Binding Events of (S)-N-(3-Oxo-octanoyl)-homoserine Lactone with Agrobacterium tumefaciens Mutant Cells Studied by Saturation Transfer Difference NMR

Abstract

Quorum-sensing é um fenômeno de comunicação amplamente estudado em bactérias, o qual envolve a produção e detecção de substâncias sinalizadoras que dependem do comportamento da colônia e da densidade celular. No presente trabalho, a interação entre a substância sinalizadora (S)-N-(3-oxo-octanoil)-HSL e a membrana celular da A. tumefaciens NTL4(pZLR4) foi estudada utilizando a espectroscopia de RMN da diferença de transferência de saturação (STD-NMR). O mapa do epitopo da substância foi obtido, mostrando que a cadeia hidrofóbica acila apresentou o mais importante ponto de interação. Os resultados foram interpretados em comparação a um sistema modelo de membrana, os lipossomas. Também foi analisado o uso da b-ciclodextrina como carreador da acil-HSL.Quorum-sensing is a widely studied communication phenomenon in bacteria, which involves the production and detection of signaling substances in relation with cell density and colony behavior. Herein, the membrane binding interactions of the signal (S)-N-(3-oxo-octanoyl)-HSL with A. tumefaciens NTL4(pZLR4) cells were studied using saturation transfer difference NMR spectroscopy (STD-NMR). The substance epitope map was obtained showing that the hydrophobic acyl chain is the most important interacting site for the signal and the cell membrane. Results were interpreted upon comparisons with a simpler system, using liposomes as membrane models. Some insights on the use of b-cyclodextrin as acyl-HSL carrier were also provided. Keywords: STD-NMR, acyl-homoserine lactone, membrane interactions IntroductionIt is well recognized nowadays that bacteria use quorum-sensing communication circuits to regulate a wide array of physiological activities. These processes include symbiosis, virulence, competence, conjugation, antibiotic production, motility, sporulation and biofilm formation, among several other phenotypes crucial for bacterial survival and host infection. This phenomenon relies on the production of low molar mass signaling molecules usually related to cell density. In many Gram-negative bacteria the acyl-homoserine lactones (acyl-HSLs) are the most studied chemical signals. 1 In the quorum-sensing process, synthases (LuxI homologs) produce the signaling molecules, which diffuse from the cytoplasm to the near environment through the cellular membrane in the case of short chain acyl-HSLs or through active efflux pumping in long chain ones. 2 The acyl-HSL concentration is related to cell density, i.e., as the colony grows the metabolite concentration also increases due to the presence of new producing individuals. The process continues until a threshold concentration is reached, when the metabolites bind to transcriptional proteins (LuxR homologs) and the complex regulates gene expression. 3 Therefore, quorum-sensing allows whole bacterial populations to act coordinately, improving their chances of successful environment colonization.One of the most interesting quorum-sensing mechanisms was reported for Agrobacterium tumefaciens, a phytopathogen that induces crown gall tu...

Synthesis, Three-Dimensional Structure, Conformation and Correct Chemical Shift Assignment Determination of Pharmaceutical Molecules by NMR and Molecular Modeling

“…[7][8][9] For example, one of the most NMR used methods for this type of interaction studies is the saturation transfer difference (STD). 10,11 Several compounds have been described in the literature with impropriate chemical shifts and wrong structures, sometimes leading to their incorrect interaction with biological targets. Therefore, the complete structure determination and chemical shift assignment of new potential pharmacological agents is very important for the study for their biological activity and interaction with targets.…”