RESUMEN
Clostridioides difficile is a leading cause of antibiotic-associated diarrhea and nosocomial infection in the United States. The symptoms of C. difficile infection (CDI) are associated with the production of two homologous protein toxins, TcdA and TcdB. The toxins are considered bona fide targets for clinical diagnosis as well as the development of novel prevention and therapeutic strategies. While there are extensive studies that document these efforts, there are several gaps in knowledge that could benefit from the creation of new research tools. First, we now appreciate that while TcdA sequences are conserved, TcdB sequences can vary across the span of circulating clinical isolates. An understanding of the TcdA and TcdB epitopes that drive broadly neutralizing antibody responses could advance the effort to identify safe and effective toxin-protein chimeras and fragments for vaccine development. Further, an understanding of TcdA and TcdB concentration changes in vivo can guide research into how host and microbiome-focused interventions affect the virulence potential of C. difficile. We have developed a panel of alpaca-derived nanobodies that bind specific structural and functional domains of TcdA and TcdB. We note that many of the potent neutralizers of TcdA bind epitopes within the delivery domain, a finding that could reflect roles of the delivery domain in receptor binding and/or the conserved role of pore-formation in the delivery of the toxin enzyme domains to the cytosol. In contrast, neutralizing epitopes for TcdB were found in multiple domains. The nanobodies were also used for the creation of sandwich ELISA assays that allow for quantitation of TcdA and/or TcdB in vitro and in the cecal and fecal contents of infected mice. We anticipate these reagents and assays will allow researchers to monitor the dynamics of TcdA and TcdB production over time, and the impact of various experimental interventions on toxin production in vivo.
Asunto(s)
Toxinas Bacterianas , Clostridioides difficile , Anticuerpos de Dominio Único , Animales , Ratones , Toxinas Bacterianas/genética , Toxinas Bacterianas/química , Enterotoxinas/genética , Clostridioides difficile/genética , Clostridioides difficile/metabolismo , Epítopos/metabolismo , Proteínas Bacterianas/metabolismoRESUMEN
The relative abundance of Wnt receptors plays a crucial role in controlling Wnt signaling in tissue homeostasis and human disease. While the ubiquitin ligases that ubiquitylate Wnt receptors are well-characterized, the deubiquitylase that reverses these reactions remains unclear. Herein, we identify USP46, UAF1, and WDR20 (USP46 complex) as positive regulators of Wnt signaling in cultured human cells. We find that the USP46 complex is similarly required for Wnt signaling in Xenopus and zebrafish embryos. We demonstrate that Wnt signaling promotes the association between the USP46 complex and cell surface Wnt coreceptor, LRP6. Knockdown of USP46 decreases steady-state levels of LRP6 and increases the level of ubiquitylated LRP6. In contrast, overexpression of the USP46 complex blocks ubiquitylation of LRP6 by the ubiquitin ligases RNF43 and ZNFR3. Size exclusion chromatography studies suggest that the size of the USP46 cytoplasmic complex increases upon Wnt stimulation. Finally, we show that USP46 is essential for Wnt-dependent intestinal organoid viability, likely via its role in LRP6 receptor homeostasis. We propose a model in which the USP46 complex increases the steady-state level of cell surface LRP6 and facilitates the assembly of LRP6 into signalosomes via a pruning mechanism that removes sterically hindering ubiquitin chains.
Asunto(s)
Endopeptidasas , Vía de Señalización Wnt , beta Catenina , Animales , Humanos , beta Catenina/genética , beta Catenina/metabolismo , Ligasas/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/genética , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/metabolismo , Receptores Wnt , Ubiquitina , Pez Cebra/metabolismo , Endopeptidasas/metabolismoRESUMEN
Clostridioides difficile is linked to nearly 225,000 antibiotic-associated diarrheal infections and almost 13,000 deaths per year in the United States. Pathogenic strains of C. difficile produce toxin A (TcdA) and toxin B (TcdB), which can directly kill cells and induce an inflammatory response in the colonic mucosa. Hirota et al. (S. A. Hirota et al., Infect Immun 80:4474-4484, 2012) first introduced the intrarectal instillation model of intoxication using TcdA and TcdB purified from VPI 10463 (VPI 10463 reference strain [ATCC 43255]) and 630 C. difficile strains. Here, we expand this technique by instilling purified, recombinant TcdA and TcdB, which allows for the interrogation of how specifically mutated toxins affect tissue. Mouse colons were processed and stained with hematoxylin and eosin for blinded evaluation and scoring by a board-certified gastrointestinal pathologist. The amount of TcdA or TcdB needed to produce damage was lower than previously reported in vivo and ex vivo Furthermore, TcdB mutants lacking either endosomal pore formation or glucosyltransferase activity resemble sham negative controls. Immunofluorescent staining revealed how TcdB initially damages colonic tissue by altering the epithelial architecture closest to the lumen. Tissue sections were also immunostained for markers of acute inflammatory infiltration. These staining patterns were compared to slides from a human C. difficile infection (CDI). The intrarectal instillation mouse model with purified recombinant TcdA and/or TcdB provides the flexibility needed to better understand structure/function relationships across different stages of CDI pathogenesis.
