RESUMEN
Familial Mediterranean fever (FMF) is an autoinflammatory disease caused by homozygous or compound heterozygous gain-of-function mutations in MEFV, which encodes pyrin, an inflammasome protein. Heterozygous carrier frequencies for multiple MEFV mutations are high in several Mediterranean populations, suggesting that they confer selective advantage. Among 2,313 Turkish people, we found extended haplotype homozygosity flanking FMF-associated mutations, indicating evolutionarily recent positive selection of FMF-associated mutations. Two pathogenic pyrin variants independently arose >1,800 years ago. Mutant pyrin interacts less avidly with Yersinia pestis virulence factor YopM than with wild-type human pyrin, thereby attenuating YopM-induced interleukin (IL)-1ß suppression. Relative to healthy controls, leukocytes from patients with FMF harboring homozygous or compound heterozygous mutations and from asymptomatic heterozygous carriers released heightened IL-1ß specifically in response to Y. pestis. Y. pestis-infected MefvM680I/M680I FMF knock-in mice exhibited IL-1-dependent increased survival relative to wild-type knock-in mice. Thus, FMF mutations that were positively selected in Mediterranean populations confer heightened resistance to Y. pestis.
Asunto(s)
Resistencia a la Enfermedad/genética , Fiebre Mediterránea Familiar/genética , Peste , Pirina/genética , Selección Genética/genética , Animales , Proteínas de la Membrana Bacteriana Externa/inmunología , Proteínas de la Membrana Bacteriana Externa/metabolismo , Resistencia a la Enfermedad/inmunología , Haplotipos , Humanos , Inflamasomas/inmunología , Inflamasomas/metabolismo , Ratones , Ratones Endogámicos C57BL , Mutación , Peste/inmunología , Peste/metabolismo , Pirina/inmunología , Pirina/metabolismo , Turquía , Factores de Virulencia/inmunología , Factores de Virulencia/metabolismo , Yersinia pestisRESUMEN
Yersinia pestis, the bacterial agent of plague, secretes several proteins important for pathogenesis or host protection. The F1 protein forms a capsule on the bacterial cell surface and is a well-characterized protective antigen but is not essential for virulence. A type III secretion system that is essential for virulence exports Yop proteins, which function as antiphagocytic or anti-inflammatory factors. Yop effectors (e.g., YopE) are delivered across the host cell plasma membrane by a translocon, composed of YopB and YopD. Complexes of YopB, YopD, and YopE (BDE) secreted by Yersinia pseudotuberculosis were purified by affinity chromatography and used as immunogens to determine if antibodies to the translocon could provide protection against Y. pestis in mice. Mice vaccinated with BDE generated high-titer immunoglobulin G antibodies specific for BDE, as shown by enzyme-linked immunosorbent assay and immunoblotting, and were protected against lethal intravenous challenge with F1(-) but not F1(+) Y. pestis. Mice passively immunized with anti-BDE serum were protected from lethal challenge with F1(-) Y. pestis. The YopB protein or a complex of YopB and YopD (BD) was purified and determined by vaccination to be immunogenic in mice. Mice actively vaccinated with BD or passively vaccinated with anti-BD serum were protected against lethal challenge with F1(-) Y. pestis. These results indicate that anti-translocon antibodies can be used as immunotherapy to treat infections by F1(-) Y. pestis.
Asunto(s)
Anticuerpos Antibacterianos/inmunología , Antígenos Bacterianos/inmunología , Proteínas de la Membrana Bacteriana Externa/inmunología , Inmunización Pasiva/métodos , Peste/prevención & control , Animales , Anticuerpos Antibacterianos/uso terapéutico , Western Blotting , Ensayo de Inmunoadsorción Enzimática , Ratones , Ratones Endogámicos BALB C , Vacuna contra la Peste/inmunología , Vacunación/métodos , Yersinia pestis/inmunologíaRESUMEN
Plague is an acute infection caused by the Gram-negative bacterium Yersinia pestis. Antibodies that are protective against plague target LcrV, an essential virulence protein and component of a type III secretion system of Y. pestis. Secreted LcrV localizes to the tips of type III needles on the bacterial surface, and its function is necessary for the translocation of Yersinia outer proteins (Yops) into the cytosol of host cells infected by Y. pestis. Translocated Yops counteract macrophage functions, for example, by inhibiting phagocytosis (YopE) or inducing cytotoxicity (YopJ). Although LcrV is the best-characterized protective antigen of Y. pestis, the mechanism of protection by anti-LcrV antibodies is not fully understood. Antibodies bind to LcrV at needle tips, neutralize Yop translocation, and promote opsonophagocytosis of Y. pestis by macrophages in vitro. However, it is not clear if anti-LcrV antibodies neutralize Yop translocation directly or if they do so indirectly, by promoting opsonophagocytosis. To determine if the protective IgG1 monoclonal antibody (MAb) 7.3 is directly neutralizing, an IgG2a subclass variant, a deglycosylated variant, F(ab')2, and Fab were tested for the ability to inhibit the translocation of Yops into Y. pestis-infected macrophages in vitro. Macrophage cytotoxicity and cellular fractionation assays show that the Fc of MAb 7.3 is not required for the neutralization of YopJ or YopE translocation. In addition, the use of Fc receptor-deficient macrophages, and the use of cytochalasin D to inhibit actin polymerization, confirmed that opsonophagocytosis is not required for MAb 7.3 to neutralize translocation. These data indicate that the binding of the variable region of MAb 7.3 to LcrV is sufficient to directly neutralize Yop translocation.
