Membrane localization of the Repeats-in-Toxin (RTX) Leukotoxin (LtxA) produced by Aggregatibacter actinomycetemcomitans

Abstract

The oral bacterium, Aggregatibacter actinomycetemcomitans, which is associated with localized aggressive periodontitis, as well as systemic infections including endocarditis, produces numerous virulence factors, including a repeats-in-toxin (RTX) protein called leukotoxin (LtxA), which kills human immune cells. The strains of A. actinomycetemcomitans most closely associated with disease have been shown to produce the most LtxA, suggesting that LtxA plays a significant role in the virulence of this organism. LtxA, like many of the RTX toxins, can be divided into four functional domains: an N-terminal hydrophobic domain, which contains a significant fraction of hydrophobic residues and has been proposed to play a role in the membrane interaction of the toxin; the central domain, which contains two lysine residues that are the sites of post-translational acylation; the repeat domain that is characteristic of the RTX toxins, and a C-terminal domain thought to be involved in secretion. In its initial interaction with the host cell, LtxA must bind to both cholesterol and an integrin receptor, lymphocyte function-associated antigen-1 (LFA-1). While both interactions are essential for toxicity, the domains of LtxA involved remain unknown. We therefore undertook a series of experiments, including tryptophan quenching and trypsin digestion, to characterize the structure of LtxA upon interaction with membranes of various lipid compositions. Our results demonstrate that LtxA adopts a U-shaped conformation in the membrane, with the N- and C-terminal domains residing outside of the membrane.

Aggregatibacter actinomycetemcomitans leukotoxin: From mechanism to targeted anti‐toxin therapeutics

“…They concluded that the toxin forms a U‐shaped conformation where the N‐terminal hydrophobic region and the C‐terminal region reside on the outside of the membrane, and the central and repeat regions are inside. An immunoblot, using a panel of monoclonal antibodies with epitopes located in varying domains of the toxin, confirmed this result (Brown et al, 2018). Interestingly, the N‐terminal hydrophobic domain, so named because it contains a majority of the hydrophobic residues of LtxA, was not located within the membrane.…”

“…The toxin has been divided into four functional domains, including an N‐terminal hydrophobic domain (residues 1–420), a central domain (residues 421–730), a repeat domain (residues 731–900), and a C‐terminal domain (residues 901–1,055; Figure 1a). The hydrophobic domain contains most of the hydrophobic amino acids, and has been proposed to play a role in membrane insertion (Lally et al, 1989), although recent results (Brown et al, 2018) call that hypothesis into question, as described in Section 3.6. The repeat domain contains the calcium‐binding, glycine‐rich amino acid sequence (LXGGXGND) that is characteristic of the RTX toxins.…”

“…Rather, large‐scale changes in the tertiary structure of the protein in a model membrane environment appear to regulate the dual solubility of the toxin. To investigate more global conformational changes, Brown et al employed tryptophan quenching, trypsin digest and mass spectrometry, and immunoblotting to identify the domains of the toxin exposed to solution when the toxin is in an aqueous environment, compared to when it is embedded in a membrane (Brown et al, 2018). Tryptophan quenching is a commonly used biophysical technique to identify regions of a protein that are accessible to an aqueous quencher, often potassium iodide, thus indicating which regions of the protein are exposed to solution.…”

Membrane Permeabilization by Pore-Forming RTX Toxins: What Kind of Lesions Do These Toxins Form?

“…For E. coli HlyA several studies showed that the hydrophobic region was responsible for the insertion of the toxin into the target membrane [46,47], and recently it has been reported that its channel-forming domain might contain β-strands, in addition to α-helical structures [48]. In this regard, it has been quite surprising that in the recent publication by Brown et al [49] they suggest that Aggregatibacter actinomycetemcomitans leukotoxin (LtxA) adopts a U-shaped conformation in the membrane, with the hydrophobic N-terminal and the C-terminal (secretion signal) domains residing outside of the membrane. It is thus clear that much remains to be known about the structure of this important domain.…”

Aggregatibacter actinomycetemcomitansLtxA hijacks endocytic trafficking pathways in human lymphocytes

“…The toxin then transverses the cell membrane, binds to the cytoplasmic tails of LFA-1, and causes activation of LFA-1 [30]. Following from the results of the liposomal study LtxA adopts a U-shaped conformation in the membrane, with the N- and C-terminal domains residing outside of the membrane [31].…”