Asunto(s)
Clostridioides difficile/patogenicidad , Susceptibilidad a Enfermedades , Enterocolitis Seudomembranosa/microbiología , Enterotoxinas/administración & dosificación , Proteínas Recombinantes/administración & dosificación , Animales , Proteínas Bacterianas/administración & dosificación , Proteínas Bacterianas/genética , Toxinas Bacterianas/administración & dosificación , Toxinas Bacterianas/genética , Colon , Modelos Animales de Enfermedad , Enterotoxinas/genética , Humanos , Inmunohistoquímica , Mucosa Intestinal/patología , Ratones , Proteínas MutantesRESUMEN
Intestinal bile acids are known to modulate the germination and growth of Clostridioides difficile Here we describe a role for intestinal bile acids in directly binding and neutralizing TcdB toxin, the primary determinant of C. difficile disease. We show that individual primary and secondary bile acids reversibly bind and inhibit TcdB to varying degrees through a mechanism that requires the combined oligopeptide repeats region to which no function has previously been ascribed. We find that bile acids induce TcdB into a compact "balled up" conformation that is no longer able to bind cell surface receptors. Lastly, through a high-throughput screen designed to identify bile acid mimetics we uncovered nonsteroidal small molecule scaffolds that bind and inhibit TcdB through a bile acid-like mechanism. In addition to suggesting a role for bile acids in C. difficile pathogenesis, these findings provide a framework for development of a mechanistic class of C. difficile antitoxins.
Asunto(s)
Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Ácidos y Sales Biliares/metabolismo , Clostridioides difficile/metabolismo , Intestinos/fisiología , Receptores de Superficie Celular/metabolismo , Células CACO-2 , Clostridioides difficile/crecimiento & desarrollo , Infecciones por Clostridium/microbiología , Células HCT116 , HumanosRESUMEN
In Staphylococcus aureus-caused endocarditis, the pathogen secretes staphylocoagulase (SC), thereby activating human prothrombin (ProT) and evading immune clearance. A previous structural comparison of the SC(1-325) fragment bound to thrombin and its inactive precursor prethrombin 2 has indicated that SC activates ProT by inserting its N-terminal dipeptide Ile1-Val2 into the ProT Ile16 pocket, forming a salt bridge with ProT's Asp194, thereby stabilizing the active conformation. We hypothesized that these N-terminal SC residues modulate ProT binding and activation. Here, we generated labeled SC(1-246) as a probe for competitively defining the affinities of N-terminal SC(1-246) variants preselected by modeling. Using ProT(R155Q,R271Q,R284Q) (ProTQQQ), a variant refractory to prothrombinase- or thrombin-mediated cleavage, we observed variant affinities between â¼1 and 650 nm and activation potencies ranging from 1.8-fold that of WT SC(1-246) to complete loss of function. Substrate binding to ProTQQQ caused allosteric tightening of the affinity of most SC(1-246) variants, consistent with zymogen activation through occupation of the specificity pocket. Conservative changes at positions 1 and 2 were well-tolerated, with Val1-Val2, Ile1-Ala2, and Leu1-Val2 variants exhibiting ProTQQQ affinity and activation potency comparable with WT SC(1-246). Weaker binding variants typically had reduced activation rates, although at near-saturating ProTQQQ levels, several variants exhibited limiting rates similar to or higher than that of WT SC(1-246). The Ile16 pocket in ProTQQQ appears to favor nonpolar, nonaromatic residues at SC positions 1 and 2. Our results suggest that SC variants other than WT Ile1-Val2-Thr3 might emerge with similar ProT-activating efficiency.
