RESUMEN
Aggregatibacter actinomycetemcomitans strains that express cytolethal distending toxin (Cdt) are associated with localized aggressive periodontitis. However, the in vivo targets of Cdt in the human oral cavity have not been firmly established. Here, we demonstrate that A. actinomycetemcomitans Cdt kills proliferating and nonproliferating U937 monocytic cells at a comparable specific activity, approximately 1.5-fold lower than that against the Cdt-hypersensitive Jurkat T-cell line. Cdt functioned both as a DNase and a phosphatidylinositol 3-phosphate (PIP(3)) phosphatase, and these activities were distinguished by site-specific mutagenesis of the active site residues of CdtB. Using these mutants, we determined that the DNase activity of CdtB is required for cell cycle arrest and caspase-dependent induction of apoptosis in proliferating U937 cells. In contrast, Cdt holotoxin induced apoptosis by a mechanism independent of caspase- and apoptosis-inducing factor in nonproliferating U937 cells. Furthermore, apoptosis of nonproliferating U937 cells was unaffected by the Cdt mutant possessing reduced phosphatase activity or by the addition of a specific PIP(3) phosphatase inhibitor, suggesting that the induction of apoptosis is independent of phosphatase activity. These results indicate that Cdt intoxication of proliferating and nonproliferating U937 cells occurs by distinct mechanisms and suggest that macrophages may also be potential in vivo targets of Cdt.
Asunto(s)
Apoptosis , Toxinas Bacterianas/toxicidad , Macrófagos/efectos de los fármacos , Pasteurellaceae/patogenicidad , Monoéster Fosfórico Hidrolasas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/genética , Línea Celular , Desoxirribonucleasas/genética , Desoxirribonucleasas/metabolismo , Humanos , Mutagénesis Sitio-Dirigida , Monoéster Fosfórico Hidrolasas/genéticaRESUMEN
ExoU, a phospholipase injected into host cells by the type III secretion system of Pseudomonas aeruginosa, leads to rapid cytolytic cell death. Although the importance of ExoU in infection is well established, the mechanism by which this toxin kills host cells is less clear. To gain insight into how ExoU causes cell death, we examined its subcellular localization following transfection or type III secretion/translocation into HeLa cells. Although rapid cell lysis precluded visualization of wild-type ExoU by fluorescence microscopy, catalytically inactive toxin was readily detected at the periphery of HeLa cells. Biochemical analysis confirmed that ExoU was targeted to the membrane fraction of transfected cells. Visualization of ExoU peptides fused with green fluorescent protein indicated that the domain responsible for this targeting was in the C terminus of ExoU, between residues 550 and 687. Localization to the plasma membrane occurred within 1 h of expression, which is consistent with the kinetics of cytotoxicity. Together, these results indicate that a domain between residues 550 and 687 of ExoU targets this toxin to the plasma membrane, a process that may be important in cytotoxicity.
Asunto(s)
Proteínas Bacterianas/metabolismo , Membrana Celular/metabolismo , Fragmentos de Péptidos/metabolismo , Células 3T3 , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/toxicidad , Células HeLa , Humanos , Ratones , Fosfolipasas A/metabolismo , Transporte de Proteínas , TransfecciónRESUMEN
ExoU, a potent patatin-like phospholipase, causes rapid cell death following its injection into host cells by the Pseudomonas aeruginosa type III secretion system. To better define regions of ExoU required for cytotoxicity, transposon-based linker insertion mutagenesis followed by site-directed mutagenesis of individual residues was employed by using a Saccharomyces cerevisiae model system. Random insertion of five amino acids identified multiple regions within ExoU that are required for cell killing. Five regions were chosen for further characterization: three corresponded to the oxyanion hole, hydrolase motif, and catalytic aspartate motif of the patatin-like domain within the N-terminal half of ExoU; one corresponded to an uncharacterized part of the patatin-like domain; and one corresponded to a region near the C terminus. Specific individual amino acid substitutions in each of the four N-terminal regions prevented killing of yeast and significantly reduced phospholipase activity. Whereas five amino acid insertions in the fifth region near the C terminus markedly reduced cytotoxicity and phospholipase activity, substitution of individual amino acids did not abolish either activity. To determine whether each of the five identified regions of ExoU was also essential for cytotoxicity in human cells, representative mutant forms of ExoU fused to green fluorescent protein were expressed in HeLa cells. These variants of ExoU were readily visualized and caused minimal cytotoxicity to HeLa cells, while wild-type ExoU fused to green fluorescent protein induced significant cell lysis and no detectable fluorescence. Thus, a minimum of five regions, including one which is well removed from the patatin-like domain, are required for the cytotoxicity and phospholipase activity of ExoU.
Asunto(s)
Proteínas Bacterianas/química , Secuencia de Aminoácidos , Proteínas Bacterianas/fisiología , Células HeLa , Humanos , Datos de Secuencia Molecular , Fosfolipasas A/metabolismo , Relación Estructura-ActividadRESUMEN
Overall, hospital-acquired pneumonia (HAP) caused by Pseudomonas aeruginosa is associated with high attributable mortality. Although the intrinsic virulence of P. aeruginosa undoubtedly contributes to this phenomenon, it is unclear whether all strains share this property or whether only a subpopulation of strains are capable of causing such severe disease. In this study, the virulence of 35 P. aeruginosa isolates obtained from patients with HAP by use of a cytolytic cell-death assay, an apoptosis assay, and a mouse model of pneumonia. The virulence of individual isolates differed significantly from one to another in each of these assays. Increased virulence was associated with the secretion of ExoU, a toxin transported by the P. aeruginosa type III secretion system. Secretion of ExoS or ExoY, 2 other proteins transported by this system, was not consistently associated with increased virulence. Together, these findings suggest that secretion of ExoU is a marker for highly virulent strains of P. aeruginosa.
Asunto(s)
Proteínas Bacterianas/toxicidad , Infección Hospitalaria/microbiología , Neumonía Bacteriana/microbiología , Pseudomonas aeruginosa/patogenicidad , Animales , Apoptosis , Glucosiltransferasas/toxicidad , Ratones , Ratones Endogámicos BALB C , VirulenciaRESUMEN
ExoU, a protein transported by the type III secretion system of Pseudomonas aeruginosa, is an important cytotoxin, though its mechanism of action is unclear. Here we show that the intracellular expression of ExoU is cytotoxic to Saccharomyces cerevisiae. Furthermore, internal amino- and carboxyl-terminal deletions confirmed that regions of ExoU previously shown to be essential for killing mammalian cells were also required for killing yeast cells. These findings indicate that S. cerevisiae is a useful model organism for the study of ExoU.