RESUMEN
The quorum-sensing signaling systems in Vibrio bacteria converge to control levels of the master transcription factors LuxR/HapR, a family of highly conserved proteins that regulate gene expression for bacterial behaviors. A compound library screen identified 2-thiophenesulfonamide compounds that specifically inhibit Vibrio campbellii LuxR but do not affect cell growth. We synthesized a panel of 50 thiophenesulfonamide compounds to examine the structure-activity relationship effects on Vibrio quorum sensing. The most potent molecule identified, PTSP (3-phenyl-1-(thiophen-2-ylsulfonyl)-1H-pyrazole), inhibits quorum sensing in multiple strains of V. vulnificus, V. parahaemolyticus, and V. campbellii at nanomolar concentrations. However, thiophenesulfonamide inhibition efficacy varies significantly among Vibrio species: PTSP is most inhibitory against V. vulnificus SmcR, but V. cholerae HapR is completely resistant to all thiophenesulfonamides tested. Reverse genetics experiments show that PTSP efficacy is dictated by amino acid sequence in the putative ligand-binding pocket: F75Y and C170F SmcR substitutions are each sufficient to eliminate PTSP inhibition. Further, in silico modeling distinguished the most potent thiophenesulfonamides from less-effective derivatives. Our results revealed the previously unknown differences in LuxR/HapR proteins that control quorum sensing in Vibrio species and underscore the potential for developing thiophenesulfonamides as specific quorum sensing-directed treatments for Vibrio infections.
Asunto(s)
Percepción de Quorum/efectos de los fármacos , Proteínas Represoras/antagonistas & inhibidores , Proteínas Represoras/metabolismo , Sulfonamidas/metabolismo , Sulfonamidas/farmacología , Transactivadores/antagonistas & inhibidores , Transactivadores/metabolismo , Vibrio/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Ligandos , Simulación del Acoplamiento Molecular , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas Represoras/química , Especificidad de la Especie , Relación Estructura-Actividad , Sulfonamidas/química , Transactivadores/química , Vibrio/química , Vibrio/genéticaRESUMEN
Quorum sensing gene expression in vibrios is regulated by the LuxR/HapR family of transcriptional factors, which includes Vibrio vulnificus SmcR. The consensus binding site of Vibrio LuxR/HapR/SmcR proteins is palindromic but highly degenerate with sequence variations at each promoter. To examine the mechanism by which SmcR recognizes diverse DNA sites, we generated SmcR separation-of-function mutants that either repress or activate transcription but not both. SmcR N55I is restricted in recognition of single base-pair variations in DNA binding site sequences and thus is defective at transcription activation but retains interaction with RNA polymerase (RNAP) alpha. SmcR S76A, L139R and N142D substitutions disrupt the interaction with RNAP alpha but retain functional DNA binding activity. X-ray crystallography and small angle X-ray scattering data show that the SmcR DNA binding domain exists in two conformations (wide and narrow), and the protein complex forms a mixture of dimers and tetramers in solution. The three RNAP interaction-deficient variants also have two DNA binding domain conformations, whereas SmcR N55I exhibits only the wide conformation. These data support a model in which two mechanisms drive SmcR transcriptional activation: interaction with RNAP and a multi-conformational DNA binding domain that permits recognition of variable DNA sites.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas de Unión al ADN/química , Transactivadores/química , Transactivadores/genética , Factores de Transcripción/química , Vibrio vulnificus/química , Sitios de Unión , Cristalografía por Rayos X , Proteínas de Unión al ADN/genética , Dimerización , Expresión Génica , Regulación Bacteriana de la Expresión Génica/genética , Modelos Moleculares , Mutación , Regiones Promotoras Genéticas , Conformación Proteica , Percepción de Quorum/genética , Proteínas Recombinantes , Proteínas Represoras/química , Proteínas Represoras/genética , Dispersión del Ángulo Pequeño , Factores de Transcripción/genética , Vibrio vulnificus/genéticaRESUMEN
Quorum sensing is a cell density-dependent form of cellular communication among bacteria. This signaling process has been heavily studied in vibrios due to their diverse and complex phenotypes and relevance to human and aquaculture disease. Mechanistic studies of Vibrio quorum sensing have required optimization of protein purification techniques to examine the role of key proteins, such as the LuxR/HapR family of transcription factors that control quorum-sensing gene expression. Protein purification is the cornerstone of biochemistry, and it is crucial to consistently produce batches of protein that are pure, active, and concentrated to perform various assays. The methods described here are optimized for purification of the Vibrio master quorum-sensing regulators, LuxR (Vibrio harveyi), HapR (Vibrio cholerae), and SmcR (Vibrio vulnificus). We anticipate that these methods can be applied to other proteins in this family of transcription factors.
Asunto(s)
Proteínas Bacterianas/aislamiento & purificación , Factores de Transcripción/aislamiento & purificación , Vibrio/química , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Percepción de Quorum , Factores de Transcripción/química , Factores de Transcripción/metabolismoRESUMEN
The marine facultative pathogen Vibrio cholerae forms complex multicellular communities on the chitinous shells of crustacean zooplankton in its aquatic reservoir. V. cholerae-chitin interactions are critical for the growth, evolution, and waterborne transmission of cholera. This is due, in part, to chitin-induced changes in gene expression in this pathogen. Here, we sought to identify factors that influence chitin-induced expression of one locus, the chitobiose utilization operon (chb), which is required for the uptake and catabolism of the chitin disaccharide. Through a series of genetic screens, we identified that the master regulator of quorum sensing, HapR, is a direct repressor of the chb operon. We also found that the levels of HapR in V. cholerae are regulated by the ClpAP protease. Furthermore, we show that the canonical quorum sensing cascade in V. cholerae regulates chb expression in an HapR-dependent manner. Through this analysis, we found that signaling via the species-specific autoinducer CAI-1, but not the interspecies autoinducer AI-2, influences chb expression. This phenomenon of species-specific regulation may enhance the fitness of this pathogen in its environmental niche.IMPORTANCE In nature, bacteria live in multicellular and multispecies communities. Microbial species can sense the density and composition of their community through chemical cues using a process called quorum sensing (QS). The marine pathogen Vibrio cholerae is found in communities on the chitinous shells of crustaceans in its aquatic reservoir. V. cholerae interactions with chitin are critical for the survival, evolution, and waterborne transmission of this pathogen. Here, we show that V. cholerae uses QS to regulate the expression of one locus required for V. cholerae-chitin interactions.