Asunto(s)
Proteínas Bacterianas/metabolismo , Coagulasa/metabolismo , Protrombina/metabolismo , Staphylococcus aureus/metabolismo , Proteínas Bacterianas/química , Sitios de Unión , Coagulasa/química , Humanos , Modelos Moleculares , Unión Proteica , Protrombina/química , Infecciones Estafilocócicas/metabolismo , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/química , Especificidad por SustratoRESUMEN
The mitochondrial antiviral signaling protein (MAVS) orchestrates host antiviral innate immune response to RNA virus infection. However, how MAVS signaling is controlled to eradicate virus while preventing self-destructive inflammation remains obscure. Here, we show that protein geranylgeranylation, a posttranslational lipid modification of proteins, limits MAVS-mediated immune signaling by targeting Rho family small guanosine triphosphatase Rac1 into the mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) at the mitochondria-ER junction. Protein geranylgeranylation and subsequent palmitoylation promote Rac1 translocation into MAMs upon viral infection. MAM-localized Rac1 limits MAVS' interaction with E3 ligase Trim31 and hence inhibits MAVS ubiquitination, aggregation, and activation. Rac1 also facilitates the recruitment of caspase-8 and cFLIPL to the MAVS signalosome and the subsequent cleavage of Ripk1 that terminates MAVS signaling. Consistently, mice with myeloid deficiency of protein geranylgeranylation showed improved survival upon influenza A virus infection. Our work revealed a critical role of protein geranylgeranylation in regulating antiviral innate immune response.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Inmunidad Innata/fisiología , Neuropéptidos/metabolismo , Infecciones por Orthomyxoviridae/inmunología , Prenilación de Proteína/inmunología , Proteína de Unión al GTP rac1/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Transferasas Alquil y Aril/genética , Transferasas Alquil y Aril/metabolismo , Animales , Retículo Endoplásmico/inmunología , Retículo Endoplásmico/metabolismo , Femenino , Humanos , Macrófagos Alveolares/inmunología , Macrófagos Alveolares/metabolismo , Masculino , Ratones Noqueados , Neuropéptidos/genética , Infecciones por Orthomyxoviridae/metabolismo , Infecciones por Orthomyxoviridae/mortalidad , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Proteínas de Motivos Tripartitos/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas de Unión al GTP rac/genética , Proteínas de Unión al GTP rac/metabolismo , Proteína de Unión al GTP rac1/genética , Proteína RCA2 de Unión a GTPRESUMEN
Current thought holds that factor Xa (FXa) bound in the prothrombinase complex is resistant to regulation by protein protease inhibitors during prothrombin activation. Here we provide evidence that, contrary to this view, the FXa-specific serpin inhibitor, protein Z-dependent protease inhibitor (ZPI), complexed with its cofactor, protein Z (PZ), functions as a physiologically significant inhibitor of prothrombinase-bound FXa during prothrombin activation. Kinetics studies showed that the rapid rate of inhibition of FXa by the ZPI-PZ complex on procoagulant membrane vesicles (ka(app) â¼107 m-1 s-1) was decreased â¼10-fold when FXa was bound to FVa in prothrombinase and a further â¼3-4-fold when plasma levels of S195A prothrombin were present (ka(app) 2 × 105 m-1 s-1). Nevertheless, the ZPI-PZ complex produced a major inhibition of thrombin generation during prothrombinase-catalyzed activation of prothrombin under physiologically relevant conditions. The importance of ZPI-PZ complex anticoagulant regulation of FXa both before and after incorporation into prothrombinase was supported by thrombin generation assays in plasma. These showed enhanced thrombin generation when the inhibitor was neutralized with a PZ-specific antibody and decreased thrombin generation when exogenous ZPI-PZ complex was added whether prothrombin was activated directly by FXa or through extrinsic or intrinsic pathway activators. Moreover, the PZ antibody enhanced thrombin generation both in the absence and presence of activated protein C (APC) anticoagulant activity. Taken together, these results suggest an important anticoagulant role for the ZPI-PZ complex in regulating both free FXa generated in the initiation phase of coagulation as well as prothrombinase-bound FXa in the propagation phase that complement prothrombinase regulation by APC.
Asunto(s)
Coagulación Sanguínea , Factor V/química , Factor Xa/química , Protrombina/química , Serpinas/química , Trombina/química , Sustitución de Aminoácidos , Anticuerpos/química , Factor V/genética , Factor V/metabolismo , Factor Xa/genética , Factor Xa/metabolismo , Humanos , Cinética , Mutación Missense , Proteína C/química , Proteína C/metabolismo , Protrombina/genética , Protrombina/metabolismo , Serpinas/genética , Serpinas/metabolismo , Trombina/genética , Trombina/metabolismoRESUMEN
Clostridium difficile infection is the leading cause of hospital-acquired diarrhea and is mediated by the actions of two toxins, TcdA and TcdB. The toxins perturb host cell function through a multistep process of receptor binding, endocytosis, low pH-induced pore formation, and the translocation and delivery of an N-terminal glucosyltransferase domain that inactivates host GTPases. Infection studies with isogenic strains having defined toxin deletions have established TcdB as an important target for therapeutic development. Monoclonal antibodies that neutralize TcdB function have been shown to protect against C. difficile infection in animal models and reduce recurrence in humans. Here, we report the mechanism of TcdB neutralization by PA41, a humanized monoclonal antibody capable of neutralizing TcdB from a diverse array of C. difficile strains. Through a combination of structural, biochemical, and cell functional studies, involving X-ray crystallography and EM, we show that PA41 recognizes a single, highly conserved epitope on the TcdB glucosyltransferase domain and blocks productive translocation and delivery of the enzymatic cargo into the host cell. Our study reveals a unique mechanism of C. difficile toxin neutralization by a monoclonal antibody, which involves targeting a process that is conserved across the large clostridial glucosylating toxins. The PA41 antibody described here provides a valuable tool for dissecting the mechanism of toxin pore formation and translocation across the endosomal membrane.
