RESUMO
Multifunctional autoprocessing repeats-in-toxin (MARTX) toxins are pore-forming bacterial toxins that translocate multiple functionally independent effector domains into a target eukaryotic cell. Vibrio cholerae colonizes intestinal epithelial cells (IECs) and uses a MARTX toxin with three effector domains-an actin cross-linking domain (ACD), a Rho inactivation domain (RID), and an α/ß hydrolase domain (ABH)-to suppress innate immunity and enhance colonization. We investigated whether these multiple catalytic enzymes delivered from a single toxin functioned in a coordinated manner to suppress intestinal innate immunity. Using cultured human IECs, we demonstrated that ACD-induced cytoskeletal collapse activated extracellular signal-regulated kinase, p38, and c-Jun amino-terminal kinase mitogen-activated protein kinase (MAPK) signaling to elicit a robust proinflammatory response characterized by the secretion of interleukin-8 (IL-8; also called CXCL8) and the expression of CXCL8, tumor necrosis factor (TNF), and other proinflammatory genes. However, RID and ABH, which are naturally delivered together with ACD, blocked MAPK activation through Rac1 and thus prevented ACD-induced inflammation. RID also abolished IL-8 secretion induced by heat-killed bacteria, TNF, or latrunculin A. Thus, MARTX toxins use enzymatic multifunctionality to silence the host response to bacterial factors and to the damage caused by the toxins. Furthermore, these data show how V. cholerae MARTX toxin suppresses intestinal inflammation and contributes to cholera being classically defined as a noninflammatory diarrheal disease.
Assuntos
Citoesqueleto de Actina/metabolismo , Toxinas Bacterianas/metabolismo , Citocinas/metabolismo , Citoesqueleto/metabolismo , Mediadores da Inflamação/metabolismo , Vibrio cholerae/metabolismo , Citoesqueleto de Actina/imunologia , Toxinas Bacterianas/imunologia , Linhagem Celular Tumoral , Citocinas/genética , Citocinas/imunologia , Citoesqueleto/imunologia , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Regulação Neoplásica da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/imunologia , Células HeLa , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Mediadores da Inflamação/imunologia , Mutação , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Vibrio cholerae/genética , Vibrio cholerae/imunologiaRESUMO
Protein purification is the most basic and critical step for protein biophysical and biochemical studies to understand its function and structure. Various fusion tags and proteases have been developed and assembled in expression and purification system. However, it is one of the fields that continues to innovate to develop improved systems that are more efficient, simpler, and less expensive. An efficient self-cleavage C-terminal fusion system was developed using the inositol hexakisphosphate-inducible Vibrio cholerae MARTXVc toxin cysteine protease domain (CPD). CPD fusion proteins are expressed from the T7 promoter and purified using a 6xHis-tag with immobilized-metal affinity chromatography. The C-terminal CPD-tag is removed by self-cleavage at the final purification stage. Here, we describe an efficient cloning method using Gibson assembly, followed by expression and purification of tagless recombinant proteins of interest using CPD self-cleavage.
Assuntos
Toxinas Bacterianas/química , Clonagem Molecular/métodos , Ácido Fítico/metabolismo , Vibrio cholerae/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/genética , Cromatografia de Afinidade , Histidina/química , Domínios Proteicos , Engenharia de Proteínas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
The Ras-extracellular signal-regulated kinase pathway is critical for controlling cell proliferation, and its aberrant activation drives the growth of various cancers. Because many pathogens produce toxins that inhibit Ras activity, efforts to develop effective Ras inhibitors to treat cancer could be informed by studies of Ras inhibition by pathogens. Vibrio vulnificus causes fatal infections in a manner that depends on multifunctional autoprocessing repeats-in-toxin, a toxin that releases bacterial effector domains into host cells. One such domain is the Ras/Rap1-specific endopeptidase (RRSP), which site-specifically cleaves the Switch I domain of the small GTPases Ras and Rap1. We solved the crystal structure of RRSP and found that its backbone shares a structural fold with the EreA/ChaN-like superfamily of enzymes. Unlike other proteases in this family, RRSP is not a metalloprotease. Through nuclear magnetic resonance analysis and nucleotide exchange assays, we determined that the processing of KRAS by RRSP did not release any fragments or cause KRAS to dissociate from its bound nucleotide but instead only locally affected its structure. However, this structural alteration of KRAS was sufficient to disable guanine nucleotide exchange factor-mediated nucleotide exchange and prevent KRAS from binding to RAF. Thus, RRSP is a bacterial effector that represents a previously unrecognized class of protease that disconnects Ras from its signaling network while inducing limited structural disturbance in its target.