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Virtual Screening to Identify the Protein Network Interactions of Triclosan with Streptococcus mutans and Enterococcus faecalis

Introduction: Triclosan is considered to be an important ingredient in toothpastes and mouth rinses. Several studies have reported contradictory results regarding the antimicrobial effect of triclosan. Hence, the present in silico study intends to identify the potential targets of triclosan in two common dental pathogens Streptococcus mutans and Enterococcus faecalis. Aim: To identify the protein network interactions of triclosan in Streptococcus mutans and Enterococcus faecalis by virtual screening method. Materials and Methods: The STITCH v5.0 database was initially used for identifying drug-protein interactions followed by VICMPred and VirulentPred which was employed to identify functional class of the proteins and its virulence property. Finally, BepiPred v1.0 Linear Epitope Prediction tool was used to identify the potential epitopes of the virulent proteins. Results: Triclosan was found to interact with crucial proteins in S. mutans and E. faecalis which could contribute to severe forms of periodontitis and endodontic diseases. Original Research Article Hariprasanth et al.; JPRI, 33(47B): 677-683, 2021; Article no.JPRI.74465 678 Conclusion: Taken together, the present study provides the preliminary data on the potential targets of triclosan in common dental pathogens. Further experimental validation is warranted to provide concrete evidence on the molecular targets of dental pathogens.


INTRODUCTION
Triclosan (TCS), an antimicrobial agent, is present in products such as toothpaste, soaps, detergents, toys, and surgical cleansing solutions [1]. It is a common ingredient in soaps, shampoos, deodorants, toothpastes, mouthwashes, cleaning supplies, and pesticides, and part of consumer products like kitchen utensils, toys, bedding, socks, and trash bags etc [2]. Antimicrobial ingredients have long been used in various products to slow down or inhibit the growth of bacteria, fungi [3]. In hospital settings, 2% triclosan has been used in surgical units for the decolonization of a patient's skin carrying methicillin-resistant Staphylococcus aureus (MRSA) [4,5]. A study using commercially available toothpaste containing triclosan indicated a significant reduction in gingivitis, bleeding, and plaque[6] other systematic analysis by Cochrane group suggested that reduction in gingivitis, bleeding, and plaque may be statistically significant under in vitro conditions, but the same could not be replicated in a clinical setting, in other words, triclosan might not attain clinical significance.Our team has extensive knowledge and research experience that has translate into high quality publications[7-11].
Such contradictory results have posed queries about the use of triclosan as an effective antimicrobial agent. Hence, the present study was intended to identify the potential molecular targets of triclosan in common dental pathogens such as S.mutans and E.faecalis. Computational techniques are widely employed to screen for drug molecules and their targets in microbes and host. Several studies have been initiated by the authors previously to deduce the potential targets of synthetic [12] and phytocompounds [13], [14] against red complexes and other common dental pathogens. Virtual screening methods have been used to reduce cost and time. The prediction tools would provide preliminary information about the molecular targets of the drug in the microbial pathogens, which will be of great use to the researchers to identify drug molecules that would best suit their needs and be less toxic to the hosts.

MATERIALS AND METHODS
The present observational study aims to screen for those proteins of Streptococcus mutans and Enterococcus faecalis interacting with Triclosan. The protein-drug interaction of bacteria was analyzed using STITCH v.5 pipelines [15] and the functional class and virulence property of the interacting proteins was detected using VICMPred and VirulentPred softwares.

Prediction of Drug-Protein Interactions
The STITCH database helps in providing an exhaustive platform for known and predicted interactions between chemicals and proteins. The interactions could be direct or physical and indirect or functional associations.

Identification of Virulent Protein and Functional Class
VICMpred [16] and VirulentPred [17] software were used for the identification of virulence factors targeted by Triclosan among the Streptococcus mutans and Enterococcus faecalis. These tools employed a support vector machine (SVM)-based five-fold cross-validation 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 Epitopes
The BepiPred v1.0 Linear Epitope Prediction tool predicts B-cell epitopes from a protein sequence, using a Random Forest regression algorithm with trained five fold cross-validation approach was used to discriminate between epitopes and nonepitope amino acid residues from crystal structures. The residues with scores above the threshold (>0.5) are predicted to be part of an epitope and colored in yellow on the graph [18,19].

RESULTS AND DISCUSSION
Stitch software was used to classify protein interactions between Streptococcus mutans and Enterococcus faecalis against triclosan (Fig. 1). Furthermore, each of the proteins interacting with the drug was tested for their virulence properties using VirulentPred and functional properties via VICMpred. The scores provided by these algorithms were verified based on their amino acid sequences and patterns which were divided into two groups. i.e., virulent and avirulent. Triclosan was found to interact with crucial proteins in S. mutans and E. faecalis. Triclosan interacts with more than ten important proteins in both the pathogen, as shown by the virulence properties of 8 virulent proteins in S. mutans and 5 virulent proteins in E. faecalis in the VirulentPred findings. Most of the virulent proteins identified were transcriptional regulators in S. mutans and proteins involved in cellular processes in case of E. faecalis (Table 1; Fig 1). Several epitopes were identified in the virulent protein, conserved hypothetical protein PksD of Streptococcus mutans (Figs 2 and Chart 1).
Target identification is an important component for the development of therapeutic and diagnostic markers for metabolic, infectious [20][21][22][23][24][25], autoimmune [26,27,28] and systemic disorders. Computational tools have long been used for the purpose of rapid identification of these potential targets from several sources [29][30]. Dental caries being a serious illness in children has to be identified at the right time and treated using drugs that do not give rise to resistant forms [31][32][33][34]. Hence, proper identification of the potential targets has become the need of the hour. We investigated the molecular targets of triclosan and their associations with pathogens in this research, which shows that there are interactions with microbes and virulence factors associated with its functions. Triclosan has been integrated with a number of other dental materials to improve inhibitory effects on microbial metabolism in plaque, calculus, and gingivitis accumulation. As triclosan is used at low concentrations, it inhibits microorganism development; however, when used at higher concentrations, it can disrupt the growth of microorganisms. While the in silico methods used provide preliminary evidence on the underlying molecular interaction between the compound and protein network of dental pathogens such as S. mutans and E. faecalis. The analysis has some limitations, such as (a) the bonding between the compound and the pathogen's protein may be purely physical and (b) the proteins of the red-complex bacteria attacked by the compound may resemble host proteins. To prevent undesirable associations of triclosan with host proteins, in vitro and in vivo studies must be performed to obtain clarification on the healthy use of chemical-compounds on human hosts.

CONCLUSION
This study identified molecular targets of triclosan on S. mutans and E. faecalis which have to be further validated to confirm the critical pathway triggered by the drugs in the physiological conditions. To the best of our knowledge, this study is the first of its kind which aims in understanding the molecular targets of the pathogens against specific drug compounds. The dosage of the drug, minimum inhibitory concentration, and minimum bactericidal concentration against specific microbes should be ascertained by in vitro and in vivo studies.

FUNDING
We thank Saveetha Dental College and Hospitals, Saveetha Institute of Medical and Technical Sciences (SIMATS), Saveetha University, Chennai for funding the project.

CONSENT
Not applicable.

ETHICAL APPROVAL
As per international standard or university standard ethical approval has been collected and preserved by the authors.