Effect of a salivary peptide on the structure of bacterial pili and adhesion to host cells
Badahdah, Arwa Abubakr
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Pathogenic bacteria and commensal bacterial flora have surface adhesive structures, which enable them to adhere to and colonize host tissue. Enterotoxigenic Escherichia coli bacteria expressing colonization factor antigen I (CFA/I) pili and related pili are the most frequently isolated cause of childhood diarrhea in the developing world. CFA/I pili are micron long helical filaments 7-8 nm in diameter. Histatin 5 is a member of a family of low-molecular-weight histidine rich salivary peptides. Its major known function is the antifungal activity against the opportunistic yeast Candida albicans. Also, histatin 5 has bacteriostatic and bactericidal effects against several species of oral bacteria. In this study, it was investigated if histatin 5 could exert an antimicrobial effect through interactions with bacterial pili to deter disease not only in the oral cavity, but also in more distal regions in the GI tract. Material and methods: CFA/I pili were purified from Enterotoxigenic Escherichia coli (ETEC) bacteria expressing CFA/I. The effect of histatin 5 on the structure of the pili was tested by incubating pili with histatin 5 at a molar ratio of 1 mole of CFA/I monomer to 4 moles of histatin 5, at pH 7.4-7.6 and temperature of 37°C. Pili were visualized by transmission electron microscopy (EM) and images were processed to measure pili diameter and overall density, pili length, and the rigidity of pili by the mean of their persistence length. Association of histatin 5 with pili positive ETEC and pili negative E. coli as well as histatin 5 degradation were investigated using reversed phase high performance liquid chromatography and cationic PAGE and SDS PAGE. Binding of histatin 5 to CFA/I was tested by crosslinking experiments and initial isothermal calorimetric ITC studies. The effect of histatin 5 on the binding of bacteria to human cells was tested by incubating ETEC with histatin 5 and adding the suspension to cultured human intestinal cell line, Caco-2. Binding was visualized using epifluorescence microscopy. Results: The effect of binding was observed in the EM images of CFA/I pili that had been treated with histatin 5; pili were straighter than untreated pili, and wider in diameter. The persistence length of CFA/I pili was remarkably increased in the presence of histatin 5, by a factor of eight. Histatin 5 was associated with the pili positive ETEC and degradation products of histatin 5 started to appear after 30 min of incubation. The cross linking experiments showed high molecular weight bands in tricine SDS PAGE when CFA/I was incubated with histatin 5. Initial isothermal calorimetry results showed that the dissociation constant for histatin 5 binding to CFA/I was 35 [MU]M. Finally, binding of ETEC to Caco-2 cells was reduced by up to 35% when incubated with histatin 5. Conclusion: Histatin 5 reduced the ability of ETECs binding to intestinal epithelial cells through binding to CFA/I pili by altering their structural properties. Histatin 5 increased the rigidity of CFA/I pili, which could render the pili unable to function as a spring to maintain adhesion in the environment of the small intestine. Since infection cannot be established if a pathogen fails to adhere and maintain adhesion to its target cell, the results of this study suggest that histatin 5 could be used to deter establishment of infection through interference with adhesion.
PLEASE NOTE: This work is protected by copyright. Downloading is restricted to the BU community: please click Download and log in with a valid BU account to access. If you are the author of this work and would like to make it publicly available, please contact firstname.lastname@example.org.Dissertation (DScD) --Boston University, Henry M. Goldman School of Dental Medicine, 2014 (Department of Molecular and Cell Biology).Includes bibliographic references: leaves 150-163.
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