Generation of a recombinant Aggregatibacter actinomycetemcomitans RTX toxin in Escherichia coli

Abstract

A leukotoxin (LtxA) that is produced by Aggregatibacter actinomycetemcomitans (Aa) is an important virulence determinant in an aggressive form of periodontitis in adolescents. Understanding the function of this protein at the molecular level is critical to elucidating its role in the disease process. To accomplish genetic analysis of the protein structure and relating these observations to toxin function, we have developed an E. coli expression system for the generation and rapid purification of LtxA. Cloning the structural toxin gene, ltxA, from Aa strain JP2 under control of T7 promoter-1 of pCDFDuet-1 vector resulted in expression of a 114 KDa protein which could be easily purified by the presence of a carboxy-terminal engineered double hexahistidine (double-His) tag and was immunologically reactive with an anti-LtxA monoclonal antibody, but was not cytotoxic. Cloning a second gene, ltxC, an acyltransferase gene, into the vector under control of T7 promoter-2, resulted in expression of the biologically active LtxA. The toxin was extracted from E. coli inclusion bodies, purified by immobilized metal affinity chromatography, and refolded by dialysis. When compared by circular dichroism (CD) spectroscopy analysis, acylated recombinant LtxA has a secondary structure consistent with wt LtxA, while variations in α-helical structure of nonacylated LtxA were observed. No modifications in α-helix were found upon the toxin's binding with liposome-incorporated cholesterol. Our results suggest that pure, biologically active recombinant LtxA can be isolated by a one-step affinity chromatography from E. coli. The toxic and structural properties of the recombinant LtxA are similar to its wt counterpart.

Aggregatibacter actinomycetemcomitansLtxA hijacks endocytic trafficking pathways in human lymphocytes

“…3B and C shows a histogram obtained from 47 LtxA channels recorded under these conditions. A fit of the histogram with a Gaussian function yielded an average single-channel conductance of 1.1 ± 0.3 nS somewhat smaller than that at pH 6.0 (G = 1.2 ± 0.3 nS) [39]. Again we found that the single-channel distribution was quite broad, similar to the conditions at pH 6.0 (Fig.…”

“…Here we demonstrate that LtxA causes changes in lysosomal pH in T lymphocytes, however to a leser extent. The pore-forming properties of LtxA are well established [33,60]. Therefore, we propose that LtxA can cause permeabilization of lysosomal membrane, and possibly other intracellular organelles after the toxin is released from lysosomes.…”

“…Channel formation by LtxA was studied in detail at neutral pH [33,39]. In lipid bilayer membranes formed of asolection LtxA forms cation selective channels with a single-channel conductance of approximately 1.2 nS in 1 M KCl (pH 6.0) [39]. Since LtxA is found in endocytic vesicles, we asked whether LtxA is also able to form ion-permeable channels at acidic pH.…”

“…6 shows a single channel recording of LtxA in 1 M KCl, 10 mM MES-KOH, pH 4.7. The channel had a somewhat reduced lifetime at this pH as compared with that a neutral pH [39]. Fig.…”

“…The planar lipid bilayer assay is a highly sensitive method that allows characterization of membrane damaging activity of RTX-toxins in different physical conditions [66]. A current model proposes that RTX-toxins form cation-selective channels with diameter 0.6 – 2.6 nm in artificial membranes formed of lipid mixtures such as asolectin/n-decane membrane [39]. It was demonstrated that membrane-damaging activity of LtxA in artificial bilayers did not require the presence of the receptor [67].…”