Van Gerven Nani, Sleutel Mike, Deboeck Francine, De Greve Henri, Hernalsteens Jean-Pierre
Onderzoeksgroep Genetische Virologie, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium.
Structural Biology Brussels, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium.
Microbiology (Reading). 2009 Feb;155(Pt 2):468-476. doi: 10.1099/mic.0.022327-0.
Surface exposure of antigens on bacterial cells can be critical for eliciting an effective antibody response. Therefore, we investigated the cellular localization of the fimbrial F17a-G receptor-binding domain, fused to the translocator domain of the AIDA-I autotransporter. Synthesis of the fusion protein, under the control of the L-arabinose-inducible PBAD promoter, was shown to permeabilize Escherichia coli K-12 and Salmonella enterica serovar Typhimurium cells. The presence of permeable cells interfered with several methods that are typically used to determine surface exposure of proteins, such as protease treatment and whole-cell ELISA. Double immunofluorescence microscopy, using a second antibody directed against beta-galactosidase, a bacterial protein expressed in the cytoplasm, allowed the simultaneous detection of antigen expression and permeability in individual cells.
细菌细胞上抗原的表面暴露对于引发有效的抗体反应可能至关重要。因此，我们研究了与AIDA-I自转运蛋白的转运结构域融合的菌毛F17a-G受体结合结构域的细胞定位。在L-阿拉伯糖诱导型PBAD启动子的控制下，融合蛋白的合成显示可使大肠杆菌K-12和鼠伤寒沙门氏菌细胞通透性增加。通透性细胞的存在干扰了几种通常用于确定蛋白质表面暴露的方法，如蛋白酶处理和全细胞ELISA。使用针对β-半乳糖苷酶（一种在细胞质中表达的细菌蛋白）的二抗进行双重免疫荧光显微镜检查，可以同时检测单个细胞中的抗原表达和通透性。