Asunto(s)
Anticuerpos Neutralizantes/metabolismo , Toxinas Bacterianas/metabolismo , Clostridioides difficile/metabolismo , Enterotoxinas/metabolismo , Anticuerpos Monoclonales/metabolismo , Toxinas Bacterianas/química , Células CACO-2 , Clostridioides difficile/enzimología , Cristalografía por Rayos X , Citosol/metabolismo , Enterotoxinas/química , Humanos , Concentración de Iones de Hidrógeno , Microscopía Electrónica , Rubidio/química , Proteína de Unión al GTP rac1/química , Proteína de Unión al GTP rac1/metabolismoRESUMEN
Clostridium difficile is a major nosocomial pathogen that produces two exotoxins, TcdA and TcdB, with TcdB thought to be the primary determinant in human disease. TcdA and TcdB are large, multidomain proteins, each harboring a cytotoxic glucosyltransferase domain that is delivered into the cytosol from endosomes via a translocation domain after receptor-mediated endocytosis of toxins from the cell surface. Although there are currently no known host cell receptors for TcdA, three cell-surface receptors for TcdB have been identified: CSPG4, NECTIN3, and FZD1/2/7. The sites on TcdB that mediate binding to each receptor are not defined. Furthermore, it is not known whether the combined repetitive oligopeptide (CROP) domain is involved in or required for receptor binding. Here, in a screen designed to identify sites in TcdB that are essential for target cell intoxication, we identified a region at the junction of the translocation and the CROP domains that is implicated in CSPG4 binding. Using a series of C-terminal truncations, we show that the CSPG4-binding site on TcdB extends into the CROP domain, requiring three short repeats for binding and for full toxicity on CSPG4-expressing cells. Consistent with the location of the CSPG4-binding site on TcdB, we show that the anti-TcdB antibody bezlotoxumab, which binds partially within the first three short repeats, prevents CSPG4 binding to TcdB. In addition to establishing the binding region for CSPG4, this work ascribes for the first time a role in TcdB CROPs in receptor binding and further clarifies the relative roles of host receptors in TcdB pathogenesis.
Asunto(s)
Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/metabolismo , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Clostridioides difficile/enzimología , Glucosiltransferasas/metabolismo , Proteínas de la Membrana/metabolismo , Animales , Anticuerpos Monoclonales/química , Anticuerpos Neutralizantes/química , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/genética , Toxinas Bacterianas/antagonistas & inhibidores , Toxinas Bacterianas/genética , Anticuerpos ampliamente neutralizantes , Células CHO , Células CACO-2 , Chlorocebus aethiops , Proteoglicanos Tipo Condroitín Sulfato/genética , Clostridioides difficile/genética , Clostridioides difficile/patogenicidad , Cricetinae , Cricetulus , Glucosiltransferasas/antagonistas & inhibidores , Glucosiltransferasas/genética , Células HEK293 , Humanos , Proteínas de la Membrana/genética , Unión Proteica , Dominios ProteicosRESUMEN
Clostridium difficile is a clinically significant pathogen that causes mild-to-severe (and often recurrent) colon infections. Disease symptoms stem from the activities of two large, multidomain toxins known as TcdA and TcdB. The toxins can bind, enter, and perturb host cell function through a multistep mechanism of receptor binding, endocytosis, pore formation, autoproteolysis, and glucosyltransferase-mediated modification of host substrates. Monoclonal antibodies that neutralize toxin activity provide a survival benefit in preclinical animal models and prevent recurrent infections in human clinical trials. However, the molecular mechanisms involved in these neutralizing activities are unclear. To this end, we performed structural studies on a neutralizing monoclonal antibody, PA50, a humanized mAb with both potent and broad-spectrum neutralizing activity, in complex with TcdA. Electron microscopy imaging and multiangle light-scattering analysis revealed that PA50 binds multiple sites on the TcdA C-terminal combined repetitive oligopeptides (CROPs) domain. A crystal structure of two PA50 Fabs bound to a segment of the TcdA CROPs helped define a conserved epitope that is distinct from previously identified carbohydrate-binding sites. Binding of TcdA to the host cell surface was directly blocked by either PA50 mAb or Fab and suggested that receptor blockade is the mechanism by which PA50 neutralizes TcdA. These findings highlight the importance of the CROPs C terminus in cell-surface binding and a role for neutralizing antibodies in defining structural features critical to a pathogen's mechanism of action. We conclude that PA50 protects host cells by blocking the binding of TcdA to cell surfaces.
