Aptamers that preferentially bind type IVB pili and inhibit human monocytic-cell invasion by Salmonella enterica serovar Typhi
Aptamers that preferentially bind type IVB pili and inhibit human monocytic-cell invasion by Salmonella enterica serovar Typhi
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DOI：
10.1128/aac.49.10.4052-4060.2005
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发表时间：
2005-10-01
影响因子：
4.9
通讯作者：
Wu, JG
中科院分区：
文献类型：
--
作者：
Pan, Q;Zhang, XL;Wu, JG
伤寒沙门氏菌是人类特有的重要病原体，可引起伤寒或肠热病。已证明由S.伤寒沙门菌操纵子位于沙门菌致病岛7，在致病过程中起重要作用。在本研究中，通过粘附-侵袭实验和荧光定量PCR分析，我们证明了IVB型纤毛S。伤寒沙门氏菌A_（21 -6）对人THP-1单核细胞的Km（r）大于无菌毛沙门氏菌。肠伤寒血清型pilS：：Km（r）（pilS突变体）菌株。我们应用了一个系统的配体进化指数富集方法来选择寡核苷酸（适体）作为配体，特异性结合到IVB型皮利。使用这种方法，我们确定了一个高亲和力的单链RNA适体（S-PS 8.4）作为IVB型菌毛特异性配体，并进一步发现，选择的适体（S-PS 8.4）可以显着抑制进入的菌毛菌株（但不是无菌毛菌株）进入人THP-1细胞。通过硝酸纤维素滤膜结合测定法测定适体和pre-PilS（IVB型皮利的结构蛋白）之间的结合亲和力，并且测定单独的S-PS 8.4适体的K-d值为8.56 nM。作为针对沙门氏菌IVB型皮利的适体的实例，对于肠道伤寒血清型，适体S-PSIA可以作为分析IVB型细菌菌毛与宿主细胞相互作用的工具，并可能为开发针对伤寒杆菌的推定新药提供信息。肠道伤寒血清型细菌感染，可用于预防感染和治疗。
Salmonella enterica serovar Typhi is an important pathogen exclusively for humans and causes typhoid or enteric fever. It has been shown that type IVB pili, encoded by the S. enterica serovar Typhi pil operon located in Salmonella pathogenicity island 7, are important in the pathogenic process. In this study, by using both an adhesion-invasion assay and fluorescence quantitative PCR analysis, we demonstrated that the entry of type IVB piliated S. enterica serovar Typhi A21-6 (pil(+) Km(r)) into human THP-1 monocytic cells was greater than that of a nonpiliated S. enterica serovar Typhi pilS::Km(r) (pil mutant) strain. We have applied a systematic evolution of ligands by exponential enrichment approach to select oligonucleotides (aptamers) as ligands that specifically bind to type IVB pili. Using this approach, we identified a high-affinity single-stranded RNA aptamer (S-PS8.4) as a type IVB pilus-specific ligand and further found that the selected aptamer (S-PS8.4) could significantly inhibit the entry of the piliated strain (but not that of the nonpiliated strain) into human THP-1 cells. The binding affinities between aptamers and pre-PilS (structural protein of type IVB pili) were determined by nitrocellulose filter-binding assays, and the K-d value was determined to be 8.56 nM for the S-PS8.4 aptamer alone. As an example of an aptamer against type IVB pili of S. enterica serovar Typhi, the aptamer S-PSIA can serve as a tool for analysis of bacterial type IVB pilus-host cell interactions and may yield information for the development of putative new drugs against S. enterica serovar Typhi bacterial infections, useful both in prevention of infection and in therapeutic treatment.