Receptor binding domain of Escherichia coli F18 fimbrial adhesin FedF can be both efficiently secreted and surface displayed in a functional form in Lactococcus lactis
Receptor binding domain of Escherichia coli F18 fimbrial adhesin FedF can be both efficiently secreted and surface displayed in a functional form in Lactococcus lactis
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DOI：
10.1128/aem.70.4.2061-2071.2004
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发表时间：
2004-04-01
影响因子：
4.4
通讯作者：
Palva, A
中科院分区：
文献类型：
--
作者：
Lindholm, A;Smeds, A;Palva, A
F18菌毛粘附素（FedF）介导F18菌毛大肠杆菌对猪小肠上皮细胞的粘附。在先前的研究中，我们证明了60和109之间的氨基酸残基作为FedF的受体结合结构域的特异性。在这项研究中，不同的表达，分泌，和锚定系统的受体结合结构域的FedF粘附素乳酸乳球菌进行了评估。两个部分重叠的受体结合结构域（42和62个氨基酸残基）表达为与L。lactis subsp. Cremoris蛋白PrtP用于评价分泌效率。为了评估这些FedF-PrtP融合体的细胞表面展示，将它们进一步与不同长度的PrtP间隔区组合，所述PrtP间隔区与L. lactis AcmA锚或PAP细胞壁结合结构域。HtrA缺陷型L.构建乳酸菌NZ 9000突变体以确定其对分泌或锚定融合蛋白水平的影响。重组L.分泌FIS菌毛的受体结合结构域作为与L.乳酸菌蛋白PrtP首先通过使用两种不同的信号肽构建。FedF-PrtP融合蛋白，由L. brevis SlpA的融合蛋白的分泌量显著高于与L.乳酸菌Usp 45.在所测试的表面显示系统中，L。lactis AcmA锚的性能明显更好，特别是在L. lactis NZ 9000 DeltahtrA菌株与L. lactis PrtP锚区。在具有AcmA锚的细胞表面展示构建体中，仅具有最长PrtP间隔区的那些构建体导致重组L.乳酸细胞转化为猪肠上皮细胞。这些结果证实了在L.乳酸菌。
Adherence of F18 fimbrial Escherichia coli to porcine intestinal epithelial cells is mediated by the adhesin (FedF) of F18 fimbriae. In a previous study, we demonstrated the specificity of the amino acid residues between 60 and 109 as the receptor binding domain of FedF. In this study, different expression, secretion, and anchoring systems for the receptor binding domain of the FedF adhesin in Lactococcus lactis were evaluated. Two partially overlapping receptor binding domains (42 and 62 amino acid residues) were expressed as fusions with L. lactis subsp. cremoris protein PrtP for evaluation of secretion efficiency. To evaluate the cell surface display of these FedF-PrtP fusions, they were further combined with different lengths of PrtP spacers fused with either the L. lactis AcmA anchor or the PAP cell wall binding domain. An HtrA-defective L. lactis NZ9000 mutant was constructed to determine its effect on the level of secreted or anchored fusion proteins. Recombinant L. lactis clones secreting the receptor binding domain of FIS fimbriae as a fusion with the H domains of L. lactis protein PrtP were first constructed by using two different signal peptides. FedF-PrtP fusions, directed by the signal sequence of L. brevis SlpA, were throughout found to be secreted at significantly higher quantities than corresponding fusions with the signal peptide of L. lactis Usp45. In the surface display systems tested, the L. lactis AcmA anchor performed significantly better, particularly in the L. lactis NZ9000DeltahtrA strain, compared to the L. lactis PrtP anchor region. Of the cell surface display constructs with the AcmA anchor, only those with the longest PrtP spacer regions resulted in efficient binding of recombinant L. lactis cells to porcine intestinal epithelial cells. These results confirmed that it is possible to efficiently produce the receptor binding domain of the F18 adhesin in a functionally active form in L. lactis.