Epigallocatechin Gallate with Potent Anti-Helicobacter pylori Activity Binds Efficiently to Its Histone-like DNA Binding Protein

Abstract

Helicobacter pylori (H. pylori)a human gastric pathogenforms a major risk factor for the development of various gastric pathologies such as chronic inflammatory gastritis, peptic ulcer, lymphomas of mucosa-associated lymphoid tissues, and gastric carcinoma. The complete eradication of infection is the primary objective of treating any H. pylori-associated gastric condition. However, declining eradication efficiencies, off-target effects, and patient noncompliance to prolong and broad-spectrum antibiotic treatments has spurred the clinical interest to search for alternative effective and safer therapeutic options. As natural compounds are safe and privileged with high levels of antibacterial-activity, previous studies have tested and reported a plethora of such compounds with potential in vitro/in vivo anti-H. pylori activity. However, the mode of action of majority of these natural compounds is unclear. The present study has been envisaged to compile the information of various such natural compounds and to evaluate their binding with histone-like DNAbinding proteins of H. pylori (referred here as Hup) using in silico molecular docking-based virtual screening experiments. Hupbeing a major nucleoid-associated protein expressed by H. pylori plays a strategic role in its survival and persistent colonization under hostile stress conditions. The ligand with highest binding energy with Hupthat is, epigallocatechin-(−)gallate (EGCG)was rationally selected for further computational and experimental testing. The best docking poses of EGCG with Hup were first evaluated for their solution stability using long run molecular dynamics simulations and then using fluorescence and nuclear magnetic resonance titration experiments which demonstrated that the binding of EGCG with Hup is fairly strong (the resultant apparent dissociation constant (k D ) values were equal to 2.61 and 3.29 ± 0.42 μM, respectively).

Molecular Insights into Conformational Heterogeneity and Enhanced Structural Integrity of Helicobacter pylori DNA Binding Protein Hup at Low pH

“…Tyr fluorescence intensity maxima values at 306 nm were used to calculate the dissociation constants using the Stern−Volmer relationship and double log plots as described elsewhere. 48,49 Fluorescence Lifetime Spectroscopy. Fluorescence lifetime spectroscopy (FLS) measurements were performed on a time-correlated single-photon counting (TCSPC) spectrometer (Horiba Jobin Yvon Fluoro Cube Fluorescence Lifetime System) equipped with a Nano LED as pulse excitation source.…”

“…Further, the trajectory parameters such as root-mean-square deviations (RMSDs), radius of gyration (R g ), root-mean-square fluctuation (RMSF), and solvent accessible surface area (SASA) were analyzed. 48,59 DNA Binding Assay. The agarose gel electrophoresisbased DNA binding assay was performed with Hup (at pH 6 and 2) using ss-DNA and ds-DNA as described above in the fluorescence titration experiments.…”

“…The ds-DNA thus formed was titrated at various concentrations to attain a final molar ratio of Hup with DNA in the range of 1:0.1 to 1:5. Tyr fluorescence intensity maxima values at 306 nm were used to calculate the dissociation constants using the Stern–Volmer relationship and double log plots as described elsewhere. , …”

“…The structural ensembles of the Hup protein (pH 6 and 2) derived from MD simulation at time intervals of 0, 100, 200, 300, 400, and 500 ns were plotted using PYMOL graphics software. Further, the trajectory parameters such as root-mean-square deviations (RMSDs), radius of gyration ( R g ), root-mean-square fluctuation (RMSF), and solvent accessible surface area (SASA) were analyzed. , …”

Bacterial nucleoid-associated protein HU as an extracellular player in host-pathogen interaction

“…EGCG is a component of a green tea and its antimicrobial effect has been proven ( Mabe et al., 1999 ; Taylor et al., 2005 ; Cui et al., 2012 ; Jeon et al., 2014 ; Nakayama et al., 2015 ; Lee et al., 2017 ; Parvez et al., 2019 ). A recent study on H. pylori showed that EGCG can bind HU protein and alter its function ( Raj et al., 2021 ). Moreover, we have also confirmed the negative effect of EGCG on the function of the HU protein in F. tularensis which led to the overall attenuation of bacterial virulence both in vitro and in vivo (Stojkova et al., under review).…”