Detachment of Actinobacillus actinomycetemcomitans biofilm cells by an endogenous β-hexosaminidase activity
Detachment of Actinobacillus actinomycetemcomitans biofilm cells by an endogenous β-hexosaminidase activity
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DOI：
10.1128/jb.185.16.4693-4698.2003
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发表时间：
2003-08-01
影响因子：
3.2
通讯作者：
Fine, DH
中科院分区：
文献类型：
--
作者：
Kaplan, JB;Ragunath, C;Fine, DH
当在肉汤中培养时，革兰氏阴性牙周病原体伴放线放线杆菌的新鲜临床分离物在塑料和玻璃等表面上形成牢固粘附的生物膜菌落。这些生物膜集落将贴壁细胞释放到培养基中，释放的细胞可以附着在培养容器的表面并形成新的集落，从而使生物膜能够扩散。我们用转座子 IS903phikan 诱变 A. actinomycetemcomitans 临床菌株 CU1000，并分离出转座子插入突变体，该突变体形成紧密粘附在表面但缺乏将细胞释放到培养基中和分散的能力的生物膜集落。突变菌株中的转座子插入映射到一个名为 dspB 的基因，预计该基因编码与 20 糖基水解酶家族的催化结构域同源的分泌蛋白。携带野生型 dspB 基因的质粒恢复了突变菌株生物膜菌落的分散能力。我们在大肠杆菌中表达了 A. actinomycetemcomitans DspB 蛋白，该蛋白经过工程改造，在其 C 末端含有六组氨酸金属结合位点，并使用 Ni 亲和层析纯化了该蛋白。用 4-硝基苯基标记的单糖进行的底物特异性研究表明，DspB 水解 β-取代的 N-乙酰葡糖胺的 1-->4 糖苷键，这与其他家族 20 糖基水解酶的已知功能一致。当添加到培养基中时，纯化的 DspB 蛋白（而非热灭活的 DspB）恢复了突变株释放细胞和分散的能力。 DspB 蛋白还导致细胞从附着在塑料上生长的菌株 CU1000 的预形成生物膜菌落中分离，并导致溶液中高度自聚集的 CU1000 细胞团块解聚。我们得出的结论是，dspB 编码一种可溶性 β-N-乙酰氨基葡萄糖苷酶，该酶导致 A. actinomycetemcomitans 生物膜细胞的分离和分散。
When cultured in broth, fresh clinical isolates of the gram-negative periodontal pathogen Actinobacillus actinomycetemcomitans form tenaciously adherent biofilm colonies on surfaces such as plastic and glass. These biofilm colonies release adherent cells into the medium, and the released cells can attach to the surface of the culture vessel and form new colonies, enabling the biofilm to spread. We mutagenized A. actinomycetemcomitans clinical strain CU1000 with transposon IS903phikan and isolated a transposon insertion mutant that formed biofilm colonies which were tightly adherent to surfaces but which lacked the ability to release cells into the medium and disperse. The transposon insertion in the mutant strain mapped to a gene, designated dspB, that was predicted to encode a secreted protein homologous to the catalytic domain of the family 20 glycosyl hydrolases. A plasmid carrying a wild-type dspB gene restored the ability of biofilm colonies of the mutant strain to disperse. We expressed A. actinomycetemcomitans DspB protein engineered to contain a hexahistidine metal-binding site at its C terminus in Escherichia coli and purified the protein by using Ni affinity chromatography. Substrate specificity studies performed with monosaccharides labeled with 4-nitrophenyl groups showed that DspB hydrolyzed the 1-->4 glycosidic bond of beta-substituted N-acetylglucosamine, which is consistent with the known functions of other family 20 glycosyl hydrolases. When added to culture medium, purified DspB protein, but not heat-inactivated DspB, restored the ability of the mutant strain to release cells and disperse. DspB protein also caused the detachment of cells from preformed biofilm colonies of strain CU1000 grown attached to plastic and the disaggregation of highly autoaggregated clumps of CU1000 cells in solution. We concluded that dspB encodes a soluble beta-N-acetylglucosaminidase that causes detachment and dispersion of A. actinomycetemcomitans biofilm cells.