Structural probing of HapR to identify potent phytochemicals to control Vibrio cholera through integrated computational approaches

Highlights

• HapR is critical for expression regulation of several genes of V. cholera.

• Medicinal compounds were found inhibiting HapR.

• Strychnogucine A and Galluflavanone made stable bindings with critical residues.

• Screened hits can be used as antibacterial agents in further research.

Abstract

Cholera is a severe small intestine bacterial disease caused by consumption of water and food contaminated with Vibrio cholera. The disease causes watery diarrhea leading to severe dehydration and even death if left untreated. In the past few decades, V. cholerae has emerged as multidrug-resistant enteric pathogen due to its rapid ability to adapt in detrimental environmental conditions. This research study aimed to design inhibitors of a master virulence gene expression regulator, HapR. HapR is critical in regulating the expression of several set of V. cholera virulence genes, quorum-sensing circuits and biofilm formation. A blind docking strategy was employed to infer the natural binding tendency of diverse phytochemicals extracted from medicinal plants by exposing the whole HapR structure to the screening library. Scoring function criteria was applied to prioritize molecules with strong binding affinity (binding energy < −11 kcal/mol) and as such two compounds: Strychnogucine A and Galluflavanone were filtered. Both the compounds were found favourably binding to the conserved dimerization interface of HapR. One rare binding conformation of Strychnogucine A was noticed docked at the elongated cavity formed by α1, α4 and α6 (binding energy of −12.5 kcal/mol). The binding stability of both top leads at dimer interface and elongated cavity was further estimated using long run of molecular dynamics simulations, followed by MMGB/PBSA binding free energy calculations to define the dominance of different binding energies. In a nutshell, this study presents computational evidence on antibacterial potential of phytochemicals capable of directly targeting bacterial virulence and highlight their great capacity to be utilized in the future experimental studies to stop the evolution of antibiotic resistance evolution.

Introduction

Cholera is a commonly occurring deadly disease caused by the gram-negative bacterium Vibrio cholerae. The expression of various virulence factors such as cholera toxin, toxin co-regulated pilus and a critical colonization factor, play key role in enabling the bacterium to cause the disease [1,2]. A transcriptional cascade is responsible for the expression of these factors that starts at the tcpPH promoter [[3], [4], [5]]. AphA and AphB are the regulators in this process, where AphA belongs to a relatively uncharacterized family of regulators. It activates the expression by helping AphB through LysR-type regulator, which binds to its promoter region. Specifically, AphA is a winged helical DNA binding protein and its crystal structure shows that it has an unusual C-terminal anti-parallel coiled-coil dimerization domain [6]. During bacterial cell to cell communication, gene expression is influenced in a density-dependent manner [7]. This process is termed as quorum sensing, used by both gram-positive and gram-negative bacteria for the regulation of various physiological processes such as, biofilm development, virulence factor expression, bioluminescence and antibiotic production [8]. The quorum-sensing circuit in most gram-negative bacteria is similar to that of Vibrio fischeri, a symbiotic bacterium [9]. It has an autoinducer, a specific acylated homoserine lactone signalling molecule, synthesized by a LuxI type protein. The concentration of autoinducer increases with the increase in cell density. When a critical threshold concentration is attained both intra and extracellularly, a soluble LuxR type protein binds to the autoinducer resulting in gene expression activation. The quorum-sensing circuit of V. cholerae is similar to free-living marine bacteria Vibrio harveyi and notably complex than V. fischeri quorum-sensing circuit. The enzymes CqsA and LuxS synthesize two different autoinducers CAI-1 and AI-2, respectively [10]. With the increase in cell density, the cognate sensors CqsS and LuxPQ detect the autoinducers. These sensors are protein kinases in nature and perform phosphorylation to control the activity of the response regulator LuxO, which in turn activates the expression of four small RNAs (qrr1 to 4) [11,12]. The expression of transcriptional factor HapR is controlled by these RNAs by effecting the stability of its mRNA. HapR is a quorum sensing mediated transcription factor in V. cholerae, shares homology with LuxR of V. harveyi and differences with V. fischeri LuxR protein [6,13].

LuxO unphosphorylate at high cell density and consequently unable to perform activation of small RNAs expression. This assists HapR and LuxR to get accumulated and regulate the gene expression. Similar to V. harveyi, HapR and LuxR proteins of V. cholerae use quorum sensing for the regulation of several vital cellular processes. V. cholerae HapR protein inhibits both gene expression and biofilm formation at high cell density, which is inverse of other quorum sensing systems where both processes are induced at high cell density [14]. The virulence gene expression is suppressed by binding to a particular site in the AphA promoter between −85 and −58 positions [15]. This helps in reduction of AphA intracellular levels and prevention of virulence cascade activation. All the mentioned process is believed to play a role in the self-limiting nature of cholera infections. The mechanism is yet to be explored for the suppression of biofilm formation [16]. HapR is also involved in the expression of the other two proteins: a hemaglutinin, also referred to as HA protease, and novel transcriptional regulator for DNA uptake natural competence [17]. The structural analysis of V. cholerae HapR reveals that it is a 203 residues, all helical dimeric protein, 71% identical to V. harveyi LuxR [6]. The crystal structure was determined at 2.2 Å resolution to further understand HapR mediated regulation of virulence gene expression and biofilm formation at the molecular level. HapR has an N-terminal helix turn helix DNA binding motif and a dimerization domain at C-terminal, same as a number of other TetR family regulators [18]. There is a unique solvent-accessible tunnel at the dimerization interface that joins to an amphipathic hollow in each monomer which may serve as small molecule effector or ligand-binding pocket [6].

In silico drug design and discovery methods have become reliable, cheaper and time-effective strategy for the establishment of new leads [19]. The treatment history of diseases especially associated with microbes using natural products is as long as a human being has inhabited the earth [[20], [21], [22]]. Medicinal plants contains various phytochemicals with important pharmaceutical properties and potential to facilitate novel anti-microbial development [23,24]. These phytochemcials have revolutionized the modern medicinal system and recent World Health Organization reports anticipated that around 80% of global population is dependent on medincinal plants based therapeutics [25]. In addition, it has laso been reported that over the past four decades, >60% commercial drugs are produced from natural products or their derivatives [21,26]. Therefore, for virtual screening of lead compounds against V. cholerae HapR, our own developed Medicinal Plant Database for Drug Designing (MPD3) was employed, considering its effectiveness and vastness [26]. In order to further understand the mechanistic details, molecular dynamics (MD) simulations supported with MMPB/GBSA binding free energy estimations were carried out on potential compounds to record complexes stability [27,28]. The key residues were also identified through per residue decomposition energy analysis.