Asunto(s)
Antibacterianos/metabolismo , Anticuerpos Neutralizantes/metabolismo , Toxinas Bacterianas/metabolismo , Clostridioides difficile/enzimología , Enterocitos/metabolismo , Enterotoxinas/metabolismo , Glucosiltransferasas/metabolismo , Modelos Moleculares , Secuencia de Aminoácidos , Antibacterianos/química , Anticuerpos Monoclonales Humanizados/química , Anticuerpos Monoclonales Humanizados/metabolismo , Anticuerpos Neutralizantes/química , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/toxicidad , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Toxinas Bacterianas/toxicidad , Sitios de Unión de Anticuerpos , Células CACO-2 , Secuencia Conservada , Cristalografía por Rayos X , Enterocitos/efectos de los fármacos , Enterotoxinas/química , Enterotoxinas/genética , Enterotoxinas/toxicidad , Mapeo Epitopo , Glucosiltransferasas/química , Glucosiltransferasas/genética , Glucosiltransferasas/toxicidad , Humanos , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/toxicidad , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/toxicidad , Secuencias Repetitivas de AminoácidoRESUMEN
The exotoxins toxin A (TcdA) and toxin B (TcdB) are produced by the bacterial pathogen Clostridium difficile and are responsible for the pathology associated with C. difficile infection (CDI). The antitoxin antibodies actoxumab and bezlotoxumab bind to and neutralize TcdA and TcdB, respectively. Bezlotoxumab was recently approved by the FDA for reducing the recurrence of CDI. We have previously shown that a single molecule of bezlotoxumab binds to two distinct epitopes within the TcdB combined repetitive oligopeptide (CROP) domain, preventing toxin binding to host cells. In this study, we characterize the binding of actoxumab to TcdA and examine its mechanism of toxin neutralization. Using a combination of approaches including a number of biophysical techniques, we show that there are two distinct actoxumab binding sites within the CROP domain of TcdA centered on identical amino acid sequences at residues 2162-2189 and 2410-2437. Actoxumab binding caused the aggregation of TcdA especially at higher antibody:toxin concentration ratios. Actoxumab prevented the association of TcdA with target cells demonstrating that actoxumab neutralizes toxin activity by inhibiting the first step of the intoxication cascade. This mechanism of neutralization is similar to that observed with bezlotoxumab and TcdB. Comparisons of the putative TcdA epitope sequences across several C. difficile ribotypes and homologous repeat sequences within TcdA suggest a structural basis for observed differences in actoxumab binding and/or neutralization potency. These data provide a mechanistic basis for the protective effects of the antibody in vitro and in vivo, including in various preclinical models of CDI.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Anticuerpos Neutralizantes/metabolismo , Toxinas Bacterianas/antagonistas & inhibidores , Enterotoxinas/antagonistas & inhibidores , Epítopos/metabolismo , Sitios de Unión , Anticuerpos ampliamente neutralizantes , Agregado de Proteínas , Unión ProteicaRESUMEN
Clostridium difficile strains within the hypervirulent clade 2 are responsible for nosocomial outbreaks worldwide. The increased pathogenic potential of these strains has been attributed to several factors but is still poorly understood. During a C. difficile outbreak, a strain from this clade was found to induce a variant cytopathic effect (CPE), different from the canonical arborizing CPE. This strain (NAP1V) belongs to the NAP1 genotype but to a ribotype different from the epidemic NAP1/RT027 strain. NAP1V and NAP1 share some properties, including the overproduction of toxins, the binary toxin, and mutations in tcdC. NAP1V is not resistant to fluoroquinolones, however. A comparative analysis of TcdB proteins from NAP1/RT027 and NAP1V strains indicated that both target Rac, Cdc42, Rap, and R-Ras but only the former glucosylates RhoA. Thus, TcdB from hypervirulent clade 2 strains possesses an extended substrate profile, and RhoA is crucial for the type of CPE induced. Sequence comparison and structural modeling revealed that TcdBNAP1 and TcdBNAP1V share the receptor-binding and autoprocessing activities but vary in the glucosyltransferase domain, consistent with the different substrate profile. Whereas the two toxins displayed identical cytotoxic potencies, TcdBNAP1 induced a stronger proinflammatory response than TcdBNAP1V as determined in ex vivo experiments and animal models. Since immune activation at the level of intestinal mucosa is a hallmark of C. difficile-induced infections, we propose that the panel of substrates targeted by TcdB is a determining factor in the pathogenesis of this pathogen and in the differential virulence potential seen among C. difficile strains.
Asunto(s)
Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/metabolismo , Clostridioides difficile/metabolismo , Clostridioides difficile/patogenicidad , Enterocolitis Seudomembranosa/enzimología , Enterocolitis Seudomembranosa/microbiología , Proteína de Unión al GTP rhoA/metabolismo , Animales , Proteínas Bacterianas/genética , Toxinas Bacterianas/genética , Clostridioides difficile/clasificación , Clostridioides difficile/genética , Enterocolitis Seudomembranosa/genética , Genotipo , Glicosilación , Interacciones Huésped-Patógeno , Humanos , Masculino , Ratones , Virulencia , Proteína de Unión al GTP rhoA/genéticaRESUMEN
Prothrombin is conformationally activated by von Willebrand factor-binding protein (vWbp) from Staphylococcus aureus through insertion of the NH(2)-terminal residues of vWbp into the prothrombin catalytic domain. The rate of prothrombin activation by vWbp(1-263) is controlled by a hysteretic kinetic mechanism initiated by substrate binding. The present study evaluates activation of prothrombin by full-length vWbp(1-474) through activity progress curve analysis. Additional interactions from the COOH-terminal half of vWbp(1-474) strengthened the initial binding of vWbp to prothrombin, resulting in higher activity and an â¼100-fold enhancement in affinity. The affinities of vWbp(1-263) or vWbp(1-474) were compared by equilibrium binding to the prothrombin derivatives prethrombin 1, prethrombin 2, thrombin, meizothrombin, and meizothrombin(des-fragment 1) and their corresponding active site-blocked analogs. Loss of fragment 1 in prethrombin 1 enhanced affinity for both vWbp(1-263) and vWbp(1-474), with a 30-45% increase in Gibbs free energy, implicating a regulatory role for fragment 1 in the activation mechanism. Active site labeling of all prothrombin derivatives with D-Phe-Pro-Arg-chloromethyl ketone, analogous to irreversible binding of a substrate, decreased their K(D) values for vWbp into the subnanomolar range, reflecting the dependence of the activating conformational change on substrate binding. The results suggest a role for prothrombin domains in the pathophysiological activation of prothrombin by vWbp, and may reveal a function for autocatalysis of the vWbp·prothrombin complexes during initiation of blood coagulation.
