Irene R. Kieba

The interaction between RTX toxins and target cells

RTX toxins are important virulence factors produced by a wide range of Gram-negative bacteria. They fall into two categories: the hemolysins, which affect a variety of cell types, and the leukotoxins, which are cell-type- and species-specific. These toxins offer interesting models for targeting, insertion and translocation of aqueous proteins into lipid membranes.

RTX Toxins Recognize a β2 Integrin on the Surface of Human Target Cells

Actinobacillus actinomycetemcomitans leukotoxin and Escherichia coliα-hemolysin are RTX toxins that kill human immune cells. We have obtained a monoclonal antibody (295) to a cell surface molecule present on toxin-sensitive HL60 cells that can inhibit cytolysis by both RTX toxins. Utilization of this monoclonal antibody for immunoaffinity purification of detergent-solubilized target cell membranes yielded two polypeptide chains of approximate molecular masses of 100 and 170 kDa. Microsequencing of tryptic peptides from the two proteins showed complete homology with CD11a and CD18, the two subunits of the β2 integrin, lymphocyte function-associated antigen 1 (LFA-1). Anti-CD11a and CD18 monoclonal antibodies also inhibited RTX toxin-mediated cytolysis. Direct binding experiments demonstrated the ability of an immobilized RTX to bind LFA-1 heterodimers present in a detergent lysate of human HL60 target cells. Transfection of CD11a and CD18 integrin genes into a cell line (K562) that is not sensitive to either RTX toxin resulted in LFA-1 expressing cells, KL/4, that were sensitive to both toxins. These experiments identify LFA-1 as a cell surface receptor that mediates toxicity of members of this family of pore-forming toxins.

Identification and expression of the Actinobacillus actinomycetemcomitans leukotoxin gene.

The leukotoxin produced by the oral bacterium Actinobacillus actinomycetemcomitans has been implicated in the pathogenesis of juvenile periodontitis. In order to elucidate the structure of the leukotoxin, molecular cloning of the leukotoxin gene was carried out. A DNA library of A. actinomycetemcomitans, strain JP2, was constructed by partial digestion of genomic DNA with Sau3AI and ligation of 0.5 to 5.0 kilobase pair fragments into the Bam HI site of the plasmid vector pENN-vrf. After transformation into E. coli RR1 (lambda cI857), the clones were screened for the production of A. actinomycetemcomitans leukotoxin with polyclonal antibody. Six immunoreactive clones were identified. The clones expressed proteins which ranged from 21-80 kilodaltons, and the clone designated pII-2, producing the largest protein was selected for further study. Antibodies eluted from immobilized pII-2 protein also recognized the native A. actinomycetemcomitans leukotoxin molecule indicating that both molecules shared at least one epitope. DNA sequence analysis demonstrated that there are regions of significant amino acid sequence homology between the cloned A. actinomycetemcomitans leukotoxin and two other cytolysins, Escherichia coli alpha-hemolysin and Pasteurella haemolytica leukotoxin, suggesting that a family of cytolysins may exist which share a common mechanism of killing but vary in their target cell specificity.

Actinobacillus actinomycetemcomitans leukotoxin requires lipid microdomains for target cell cytotoxicity

Actinobacillus actinomycetemcomitans produces a leukotoxin (Ltx) that kills leukocyte function‐associated antigen‐1 (LFA‐1)‐bearing cells from man, the Great Apes and Old World monkeys. The unique specificity of Ltx for the β2 integrin, LFA‐1, suggests it is capable of providing insight into the pathogenic mechanisms of Ltx and other RTX toxins. Using the Jurkat T cell line and an LFA‐1‐deficient Jurkat mutant (Jβ2.7) as models, we found the initial effect of Ltx is to elevate cytosolic Ca2+[Ca2+]c, an event that is independent of the Ltx/LFA‐1 interaction. [Ca2+]c increases initiate a series of events that involve the activation of calpain, talin cleavage, mobilization to, and subsequent clustering of, LFA‐1 in cholesterol and sphingolipid‐rich regions of the plasma membrane known as lipid rafts. The association of Ltx and LFA‐1 within lipid rafts is essential for cell lysis. Jβ2.7 cells fail to accumulate Ltx in their raft fractions and are not killed, while cholesterol depletion experiments demonstrate the necessity of raft integrity for Ltx function. We propose that toxin‐induced Ca2+ fluxes mobilize LFA‐1 to lipid rafts where it associates with Ltx. These findings suggest that Ltx utilizes the raft to stimulate an integrin signalling pathway that leads to apoptosis of target cells.