Asunto(s)
Anticuerpos Antibacterianos/inmunología , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Antígenos Bacterianos/inmunología , Proteínas Citotóxicas Formadoras de Poros/antagonistas & inhibidores , Proteínas Citotóxicas Formadoras de Poros/inmunología , Yersinia pestis/inmunología , Animales , Femenino , Inmunoglobulina G/inmunología , Macrófagos/inmunología , Macrófagos/microbiología , Ratones Endogámicos C57BL , FagocitosisRESUMEN
Yersinia spp. cause gastroenteritis and the plague, representing historically devastating pathogens that are currently an important biodefense and antibiotic resistance concern. A critical virulence determinant is the Yersinia protein kinase A, or YpkA, a multidomain protein that disrupts the eukaryotic actin cytoskeleton. Here we solve the crystal structure of a YpkA-Rac1 complex and find that YpkA possesses a Rac1 binding domain that mimics host guanidine nucleotide dissociation inhibitors (GDIs) of the Rho GTPases. YpkA inhibits nucleotide exchange in Rac1 and RhoA, and mutations that disrupt the YpkA-GTPase interface abolish this activity in vitro and impair in vivo YpkA-induced cytoskeletal disruption. In cell culture experiments, the kinase and the GDI domains of YpkA act synergistically to promote cytoskeletal disruption, and a Y. pseudotuberculosis mutant lacking YpkA GDI activity shows attenuated virulence in a mouse infection assay. We conclude that virulence in Yersinia depends strongly upon mimicry of host GDI proteins by YpkA.
Asunto(s)
Proteínas Bacterianas/metabolismo , Inhibidores de Disociación de Guanina Nucleótido/metabolismo , Imitación Molecular , Proteínas Serina-Treonina Quinasas/metabolismo , Factores de Virulencia/metabolismo , Yersinia pseudotuberculosis/patogenicidad , Proteína de Unión al GTP rac1/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Línea Celular , Cristalografía , Citoesqueleto/metabolismo , Inhibidores de Disociación de Guanina Nucleótido/química , Humanos , Mucosa Intestinal/metabolismo , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Transfección , Factores de Virulencia/química , Factores de Virulencia/genética , Yersinia pseudotuberculosis/enzimología , Yersinia pseudotuberculosis/metabolismo , Proteína de Unión al GTP cdc42/química , Proteína de Unión al GTP cdc42/metabolismo , Proteína de Unión al GTP rac1/química , Inhibidores de la Disociación del Nucleótido Guanina rho-Específico , Proteína de Unión al GTP rhoA/química , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
YopH is a protein tyrosine phosphatase and an essential virulence determinant of the pathogenic bacterium Yersinia. Yersinia delivers YopH into infected host cells using a type III secretion mechanism. YopH dephosphorylates several focal adhesion proteins including p130Cas in human epithelial cells, resulting in disruption of focal adhesions and cell detachment from the extracellular matrix. How the C-terminal protein tyrosine phosphatase domain of YopH targets specific substrates such as p130Cas in the complex milieu of the host cell has not been fully elucidated. An N-terminal non-catalytic domain of YopH binds p130Cas in a phosphotyrosine-dependent manner and functions as a novel substrate-targeting site. The structure of the YopH protein tyrosine phosphatase domain bound to a model phosphopeptide substrate was solved and the resulting structure revealed a second substrate-targeting site ('site 2') within the catalytic domain. Site 2 binds to p130Cas in a phosphotyrosine-dependent manner, and co-operates with the N-terminal domain ('site 1') to promote efficient recognition of p130Cas by YopH in epithelial cells. The identification of two substrate-targeting sites in YopH that co-operate to promote epithelial cell detachment and bacterial virulence reinforces the importance of protein-protein interactions for determining protein tyrosine phosphatase specificity in vivo, and highlights the sophisticated nature of microbial pathogenicity factors.
Asunto(s)
Proteínas Tirosina Fosfatasas/metabolismo , Virulencia/genética , Infecciones por Yersinia pseudotuberculosis/microbiología , Yersinia pseudotuberculosis/patogenicidad , Proteínas Adaptadoras Transductoras de Señales , Animales , Proteínas de la Membrana Bacteriana Externa , Sitios de Unión , Células HeLa , Humanos , Ratones , Ratones Endogámicos C57BL , Proteínas Tirosina Fosfatasas/análisis , Yersinia pseudotuberculosis/enzimología , Yersinia pseudotuberculosis/genéticaRESUMEN
Yersinia are causative agents in human diseases ranging from gastrointestinal syndromes to Bubonic Plague. There is increasing risk of misuse of infectious agents, such as Yersinia pestis, as weapons of terror as well as instruments of warfare for mass destruction. YopH is an essential virulence factor whose protein-tyrosine phosphatase (PTP) activity is required for Yersinia pathogenicity. Consequently, there is considerable interest in developing potent and selective YopH inhibitors as novel anti-plague agents. We have screened a library of 720 structurally diverse commercially available carboxylic acids and identified 26 YopH inhibitors with IC50 values below 100 mum. The most potent and specific YopH inhibitor is aurintricarboxylic acid (ATA), which exhibits a Ki value of 5 nm for YopH and displays 6-120-fold selectivity in favor of YopH against a panel of mammalian PTPs. To determine whether ATA can block the activity of YopH in a cellular context, we have examined the effect of ATA on T-cell signaling in human Jurkat cells transfected with YopH. We show that YopH severely decreases the T-cell receptor-induced cellular tyrosine phosphorylation, ERK1/2 activity, and interleukin-2 transcriptional activity. We demonstrate that ATA can effectively block the inhibitory activity of YopH and restore normal T-cell function. These results provide a proof-of-concept for the hypothesis that small molecule inhibitors that selectively target YopH may be therapeutically useful. In addition, it is expected that potent and selective YopH inhibitors, such as ATA, should be useful reagents to delineate YopH's cellular targets in plague and other pathogenic conditions caused by Yersinia infection.