RESUMEN
The activation of CD40-mediated signaling in antigen-presenting cells is a promising therapeutic strategy to promote immune responses against tumors. Most agonistic anti-CD40 antibodies currently in development require the Fcγ-receptor (FcγR)-mediated crosslinking of CD40 molecules for a meaningful activation of CD40 signaling but have limitations due to dose-limiting toxicities. Here we describe the identification of CD40 antibodies which strongly stimulate antigen-presenting cells in an entirely FcγR-independent manner. These Fc-silenced anti-CD40 antibodies induce an efficient upregulation of costimulatory receptors and cytokine release by dendritic cells. Finally, the most active identified anti-CD40 antibody shows activity in humanized mice. More importantly, there are no signs of obvious toxicities. These studies thus demonstrate the potent activation of antigen-presenting cells with anti-CD40 antibodies lacking FcγR-binding activity and open the possibility for an efficacious and safe combination therapy for cancer patients.
RESUMEN
Type IV secretion systems are macromolecular assemblies in the cell envelopes of bacteria that function in macromolecular translocation. Structural biology approaches have provided insights into the interaction of core complex components, but information about proteins that undergo transient interactions with membrane components has not been forthcoming. We have pursued an unbiased approach using peptide arrays and phage display to identify interaction partners and interaction domains of type IV secretion system assembly factor VirB8. These approaches identified the globular domain from the VirB5 protein to interact with VirB8. This interaction was confirmed in cross-linking, pull-down, and fluorescence resonance energy transfer (FRET)-based interaction assays. In addition, using phage display analysis, we identified different regions of VirB6 as potential interaction partners of VirB8. Using a FRET-based interaction assay, we provide the first direct experimental evidence of the interaction of a VirB6 periplasmic domain with VirB8. These results will allow us to conduct directed structural biological work and structure-function analyses aimed at defining the molecular details and biological significance of these interactions with VirB8 in the future.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Brucella/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Proteínas de Transporte de Membrana/genética , Biblioteca de Péptidos , Periplasma/metabolismo , Multimerización de Proteína/efectos de los fármacos , Estructura Terciaria de Proteína , Transporte de Proteínas , Factores de Virulencia/metabolismoRESUMEN
Pathogenic Brucella species replicate within mammalian cells, and their type IV secretion system is essential for intracellular survival and replication. The options for biochemical studies on the Brucella secretion system are limited due to the rigidity of the cells and biosafety concerns, which preclude large-scale cell culture and fractionation. To overcome these problems, we heterologously expressed the Brucella suis virB operon in the closely related alpha(2)-proteobacterium Agrobacterium tumefaciens and showed that the VirB proteins assembled into a complex. Eight of the twelve VirB proteins were detected in the membranes of the heterologous host with specific antisera. Cross-linking indicated protein-protein interactions similar to those in other type IV secretion systems, and the results of immunofluorescence analysis supported the formation of VirB protein complexes in the cell envelope. Production of a subset of the B. suis VirB proteins (VirB3-VirB12) in A. tumefaciens strongly increased its ability to receive IncQ plasmid pLS1 in conjugation experiments, and production of VirB1 further enhanced the conjugation efficiency. Plasmid recipient competence correlated with periplasmic leakage and the detergent sensitivity of A. tumefaciens, suggesting a weakening of the cell envelope. Heterologous expression thus permits biochemical characterization of B. suis type IV secretion system assembly.
Asunto(s)
Agrobacterium tumefaciens/citología , Agrobacterium tumefaciens/inmunología , Brucella suis/inmunología , Brucelosis/inmunología , Brucelosis/microbiología , Plásmidos/genética , Brucella suis/genética , Brucelosis/metabolismo , Membrana Celular/inmunología , Técnica del Anticuerpo Fluorescente , OperónRESUMEN
VirB1-like proteins are believed to act as lytic transglycosylases, which facilitate the assembly of type IV secretion systems via localized lysis of the peptidoglycan. This paper presents the biochemical analysis of interactions of purified Brucella suis VirB1 with core components of the type IV secretion system. Genes encoding VirB1, VirB8, VirB9, VirB10 and VirB11 were cloned into expression vectors; the affinity-tagged proteins were purified from Escherichia coli, and analyses by gel filtration chromatography showed that they form monomers or homo-multimers. Analysis of protein-protein interactions by affinity precipitation revealed that VirB1 bound to VirB9 and VirB11. The results of bicistron expression experiments followed by gel filtration further supported the VirB1-VirB9 interaction. Peptide array mapping identified regions of VirB1 that interact with VirB8, VirB9 and VirB11 and underscored the importance of the C-terminus, especially for the VirB1-VirB9 interaction. The binding sites were localized on a structure model of VirB1, suggesting that different portions of VirB1 may interact with other VirB proteins during assembly of the type IV secretion machinery.
Asunto(s)
Proteínas Bacterianas/metabolismo , Brucella suis/enzimología , Glicosiltransferasas/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Sitios de Unión , Brucella suis/genética , Brucella suis/metabolismo , Dimerización , Regulación Bacteriana de la Expresión Génica , Vectores Genéticos , Glicosiltransferasas/química , Glicosiltransferasas/genética , Modelos Moleculares , Datos de Secuencia Molecular , Unión ProteicaRESUMEN
Type IV secretion systems mediate the translocation of virulence factors (proteins and/or DNA) from Gram-negative bacteria into eukaryotic cells. A complex of 11 conserved proteins (VirB1-VirB11) spans the inner and the outer membrane and assembles extracellular T-pili in Agrobacterium tumefaciens. Here we report a sequence of protein interactions required for the formation of complexes between VirB2 and VirB5, which precedes their incorporation into pili. The NTPase Walker A active site of the inner membrane protein VirB4 is required for virulence, but an active site VirB4 variant stabilized VirB3 and VirB8 and enabled T-pilus formation. Analysis of VirB protein complexes extracted from the membranes with mild detergent revealed that VirB2-VirB5 complex formation depended on VirB4, which identified a novel T-pilus assembly step. Bicistron expression demonstrated direct interaction of VirB4 with VirB8, and analyses with purified proteins showed that VirB5 bound to VirB8 and VirB10. VirB4 therefore localizes at the basis of a trans-envelope interaction sequence, and by stabilization of VirB8 it mediates the incorporation of VirB5 and VirB2 into extracellular pili.