Asunto(s)
Proteínas Portadoras/química , Precursores Enzimáticos/química , Glicoproteínas de Membrana Plaquetaria/química , Protrombina/metabolismo , Staphylococcus aureus/metabolismo , Factor de von Willebrand/química , Unión Competitiva , Coagulación Sanguínea , Dominio Catalítico , Fibrina/química , Células HEK293 , Humanos , Cinética , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Termodinámica , Trombina/química , Factores de Virulencia/químicaRESUMEN
Coagulase-positive Staphylococcus aureus (S. aureus) is the major causal pathogen of acute endocarditis, a rapidly progressing, destructive infection of the heart valves. Bacterial colonization occurs at sites of endothelial damage, where, together with fibrin and platelets, the bacteria initiate the formation of abnormal growths known as vegetations. Here we report that an engineered analog of prothrombin could be used to detect S. aureus in endocarditic vegetations via noninvasive fluorescence or positron emission tomography (PET) imaging. These prothrombin derivatives bound staphylocoagulase and intercalated into growing bacterial vegetations. We also present evidence for bacterial quorum sensing in the regulation of staphylocoagulase expression by S. aureus. Staphylocoagulase expression was limited to the growing edge of mature vegetations, where it was exposed to the host and co-localized with the imaging probe. When endocarditis was induced with an S. aureus strain with genetic deletion of coagulases, survival of mice improved, highlighting the role of staphylocoagulase as a virulence factor.
Asunto(s)
Endocarditis Bacteriana/diagnóstico , Protrombina/metabolismo , Staphylococcus aureus/metabolismo , Animales , Coagulasa/metabolismo , Ratones , Microscopía Fluorescente , Tomografía de Emisión de Positrones , Ingeniería de Proteínas , Percepción de Quorum/fisiología , Staphylococcus aureus/patogenicidadRESUMEN
Mouse and human prothrombin (ProT) active site specifically labeled with D-Phe-Pro-Arg-CH(2)Cl (FPR-ProT) inhibited tissue factor-initiated thrombin generation in platelet-rich and platelet-poor mouse and human plasmas. FPR-prethrombin 1 (Pre 1), fragment 1 (F1), fragment 1.2 (F1.2), and FPR-thrombin produced no significant inhibition, demonstrating the requirement for all three ProT domains. Kinetics of inhibition of ProT activation by the inactive ProT(S195A) mutant were compatible with competitive inhibition as an alternate nonproductive substrate, although FPR-ProT deviated from this mechanism, implicating a more complex process. FPR-ProT exhibited â¼10-fold more potent anticoagulant activity compared with ProT(S195A) as a result of conformational changes in the ProT catalytic domain that induce a more proteinase-like conformation upon FPR labeling. Unlike ProT and ProT(S195A), the pathway of FPR-ProT cleavage by prothrombinase was redirected from meizothrombin toward formation of the FPR-prethrombin 2 (Pre 2)·F1.2 inhibitory intermediate. Localization of ProT labeled with Alexa Fluor® 660 tethered through FPR-CH(2)Cl ([AF660]FPR-ProT) during laser-induced thrombus formation in vivo in murine arterioles was examined in real time wide-field and confocal fluorescence microscopy. [AF660]FPR-ProT bound rapidly to the vessel wall at the site of injury, preceding platelet accumulation, and subsequently to the thrombus proximal, but not distal, to the vessel wall. [AF660]FPR-ProT inhibited thrombus growth, whereas [AF660]FPR-Pre 1, lacking the F1 membrane-binding domain did not bind or inhibit. Labeled F1.2 localized similarly to [AF660]FPR-ProT, indicating binding to phosphatidylserine-rich membranes, but did not inhibit thrombosis. The studies provide new insight into the mechanism of ProT activation in vivo and in vitro, and the properties of a unique exosite-directed prothrombinase inhibitor.
