Virtual Screening to Identify the Protein Network Interaction of Berberine with Red Complex Pathogens

Introduction: Periodontal disease is an infection of the tissues that hold your teeth in place. It's typically caused by poor brushing and flossing habits that allow plaque, a sticky film of bacteria, to build up on the teeth and harden. Elimination of these pathogens from the site of infection remains a perplexing task, which demands the use of antibiotics. The emergence of drug resistant forms has spurred interest into identifying novel therapeutic targets against these pathogens. Aim: The present study employs virtual screening method to identify the protein network interaction of berberine with red complex pathogens. Materials and Methods: Computational tools were used to identify the targets, assess their functional role and virulence property. Further, the peptide epitopes present in the virulence factors were identified using the BepiPred tool. The subcellular location of the virulence proteins was also elucidated using PSORTb. Results: Berberine was found to target vital protein transporters such as TetR family transcriptional Original Research Article Abinaya et al.; JPRI, 33(47B): 645-652, 2021; Article no.JPRI.74460 646 regulator and MerR family transcriptional regulator, which is known to play a crucial role in the survival of bacterial cells. Conclusion: Hence the present study provides preliminary data on the protein targets of berberine against red complex pathogens. However, in vitro studies using the compound is warranted to further confirm the efficacy of the compound.


INTRODUCTION
Periodontal diseases are complex infections exerted by a multiplex of bacterial species that interchange with host tissues and cells, causing the release of an extensive array of inflammatory cytokines, chemokines and mediators, some of which lead to the destruction of the periodontal structures, including the tooth-supporting tissues, alveolar bone, and periodontal ligament [1]. Bacteria are analogous with periodontal disease that trigger inflammatory responses in the immune cells, which in later stages of the disease causes loss of both soft and hard tissue structures supporting the teeth [2]. Until now, only a handful of bacteria have been characterized as infectious agents of periodontal disease. Periodontitis is a dysbiotic disease resulting from deviation in subgingival grampositive bacteria to gram-negative bacteria. The development of periodontal dysbiosis occurs over a broadened timeframe, which slowly revolves the mutual association of the host and the microbe to pathogenic form. Among microbial complexes, the first complex is a red-complex consisting of Tannerella forsythia, Treponema denticola, and Porphyromonas gingivalis [3]. The red complex, which appears later during biofilm development, comprises species that are considered periodontal pathogens.
Porphyromonas gingivalis has long been considered to be an important member of periodontopathic microbiota interacting in periodontal disease progression with bone and tissue destruction [2,4]. When grown on a blood agar surface, they produce black pigmented colones. Treponema denticola are long, thin, corkscrew-like gram-negative anaerobic bacteria whose characteristic motility and morphology can readily be identified by darkfield and phase contrast microscopic examination [5]. The spiralshaped cells of T. denticola are covered with an outer sheath consisting of a fragile envelope-like structure [6]. Tannerella forsythia are anaerobic gram-negative members of the Cytophaga bacteroides family and were initially named Bacteroides forsythus [7]. T. forsythia is associated, more frequently and at higher levels, with various forms of periodontal disease, including gingivitis, chronic, and aggressive periodontitis, than with healthy individuals [8].
The emergence of drug resistant microbes has become a common observation in recent years in dental settings also. The in silico approach has helped researchers to drastically cut down their time and resources in screening bioactive compounds with the ability to destroy a broad spectrum of microbial pathogens [9]. Berberine is a chemical present in plants European barberry, goldenseal, and Oregon grapes. It is most commonly taken for diabetes, high levels of cholesterol or other fats in hyperlipidemia, and high blood pressure [10]. Our team has extensive knowledge and research experience that has translate into high quality publications [3][4][5][6][7] The aim of this study is to analyze to identify the protein network interaction of berberine in red complex pathogens employing the virtual screening methods.

Study Design
The present study follows an observational study design that aims to virtually screen and identify the protein network interaction of berberine in red complex pathogens. The interaction of drugs with bacteria was analyzed using STITCH five pipeline and the virulence properties of the interacting proteins were deduced by VICMPred and VirulentPred softwares. The strains are included in the STITCH database, and the query was user defined [11].

