RESUMO
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 (31) P 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.
Assuntos
Toxinas Bacterianas/farmacologia , Exotoxinas/farmacologia , Bicamadas Lipídicas/química , Lipossomos/química , Pasteurellaceae , Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Forma Celular/efeitos dos fármacos , Exotoxinas/química , Exotoxinas/metabolismo , Fluoresceínas/química , Corantes Fluorescentes/química , Humanos , Células Jurkat , Microvilosidades/efeitos dos fármacos , Microvilosidades/ultraestrutura , Transição de Fase , Fosfatidilcolinas/química , Fosfatidiletanolaminas/químicaRESUMO
Aggregatibacter actinomycetemcomitans leukotoxin (Ltx) is a repeats-in-toxin (RTX) cytolysin that kills human leukocyte function-associated antigen-1 (LFA-1; alpha(L)/beta(2))-bearing cells. In order to determine whether the alpha(L) portion of the heterodimer is involved in Ltx recognition, we transfected human, mouse and bovine alpha(L) cDNAs into J-beta(2).7, an alpha(L)-deficient cell line, and looked for restoration of Ltx susceptibility. Cells expressing either bovine or human alpha(L) in conjunction with human beta(2) were efficiently killed by Ltx, an indication that bovine alpha(L) could substitute for its human counterpart in critical regions used by Ltx for attachment to LFA-1. On the other hand, cells expressing murine alpha(L) and human beta(2) 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 alpha(L) recognized by Ltx, we constructed and evaluated a panel of chimeric human/murine alpha(L) genes in J-beta(2).7 cells. Analysis of the alpha(L) mutant panel showed that the presence of human N-terminal 128 amino acids on a mouse CD11a background, a region that includes beta-sheets 1 and 2 of the beta-propeller of the human alpha(L) chain, was sufficient for Ltx cytolysis.
Assuntos
Aggregatibacter actinomycetemcomitans/metabolismo , Antígeno CD11a/metabolismo , Exotoxinas/farmacologia , Antígeno-1 Associado à Função Linfocitária/metabolismo , Animais , Antígeno CD11a/química , Antígeno CD11a/genética , Bovinos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Citometria de Fluxo , Humanos , Antígeno-1 Associado à Função Linfocitária/genética , Antígeno-1 Associado à Função Linfocitária/fisiologia , Camundongos , Microscopia Confocal , Ligação Proteica/efeitos dos fármacos , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/fisiologiaRESUMO
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 beta2 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 (Jbeta2.7) 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. Jbeta2.7 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.
Assuntos
Toxinas Bacterianas/farmacologia , Exotoxinas/farmacologia , Microdomínios da Membrana/efeitos dos fármacos , Toxinas Bacterianas/metabolismo , Antígenos CD18/metabolismo , Cálcio/metabolismo , Calpaína/antagonistas & inibidores , Calpaína/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Colesterol/metabolismo , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Exotoxinas/metabolismo , Imunofluorescência , Humanos , Integrina alfa4beta1/metabolismo , Células Jurkat , Antígeno-1 Associado à Função Linfocitária/metabolismo , Microdomínios da Membrana/metabolismo , Microscopia Confocal , Talina/metabolismoRESUMO
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.