Asunto(s)
Dominio Catalítico , Protrombina/metabolismo , Tromboplastina/metabolismo , Trombosis/enzimología , Sustitución de Aminoácidos , Animales , Coagulación Sanguínea , Activación Enzimática/genética , Humanos , Cinética , Ratones , Mutación Missense , Estructura Terciaria de Proteína , Protrombina/química , Protrombina/genética , Tromboplastina/química , Tromboplastina/genética , Trombosis/genéticaRESUMEN
Skizzle (SkzL), secreted by Streptococcus agalactiae, has moderate sequence identity to streptokinase and staphylokinase, bacterial activators of human plasminogen (Pg). SkzL binds [Glu]Pg with low affinity (K(D) 3-16 mum) and [Lys]Pg and plasmin (Pm) with indistinguishable high affinity (K(D) 80 and 50 nm, respectively). Binding of SkzL to Pg and Pm is completely lysine-binding site-dependent, as shown by the effect of the lysine analog, 6-aminohexanoic acid. Deletion of the COOH-terminal SkzL Lys(415) residue reduces affinity for [Lys]Pg and active site-blocked Pm 30-fold, implicating Lys(415) in a lysine-binding site interaction with a Pg/Pm kringle. SkzL binding to active site fluorescein-labeled Pg/Pm analogs demonstrates distinct high and low affinity interactions. High affinity binding is mediated by Lys(415), whereas the source of low affinity binding is unknown. SkzL enhances the activation of [Glu]Pg by urokinase (uPA) approximately 20-fold, to a maximum rate indistinguishable from that for [Lys]Pg and [Glu]Pg activation in the presence of 6-aminohexanoic acid. SkzL binds preferentially to the partially extended beta-conformation of [Glu]Pg, which is in unfavorable equilibrium with the compact alpha-conformation, thereby converting [Glu]Pg to the fully extended gamma-conformation and accelerating the rate of its activation by uPA. SkzL enhances [Lys]Pg and [Glu]Pg activation by single-chain tissue-type Pg activator, approximately 42- and approximately 650-fold, respectively. SkzL increases the rate of plasma clot lysis by uPA and single-chain tissue-type Pg activator approximately 2-fold, confirming its cofactor activity in a physiological model system. The results suggest a role for SkzL in S. agalactiae pathogenesis through fibrinolytic enhancement.
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Proteínas Bacterianas/metabolismo , Fibrinolisina/biosíntesis , Fibrinolisina/metabolismo , Fibrinólisis/fisiología , Metaloendopeptidasas/metabolismo , Plasminógeno/metabolismo , Streptococcus agalactiae/metabolismo , Estreptoquinasa/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Secuencia Conservada , Humanos , Cinética , Metaloendopeptidasas/química , Metaloendopeptidasas/genética , Unión Proteica , Estreptoquinasa/química , Estreptoquinasa/genética , Especificidad por SustratoRESUMEN
Von Willebrand factor-binding protein (VWbp), secreted by Staphylococcus aureus, displays secondary structural homology to the 3-helix bundle, D1 and D2 domains of staphylocoagulase (SC), a potent conformational activator of the blood coagulation zymogen, prothrombin (ProT). In contrast to the classical proteolytic activation mechanism of trypsinogen-like serine proteinase zymogens, insertion of the first 2 residues of SC into the NH(2)-terminal binding cleft on ProT (molecular sexuality) induces rapid conformational activation of the catalytic site. Based on plasma-clotting assays, the target zymogen for VWbp may be ProT, but this has not been verified, and the mechanism of ProT activation is unknown. We demonstrate that VWbp activates ProT conformationally in a mechanism requiring its Val(1)-Val(2) residues. By contrast to SC, full time-course kinetic studies of ProT activation by VWbp demonstrate that it activates ProT by a substrate-dependent, hysteretic kinetic mechanism. VWbp binds weakly to ProT (K(D) 2.5 microM) to form an inactive complex, which is activated through a slow conformational change by tripeptide chromogenic substrates and its specific physiological substrate, identified here as fibrinogen (Fbg). This mechanism increases the specificity of ProT activation by delaying it in a slow reversible process, with full activation requiring binding of Fbg through an exosite expressed on the activated ProT*.VWbp complex. The results suggest that this unique mechanism regulates pathological fibrin (Fbn) deposition to VWF-rich areas during S. aureus endocarditis.
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Proteínas Portadoras/fisiología , Protrombina/química , Factor de von Willebrand/fisiología , Unión Competitiva , Coagulación Sanguínea , Proteínas Portadoras/química , Activación Enzimática , Humanos , Hidrólisis , Cinética , Conformación Molecular , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Staphylococcus aureus/metabolismo , Especificidad por Sustrato , Tripsinógeno/química , Factor de von Willebrand/químicaRESUMEN
The specificity of thrombin for procoagulant and anticoagulant substrates is regulated allosterically by Na+. Ordered cleavage of prothrombin (ProT) at Arg320 by the prothrombinase complex generates proteolytically active, meizothrombin (MzT), followed by cleavage at Arg271 to produce thrombin and fragment 1.2. The alternative pathway of initial cleavage at Arg271 produces the inactive zymogen form, the prethrombin 2 (Pre 2).fragment 1.2 complex, which is cleaved subsequently at Arg320. Cleavage at Arg320 of ProT or prethrombin 1 (Pre 1) activates the catalytic site and the precursor form of exosite I (proexosite I). To determine the pathway of expression of Na+-(pro)exosite I linkage during ProT activation, the effects of Na+ on the affinity of fluorescein-labeled hirudin-(54-65) ([5F]Hir-(54-65)(SO-3)) for the zymogens, ProT, Pre 1, and Pre 2, and for the proteinases, MzT and MzT-desfragment 1 (MzT(-F1)) were quantitated. The zymogens showed no significant linkage between proexosite I and Na+, whereas cleavage at Arg320 caused the affinities of MzT and MzT(-F1) for [5F]Hir-(54-65)(SO-3) to be enhanced by Na+ 8- to 10-fold and 5- to 6-fold, respectively. MzT and MzT(-F1) showed kinetically different mechanisms of Na+ enhancement of chromogenic substrate hydrolysis. The results demonstrate for the first time that MzT is regulated allosterically by Na+. The results suggest that the distinctive procoagulant substrate specificity of MzT, in activating factor V and factor VIII on membranes, and the anticoagulant, membrane-modulated activation of protein C by MzT bound to thrombomodulin are regulated by Na+-induced allosteric transition. Further, the Na+ enhancement in MzT activity and exosite I affinity may function in directing the sequential ProT activation pathway by accelerating thrombin formation from the MzT fast form.
