Departmental Papers (Dental)

Document Type

Journal Article

Date of this Version

8-9-2013

Publication Source

Journal of Biological Chemistry

Volume

288

Issue

32

Start Page

23607

Last Page

23621

DOI

10.1074/jbc.M113.486654

Abstract

Aggregatibacter actinomycetemcomitans produces a repeats-in-toxin (RTX) leukotoxin (LtxA) that selectively kills human immune cells. Binding of LtxA to its β2 integrin receptor (lymphocyte function-associated antigen-1 (LFA-1)) results in the clustering of the toxin·receptor complex in lipid rafts. Clustering occurs only in the presence of LFA-1 and cholesterol, and LtxA is unable to kill cells lacking either LFA-1 or cholesterol. Here, the interaction of LtxA with cholesterol was measured using surface plasmon resonance and differential scanning calorimetry. The binding of LtxA to phospholipid bilayers increased by 4 orders of magnitude in the presence of 40% cholesterol relative to the absence of cholesterol. The affinity was specific to cholesterol and required an intact secondary structure. LtxA contains two cholesterol recognition/amino acid consensus (CRAC) sites; CRAC336 (333LEEYSKR339) is highly conserved among RTX toxins, whereas CRAC503 (501VDYLK505) is unique to LtxA. A peptide corresponding to CRAC336 inhibited the ability of LtxA to kill Jurkat (Jn.9) cells. Although peptides corresponding to both CRAC336 and CRAC503 bind cholesterol, only CRAC336 competitively inhibited LtxA binding to this sterol. A panel of full-length LtxA CRAC mutants demonstrated that an intact CRAC336 site was essential for LtxA cytotoxicity. The conservation of CRAC336 among RTX toxins suggests that this mechanism may be conserved among RTX toxins.

Copyright/Permission Statement

This is the pre-peer reviewed version of the following article: [Brown, A. C., Balashova, N. V., Epand, R. M., Epand, R. F., Bragin, A., Kachlany, S. C., … Lally, E. T. (2013). Aggregatibacter actinomycetemcomitans Leukotoxin Utilizes a Cholesterol Recognition/Amino Acid Consensus Site for Membrane Association. The Journal of Biological Chemistry, 288(32), 23607–23621. http://doi.org/10.1074/jbc.M113.486654], which has been published in final form at [http://doi.org/10.1074/jbc.M113.486654]. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions.

Keywords

Bacterial Toxins, Cholesterol, Integrins, Lipid Raft, Microbial Pathogenesis, Lipid-Protein Interaction

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Date Posted: 01 March 2022

This document has been peer reviewed.