Reichhardt Courtney, Jacobson Amy N, Maher Marie C, Uang Jeremy, McCrate Oscar A, Eckart Michael, Cegelski Lynette
Department of Chemistry, Stanford University, Stanford, California 94305, United States of America.
Protein and Nucleic Acid Facility, Stanford University, Stanford, California 94305, United States of America.
PLoS One. 2015 Oct 20;10(10):e0140388. doi: 10.1371/journal.pone.0140388. eCollection 2015.
Microorganisms produce functional amyloids that can be examined and manipulated in vivo and in vitro. Escherichia coli assemble extracellular adhesive amyloid fibers termed curli that mediate adhesion and promote biofilm formation. We have characterized the dye binding properties of the hallmark amyloid dye, Congo red, with curliated E. coli and with isolated curli fibers. Congo red binds to curliated whole cells, does not inhibit growth, and can be used to comparatively quantify whole-cell curliation. Using Surface Plasmon Resonance, we measured the binding and dissociation kinetics of Congo red to curli. Furthermore, we determined that the binding of Congo red to curli is pH-dependent and that histidine residues in the CsgA protein do not influence Congo red binding. Our results on E. coli strain MC4100, the most commonly employed strain for studies of E. coli amyloid biogenesis, provide a starting point from which to compare the influence of Congo red binding in other E. coli strains and amyloid-producing organisms.
微生物会产生功能性淀粉样蛋白,这些蛋白可在体内和体外进行检测与操控。大肠杆菌会组装称为卷曲纤维的细胞外粘附性淀粉样纤维,这些纤维介导粘附并促进生物膜形成。我们已对标志性淀粉样染料刚果红与卷曲化大肠杆菌及分离出的卷曲纤维的染料结合特性进行了表征。刚果红与卷曲化的全细胞结合,不抑制生长,可用于比较定量全细胞的卷曲化程度。利用表面等离子体共振技术,我们测量了刚果红与卷曲纤维的结合和解离动力学。此外,我们确定刚果红与卷曲纤维的结合是pH依赖性的,并且CsgA蛋白中的组氨酸残基不影响刚果红的结合。我们对大肠杆菌菌株MC4100(用于研究大肠杆菌淀粉样蛋白生物合成最常用的菌株)的研究结果,为比较刚果红结合在其他大肠杆菌菌株和产生淀粉样蛋白的生物体中的影响提供了一个起点。