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Sitio Alostérico , Precursores Enzimáticos/química , Protrombina/química , Sodio/química , Trombina/química , Regulación Alostérica , Factores de Coagulación Sanguínea/química , Factores de Coagulación Sanguínea/metabolismo , Cationes Monovalentes/química , Cationes Monovalentes/metabolismo , Membrana Celular/metabolismo , Endopeptidasas/química , Activación Enzimática , Precursores Enzimáticos/metabolismo , Hirudinas/química , Hirudinas/metabolismo , Humanos , Cinética , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Protrombina/metabolismo , Sodio/metabolismo , Trombina/metabolismoRESUMEN
Staphylocoagulase (SC) is a potent nonproteolytic prothrombin (ProT) activator and the prototype of a newly established zymogen activator and adhesion protein family. The staphylocoagulase fragment containing residues 1-325 (SC-(1-325)) represents a new type of nonproteolytic activator with a unique fold consisting of two three-helix bundle domains. The N-terminal, domain 1 of SC (D1, residues 1-146) interacts with the 148 loop of thrombin and prethrombin 2 and the south rim of the catalytic site, whereas domain 2 of SC (D2, residues 147-325) occupies (pro)exosite I, the fibrinogen (Fbg) recognition exosite. Reversible conformational activation of ProT by SC-(1-325) was used to create novel analogs of ProT covalently labeled at the catalytic site with fluorescence probes. Analogs selected from screening 10 such derivatives were used to characterize quantitatively equilibrium binding of SC-(1-325) to ProT, competitive binding with native ProT, and SC domain interactions. The results support the conclusion that SC-(1-325) binds to a single site on fluorescein-labeled and native ProT with indistinguishable dissociation constants of 17-72 pM. The results obtained for isolated SC domains indicate that D2 binds ProT with approximately 130-fold greater affinity than D1, yet D1 binding accounts for the majority of the fluorescence enhancement that accompanies SC-(1-325) binding. The SC-(1-325).(pro)thrombin complexes and free thrombin showed little difference in substrate specificity for tripeptide substrates or with their natural substrate, Fbg. Lack of a significant effect of blockage of (pro)exosite I of (pro)thrombin by SC-(1-325) on Fbg cleavage indicates that a new Fbg substrate recognition exosite is expressed on the SC-(1-325).(pro)thrombin complexes. Our results provide new insight into the mechanism that mediates zymogen activation by this prototypical bacterial activator.
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Coagulasa/química , Colorantes Fluorescentes/química , Protrombina/química , Sitios de Unión , Unión Competitiva , Dominio Catalítico , Adhesión Celular , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Fibrina/química , Humanos , Cinética , Microscopía Fluorescente , Modelos Químicos , Modelos Moleculares , Péptidos/química , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Especificidad por Sustrato , Factores de TiempoRESUMEN
The cytoplasmic domain of the anion exchange protein (cdb3) serves as a critical organizing center for protein-protein interactions that stabilize the erythrocyte membrane. The structure of the central core of cdb3, determined by X-ray crystallography from crystals grown at pH 4.8, revealed a compact dimer for residues 55-356 and unresolved N- and C-termini on each monomer [Zhang et al. (2000) Blood 96, 2925-2933]. Given that previous studies had suggested a highly asymmetric structure for cdb3 and that pH dependent structural transitions of cdb3 have been reported, the structure of cdb3 in solution at neutral pH was investigated via site-directed spin labeling in combination with conventional electron paramagnetic resonance (EPR) and double electron electron resonance (DEER) spectroscopies. These studies show that the structure of the central compact dimer (residues 55-356) is indistinguishable from the crystal structure determined at pH 4.8. N-Terminal residues 1-54 and C-terminal residues 357-379 are dynamically disordered and show no indications of stable secondary structure. These results establish a structural model for cdb3 in solution at neutral pH which represents an important next step in characterizing structural details of the protein-protein interactions that stabilize the erythrocyte membrane.