongest biofilm formation observed in glass. This ability was mainly associated with a proteinaceous extracellular matrix, confirmed by the dispersion of the biofilms after proteinase K and trypsin treatment. The biofilm formation in SDSD isolates was also confirmed by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). Under SEM observation, VSD16 isolate formed cell aggregates during biofilm growth while VSD9 and VSD10 formed smooth and filmy layers. We show that brpA-like gene is present and expressed in SDSD biofilm-producing isolates and its expression levels correlated with the biofilm production capability, being more expressed in the late exponential phase of planktonic growth compared to biofilm growth. Fisetin, a known biofilm inhibitor and a putative BrpA binding molecule, dramatically inhibited biofilm formation by the SDSD isolates but did not affect planktonic growth, at the tested concentrations. Homology modeling was used to predict the 3D structure of BrpA-like protein. Using high throughput virtual screening and molecular docking, we selected five ligand molecules with strong binding affinity to the hydrophobic cleft of the protein, making them potential inhibitor candidates of the SDSD BrpA-like protein. These results warrant further investigations for developing novel strategies for SDSD anti-biofilm therapy.