Structure and function of the B and D genes of the Actinobacillus actinomycetemcomitans leukotoxin complex

The Actinobacillus actinomycetemcomitans leukotoxin gene complex, consisting of four genes, has been cloned and the sequence of the AaLtC and AaLtA genes reported. The present paper details the sequences of the AaLtB and AaLtD genes which, like AaLtC and AaLTA, are also homologues of genes found in other cytolytic toxin complexes of several other Gram-negative bacterial pathogens. When tested in a recombinant expression system, the AaLtB and/or AaLtD genes are required for the translocation and insertion of the A. actinomycetemcomitans leukotoxin (AaLtA) into the cell membrane of Escherichia coli.

Aggregatibacter actinomycetemcomitans leukotoxin requires β‐sheets 1 and 2 of the human CD11a β‐propeller for cytotoxicity

Aggregatibacter actinomycetemcomitans leukotoxin (Ltx) is a repeats‐in‐toxin (RTX) cytolysin that kills human leukocyte function‐associated antigen‐1 (LFA‐1; αL/β2)‐bearing cells. In order to determine whether the αL portion of the heterodimer is involved in Ltx recognition, we transfected human, mouse and bovine αL cDNAs into J‐β2.7, an αL‐deficient cell line, and looked for restoration of Ltx susceptibility. Cells expressing either bovine or human αL in conjunction with human β2 were efficiently killed by Ltx, an indication that bovine αL could substitute for its human counterpart in critical regions used by Ltx for attachment to LFA‐1. On the other hand, cells expressing murine αL and human β2 were not susceptible to the lethal effects of Ltx indicating that the toxin recognition sites are not present in the corresponding mouse sequence. To further identify the region(s) of αL recognized by Ltx, we constructed and evaluated a panel of chimeric human/murine αL genes in J‐β2.7 cells. Analysis of the αL mutant panel showed that the presence of human N‐terminal 128 amino acids on a mouse CD11a background, a region that includes β‐sheets 1 and 2 of the β‐propeller of the human αL chain, was sufficient for Ltx cytolysis.

STRUCTURE/FUNCTION ASPECTS OF ACTINOBACILLUS ACTINOMYCETEMCOMITANS LEUKOTOXIN

Actinobacillus actinomycetemcomitans has been implicated as a causative organism in early-onset periodontitis. The mechanisms by which A. actinomycetemcomitans is pathogenic are not known, but the organism produces several potential virulence factors, one of which is a leukotoxin. As a group, bacterial protein toxins are made up of structural domains which control various aspects of toxic activity, such as target cell recognition, membrane insertion, and killing. The purpose of this article is to review the structure of RTX, with special emphasis to its relation to toxin function. In addition, we will propose a model based upon other bacterial proteins whereby the water-soluble A. actinomycetemcomitans leukotoxin is able to achieve insertion into a biological membrane. J Periodontol 1996;67:298-308.

Maintenance of oxidative phosphorylation protects cells from Actinobacillus actinomycetemcomitans leukotoxin-induced apoptosis

