RESUMO
In this work we carried out an in silico analysis to understand the interaction between InvF-SicA and RNAP in the bacterium Salmonella Typhimurium strain LT2. Structural analysis of InvF allowed the identification of three possible potential cavities for interaction with SicA. This interaction could occur with the structural motif known as tetratricopeptide repeat (TPR) 1 and 2 in the two cavities located in the interface of the InvF and α-CTD of RNAP. Indeed, molecular dynamics simulations showed that SicA stabilizes the Helix-turn-Helix DNA-binding motifs, i.e., maintaining their proper conformation, mainly in the DNA Binding Domain (DBD). Finally, to evaluate the role of amino acids that contribute to protein-protein affinity, an alanine scanning mutagenesis approach, indicated that R177 and R181, located in the DBD motif, caused the greatest changes in binding affinity with α-CTD, suggesting a central role in the stabilization of the complex. However, it seems that the N-terminal region also plays a key role in the protein-protein interaction, especially the amino acid R40, since we observed conformational flexibility in this region allowing it to interact with interface residues. We consider that this analysis opens the possibility to validate experimentally the amino acids involved in protein-protein interactions and explore other regulatory complexes where chaperones are involved.
Assuntos
Proteínas de Bactérias , Chaperonas Moleculares , Proteínas de Bactérias/genética , Chaperonas Moleculares/genética , Salmonella typhimurium/genética , Aminoácidos/metabolismo , DNA/metabolismoRESUMO
Salmonella enterica serovar Typhimurium causes gastroenteritis and systemic infections in humans. For this bacterium the expression of a type III secretion system (T3SS) and effector proteins encoded in the Salmonella pathogenicity island-1 (SPI-1), is keystone for the virulence of this bacterium. Expression of these is controlled by a regulatory cascade starting with the transcriptional regulators HilD, HilC and RtsA that induce the expression of HilA, which then activates expression of the regulator InvF, a transcriptional regulator of the AraC/XylS family. InvF needs to interact with the chaperone SicA to activate transcription of SPI-1 genes including sicA, sopB, sptP, sopE, sopE2, and STM1239. InvF very likely acts as a classical activator; however, whether InvF interacts with the RNA polymerase alpha subunit RpoA has not been determined. Results from this study confirm the interaction between InvF with SicA and reveal that both proteins interact with the RNAP alpha subunit. Thus, our study further supports that the InvF/SicA complex acts as a classical activator. Additionally, we showed for the first time an interaction between a chaperone of T3SS effectors (SicA) and the RNAP.
Assuntos
Proteínas de Ligação a DNA , Salmonella typhimurium , Humanos , Salmonella typhimurium/metabolismo , Proteínas de Ligação a DNA/genética , Transativadores/genética , Transativadores/metabolismo , Proteínas de Bactérias/metabolismo , Fatores de Transcrição/metabolismo , Chaperonas Moleculares/metabolismo , Regulação Bacteriana da Expressão GênicaRESUMO
Extracts of Hibiscus sabdariffa L. (commonly called Rosselle or "Jamaica flower" in Mexico) have been shown to have antibiotic and antivirulence properties in several bacteria. Here, an organic extract of H. sabdariffa L. is shown to inhibit motility in Salmonella enterica serovars Typhi and Typhimurium. The compound responsible for this effect was purified and found to be the hibiscus acid. When tested, this compound also inhibited motility and reduced the secretion of both flagellin and type III secretion effectors. Purified hibiscus acid was not toxic in tissue-cultured eukaryotic cells, and it was able to reduce the invasion of Salmonella Typhimurium in epithelial cells. Initial steps to understand its mode of action showed it might affect membrane proton balance.
Assuntos
Antibacterianos/farmacologia , Citratos/farmacologia , Flagelos/fisiologia , Flores/química , Hibiscus/química , Extratos Vegetais/farmacologia , Salmonella enterica/efeitos dos fármacos , Flagelos/efeitos dos fármacosRESUMO
Transcriptional factors play an important role in gene regulation in all organisms, especially in Bacteria. Here special emphasis is placed in the AraC/XylS family of transcriptional regulators. This is one of the most abundant as many predicted members have been identified and more members are added because more bacterial genomes are sequenced. Given the way more experimental evidence has mounded in the past decades, we decided to update the information about this captivating family of proteins. Using bioinformatics tools on all the data available for experimentally characterized members of this family, we found that many members that display a similar functional classification can be clustered together and in some cases they have a similar regulatory scheme. A proposal for grouping these proteins is also discussed. Additionally, an analysis of surveyed proteins in bacterial genomes is presented. Altogether, the current review presents a panoramic view into this family and we hope it helps to stimulate future research in the field.