Aptamers That Preferentially Bind Type IVB Pili and Inhibit Human Monocytic-Cell Invasion by Salmonella enterica Serovar Typhi

Abstract

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-PS 8.4 ) as a type IVB pilus-specific ligand and further found that the selected aptamer (S-PS 8.4 ) 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-PS 8.4 aptamer alone. As an example of an aptamer against type IVB pili of S. enterica serovar Typhi, the aptamer S-PS 8.4 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.Of the more than 2,300 closely related Salmonella serovars identified, Salmonella enterica serovar Typhi is an important pathogen exclusively for humans and can be transmitted through contaminated food and water. It causes typhoid or enteric fever, which is a serious public health problem in developing countries. The genome of S. enterica serovar Typhi contains three large inserts (pathogenicity islands) (11), relative to the chromosome of Salmonella enterica serovar Typhimurium, which is normally noninvasive for humans. The type IVB pil operon of S. enterica serovar Typhi is located in Salmonella pathogenicity island 7 (18) and contains a pilS gene encoding the structural pilin (36, 37). It has been demonstrated that a pilS mutant of S. enterica serovar Typhi exhibited muchreduced adhesion to and invasion of human epithelial gastrointestinal cells in vitro and that purified soluble pre-PilS protein, retaining the signal sequence normally cleaved when the protein is excreted to form insoluble pili based on polymerized PilS, inhibited bacterial invasion (37). The structure of the N-terminally truncated type IVB structural pilin from S. enterica serovar Typhi was determined by nuclear magnetic resonance analysis (34). Type IVB pili, composed largely of polymerized PilS protein, also mediate bacterial self-association, but only when the...

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