Inhibition of LtxA toxicity by blocking cholesterol binding with peptides

Abstract

Summary The leukotoxin (LtxA) produced by Aggregatibacter actinomycetemcomitans kills host immune cells, allowing the bacterium to establish an ecological niche in the upper aerodigestive tract of its human host. The interaction of LtxA with human immune cells is both complex and multifaceted, involving membrane lipids as well as cell-surface proteins. In the initial encounter with the host cell, LtxA associates with lymphocyte function-associated antigen-1 (LFA-1), a cell surface adhesion glycoprotein. However, we have also demonstrated that the toxin associates strongly with the plasma membrane lipids, specifically cholesterol. This association with cholesterol is regulated by a cholesterol recognition amino acid consensus (CRAC) motif, with a sequence of 334LEEYSKR340, in the N-terminal region of the toxin. Here, we have demonstrated that removal of cholesterol from the plasma membrane or mutation of the LtxA CRAC motif inhibits the activity of the toxin in THP-1 cells. To inhibit LtxA activity, we designed a short peptide corresponding to the CRAC336 motif of LtxA (CRAC336WT). This peptide binds to cholesterol and thereby inhibits the toxicity of LtxA in THP-1 cells. Previously, we showed that this peptide inhibits LtxA toxicity against Jn.9 (Jurkat) cells, indicating that peptides derived from the cholesterol-binding site of LtxA may have a potential clinical applicability in controlling infections of RTX-producing organisms.

Use of a Cholesterol Recognition Amino Acid Consensus Peptide To Inhibit Binding of a Bacterial Toxin to Cholesterol

“…The concentration used in the long-term toxicity assay is more than 2 times greater than the half-maximal inhibitory concentration (IC 50 ), found previously for CRAC 336WT inhibition of LtxA toxicity against THP-1 cells. 23 …”

“…25 In addition, we have shown experimentally that this same peptide inhibits the activity of LtxA against target cells. 23 Here, our goal was to determine the mechanism by which the CRAC 336WT peptide interacts with membranes to understand the specific mechanism by which it inhibits LtxA activity. We found that the peptide interacts near the surface of the membrane through recognition of the hydroxyl group of Chol.…”

Inhibition of Bacterial Toxin Activity by the Nuclear Stain, DRAQ5™

“…We have previously shown that, like other bacterial toxins, e.g., (Alonso et al , 2000; Boesze-Battaglia et al , 2009; Farrand et al , 2010; Lai et al , 2013; Maxfield and Tabas, 2005; Patel et al , 2002; Schwiering et al , 2013; Zitzer et al , 2001), LtxA requires cholesterol in the plasma membrane to bind and kill target cells (Brown et al , 2013; Brown et al , 2015; Fong et al , 2006). Thus, it is possible that the cell counteracts DRAQ5 ™ -mediated decreases in the plasma membrane fluidity by decreasing the membrane cholesterol composition and that this change inhibits LtxA internalization.…”

Aggregatibacter actinomycetemcomitansLtxA hijacks endocytic trafficking pathways in human lymphocytes

“…LtxA toxicity requires the presence of the β 2 integrin LFA-1(LFA-1, CD11a/CD18 or α L /β 2 ) [18] and cholesterol enriched membrane domains [19,20]. LFA-1 is a native ligand for intercellular adhesion molecule (ICAM-1) located on vascular endothelial cells [21].…”

“…Additionally, LFA-1 activity toward the components of the extracellular matrix is regulated by the ability of these receptors to switch between active and inactive conformations [21]. Cholesterol is essential for LtxA association with plasma membrane of human lymphocytes [19] and human monocytes [20] [26]. Binding to cholesterol is mediated by a cholesterol recognition amino acid consensus (CRAC) motif, in the N-terminal region of the toxin [19].…”