RESUMO
Uropathogenic strains of Escherichia coli produce virulence factors, such as the protein toxin alpha-hemolysin (HlyA), that enable the bacteria to colonize the host and establish an infection. HlyA is synthetized as a protoxin (ProHlyA) that is transformed into the active form in the bacterial cytosol by the covalent linkage of two fatty-acyl moieties to the polypeptide chain before the secretion of HlyA into the extracellular medium. The aim of this work was to investigate the effect of the fatty acylation of HlyA on protein conformation and protein-membrane interactions. Polarization-modulated infrared reflection-absorption spectroscopy (PM-IRRAS) experiments were performed at the air-water interface, and lipid monolayers mimicking the outer leaflet of red-blood-cell membranes were used as model systems for the study of protein-membrane interaction. According to surface-pressure measurements, incorporation of the acylated protein into the lipid films was faster than that of the nonacylated form. PM-IRRAS measurements revealed that the adsorption of the proteins to the lipid monolayers induced disorder in the lipid acyl chains and also changed the elastic properties of the films independently of protein acylation. No significant difference was observed between HlyA and ProHlyA in the interaction with the model lipid monolayers; but when these proteins became adsorbed on a bare air-water interface, they adopted different secondary structures. The assumption of the correct protein conformation at a hydrophobic-hydrophilic interface could constitute a critical condition for biologic activity.
Assuntos
Proteínas de Escherichia coli/química , Proteínas Hemolisinas/química , Adsorção , Proteínas de Bactérias/química , Interações Hidrofóbicas e Hidrofílicas , Água/químicaAssuntos
Cálcio/metabolismo , Eritrócitos/química , Proteínas de Escherichia coli/farmacologia , Proteínas Hemolisinas/farmacologia , Acilação , Trifosfato de Adenosina/farmacologia , Animais , Sinalização do Cálcio/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Membrana Eritrocítica/efeitos dos fármacos , Eritrócitos/ultraestrutura , Proteínas de Escherichia coli/química , Proteínas Hemolisinas/química , Líquido Intracelular/química , Transporte de Íons , Processamento de Proteína Pós-Traducional , Coelhos , Receptores Purinérgicos/fisiologia , Imagem com Lapso de TempoRESUMO
Several toxins that act on animal cells present different, but specific, interactions with cholesterol or sphingomyelin. In the present study we demonstrate that HlyA (α-haemolysin) of Escherichia coli interacts directly with cholesterol. We have recently reported that HlyA became associated with detergent-resistant membranes enriched in cholesterol and sphingomyelin; moreover, toxin oligomerization, and hence haemolytic activity, diminishes in cholesterol-depleted erythrocytes. Considering these results, we studied the insertion process, an essential step in the lytic mechanism, by the monolayer technique, finding that HlyA insertion is favoured in cholesterol- and sphingomyelin-containing membranes. On the basis of this result, we studied the direct interaction with either of the lipids by lipid dot blotting, lysis inhibition and SPR (surface plasmon resonance) assays. The results of the present study demonstrated that an interaction between cholesterol and HlyA exists that seems to favour a conformational state of the protein that allows its correct insertion into the membrane and its further oligomerization to form pores.