Subnanomolar concentrations (3 × 10−10 M) of Actinobacillus actinomycetemcomitans leukotoxin (Ltx) trigger apoptosis of JY cells, as shown by a decrease in mitochondrial transmembrane potential (ΔΨm), hyperproduction of reactive oxygen species (ROS) and release of cytochrome c from the intermembrane space. When compared with heat‐inactivated leukotoxin (ΔI Ltx) controls, ATP levels in Ltx‐treated JY cells continued to decrease during a 24 h experiment while cytoplasmic ADP concentrations were increasing. These results suggest that a blockage occurred in ATP/ADP exchange. To maintain ATP/ADP exchange, JY cells were transfected with bcl‐2 and bcl‐xL and incubated with Ltx. ATP levels of the transfected cells decreased to 67% (JY/bcl‐2) and 73% (JY/bcl‐xL) after the experiment. Furthermore, cytochrome c remained localized to the mitochondrial fraction of Ltx‐treated JY/bcl‐2 and JY/bcl‐xL cells, whereas its presence in the cytoplasmic fraction of JY/gen cells suggests an uncoupling of electron transport. Expression of bcl‐2 and bcl‐xL in cells inhibited downstream apoptotic events such as cleavage of poly(ADP‐ribose) polymerase, DNA fragmentation and activation of a family of caspases. The results indicate that Ltx induces apoptosis through a mitochondrial pathway that involves decreased levels of the ADP in the mitochondrial matrix, a lack of substrate for ATP synthetase and arrest of oxidative phosphorylation.

Aggregatibacter actinomycetemcomitans leukotoxin is post-translationally modified by addition of either saturated or hydroxylated fatty acyl chains.

Aggregatibacter actinomycetemcomitans, a common inhabitant of the human upper aerodigestive tract, produces a repeat in toxin (RTX), leukotoxin (LtxA). The LtxA is transcribed as a 114-kDa inactive protoxin with activation being achieved by attachment of short chain fatty acyl groups to internal lysine residues. Methyl esters of LtxA that were isolated from A. actinomycetemcomitans strains JP2 and HK1651 and subjected to gas chromatography/mass spectrometry contained palmitoyl (C16:0, 27-29%) and palmitolyl (C16:1 cis Δ9, 43-44%) fatty acyl groups with smaller quantities of myristic (C14:0, 14%) and stearic (C18:0, 12-14%) fatty acids. Liquid chromatography/mass spectrometry of tryptic peptides from acylated and unacylated recombinant LtxA confirmed that Lys(562) and Lys(687) are the sites of acyl group attachment. During analysis of recombinant LtxA peptides, we observed peptide spectra that were not observed as part of the RTX acylation schemes of either Escherichia coliα-hemolysin or Bordetella pertussis cyclolysin. Mass calculations of these spectra suggested that LtxA was also modified by the addition of monohydroxylated forms of C14 and C16 acyl groups. Multiple reaction monitoring mass spectrometry identified hydroxymyristic and hydroxypalmitic acids in wild-type LtxA methyl esters. Single or tandem replacement of Lys(562) and Lys(687) with Arg blocks acylation, resulting in a >75% decrease in cytotoxicity when compared with wild-type toxin, suggesting that these post-translational modifications are playing a critical role in LtxA-mediated target cell cytotoxicity.

Aggregatibacter actinomycetemcomitans leukotoxin cytotoxicity occurs through bilayer destabilization

The Gram‐negative bacterium, Aggregatibacter actinomycetemcomitans, is a common inhabitant of the human upper aerodigestive tract. The organism produces an RTX (Repeats in ToXin) toxin (LtxA) that kills human white blood cells. LtxA is believed to be a membrane‐damaging toxin, but details of the cell surface interaction for this and several other RTX toxins have yet to be elucidated. Initial morphological studies suggested that LtxA was bending the target cell membrane. Because the ability of a membrane to bend is a function of its lipid composition, we assessed the proficiency of LtxA to release of a fluorescent dye from a panel of liposomes composed of various lipids. Liposomes composed of lipids that form nonlamellar phases were susceptible to LtxA‐induced damage while liposomes composed of lipids that do not form non‐bilayer structures were not. Differential scanning calorimetry demonstrated that the toxin decreased the temperature at which the lipid transitions from a bilayer to a nonlamellar phase, while 31P nuclear magnetic resonance studies showed that the LtxA‐induced transition from a bilayer to an inverted hexagonal phase occurs through the formation of an isotropic intermediate phase. These results indicate that LtxA cytotoxicity occurs through a process of membrane destabilization.