Prediction of Protein-drug Interactions
The STITCH database provides an exhaustive platform for known and predicted interactions between drugs and proteins. The interactions include physical and functional associations which stem from computational prediction and interactions aggregated from primary databases [11].

Virulence Prediction
VICMpred [12] and VirulentPred [13] pipelines were used for the identification of virulence factors targeted by berberine among the red complex pathogens. These tools employed a support vector machine based five fold crossvalidation process to validate results. Virulence factors were screened based on amino acid composition using the VirulentPred tool, which classified them into two groups, that is virulent and avirulent. VICMpred groups proteins into four major classes, namely, proteins involved in cellular processes, metabolism, information storage, and virulence.

Prediction of Subcellular Localization of the Virulent Proteins
Computational prediction of subcellular localization of proteins aids in designing novel drug targets or for substantiating the role of an antimicrobial drug which targets the virulent protein. An algorithm which assigns a probable localization site to a protein from an amino acid sequence is pSORTb V3.0 [14].

Prediction of Epitopes
For the prediction of B-cell epitopes from a protein sequence, the server BepiPred-2.0 was used. To be part of an epitope, the residues with scores above the threshold (>0.5) [8,9,15].

RESULTS AND DISCUSSION
Our team has extensive knowledge and research experience that has translated into high quality publications [10,[16][17][18][19][20][21]11]. Red complex pathogens, which include P. gingivalis, T. denticola, and T. forsythia, are vital contributors of periodontal infection. Berberine was found to interact with a plethora of crucial proteins in P. gingivalis, T. denticola and T. forsythia. They also possess strong mechanisms of defence, including efflux pumps, drug resistant genes, etc., to combat the selective pressure created by antibiotics. Hence, non-antibiotic drugs can be the drug of choice for multiple ailments associated with microbial infections.
The present study documents several types of metabolism molecules such as hypothetical protein, TetR family transcriptional regulator and MerR family transcriptional regulator are domain protein of T. denticola and membrane protein of T. forsythia targeted by berberine (Table 1; Fig.  1). In addition to it, many peptide epitopes were also identified in the virulence proteins, which can justify the use of berberine as an antimicrobial agent (Fig. 2). Most of the interacting proteins were found in the cytoplasm ( Table 2). Several synthetic, semisynthetic and phytocompounds have been virtually screened for the presence of protein targets which can be further probed into to ascertain the mode of action of these compounds [17][18][19][20]12]. In line with these facts, berberine was chosen for the study to identify its potential targets in red complex pathogens. Our research team has embarked on identifying the potential molecular targets of different plant compounds against dental pathogens with a special emphasis on periodontal pathogens and emerging drug resistant species [22][23][24][25][26][27][28][29][30][31][32][33][34].
Berberine hydrochloride (BH) has been checked for the antimicrobial activity against Candida albicans. Huang et al., recently report the antifungal effect of BH against C. albicans. The BH was found to alter the HOG-MAPK pathway with the simultaneous upregulation of core genes such as SLN1, SSK2, HOG1 and PBS2 [35]. A previous report by the same research group documented that BH could inhibit the formation of biofilm by destroying the cells and the adjacent structures. The molecular mechanisms involved may be the down regulation of expression of EFG1, HWP1, ECE1 and ALS1 which are involved the formation of hyphae and morphological transformation of C. albicans [36,[37][38].

CONCLUSION
Berberine, with all its potential targets addressed, can be used as an antimicrobial agent to exterminate dental pathogens which are unmanageable to treatment. The mode of action of berberine as an MerR family transcriptional regulator and TerR family transcriptional regulator will produce it a perfect drug for therapeutic uses and applications.

DISCLAIMER
The products used for this research are commonly and predominantly use products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company; rather it was funded by personal efforts of the authors.

CONSENT
It is not applicable.

ETHICAL APPROVAL
We conducted our research after obtaining proper IEC approval.

FUNDING
The present study was supported by the Saveetha Dental College, Saveetha Institute of Medical and Technical Sciences, Saveetha University & Sri Sambanda Agency Pvt. Ltd.