In Silico Destruction of Porphyromonas gingivalis Fimbriae by Streptomyces sp. Strain GMY02

. Porphyromonas gingivalis , a keystone pathogen in chronic periodontitis, has fimbria as one of the most important virulence factors because it plays a vital role in the pathogenesis of P. gingivalis infection. This bacterium has 2 fimbriae: the major (FimA) and minor (Mfa1) fimbriae. Fimbriae are attractive targets for anti-infective therapy for periodontal disease. The aim of this study was to analyze the interactions of several compounds thought to be present in Streptomyces sp. GMY02 strain against FimA and Mfa1 proteins in P. gingivalis fimbriae in silico. A total of 8 ligands were docked to FimA and Mfa1 fimbriae using AutoDock Vina in University of California, San Francisco (UCSF) Chimera 1.16. All of the selected ligands had higher free energy values than metronidazole as well. In conclusion, the compounds suspected to be present in Streptomyces sp . strain GMY02 has the potential to destruct P. gingivalis fimbriae.


Introduction
Porphyromonas gingivalis, a keystone pathogen in chronic periodontitis, has virulence factors including capsule, fimbriae, lipopolysaccharide (LPS), protease (gingipain), and outer membrane protein [1][2][3].Although P. gingivalis expresses several potential virulence factors, the fimbriae are of particular importance because they play a vital role in host cell attachment and invasion, colonization of P. gingivalis with other bacteria and host tissues, biofilm formation, bacterial motility, and protein and DNA transport across the cell membrane [4].Porphyromonas gingivalis has 2 fimbriae, namely the major (FimA) and minor (Mfa1) fimbriae, which consist of the protein polymers FimA and Mfa1, and are encoded by the fimA and mfa1 genes, respectively [5].According to its DNA sequence, the fimA genotype of P. gingivalis can be divided into six categories (I, Ib, Ⅱ, III, IV, V).Type I fimA exhibiting a solid correlation with plaque formation.The major role of type I fimbriae (FimA) of P. gingivalis is to mediate biofilm formation, adherence to saliva-coated surfaces, and adherence to gingival epithelial cells, it can also trigger an inflammatory response.FimA genotype I has also been observed at high frequency in patients with severe * Corresponding author: henisusilowati@ugm.ac.id periodontitis.Given the important role of the fimbria in the pathogenesis of P. gingivalis, the fimbria is an attractive target for anti-infective therapy to prevent or treat periodontal disease [6,7].
Several new approaches to treat bacterial infections are by inhibiting biofilms without inducing microbial dysbiosis from the oral cavity.These new approaches include the use of nanomaterials, quaternary ammonium salts, small molecules, arginine, and natural materials [8].One of the natural materials currently being developed as an anti-biofilm is Streptomyces sp.strain GMY02, is a potential bacterium isolated from marine sediment samples from Krakal Beach (8°8′44″S, 110°35′59″E), Yogyakarta, Indonesia.Identification, annotation and analysis of gene clusters involved in secondary metabolite biosynthesis in Streptomyces sp. the GMY02 strain was carried out with antiSMASH 6.0 [9].
Molecular docking is an approach used extensively in modern drug designing and development.This method is mainly used in drug design to explores the conformations of ligands within the macromolecular target binding site, providing an estimation of receptor-ligand binding free energy for all different conformations.Molecular docking is an established in silico structure-based method widely used in drug discovery.Nowadays, in silico methodologies have become a crucial part of the drug discovery process.In silico is a research method that utilizes computing and database technology to develop further research.The use of the in silico method in drug development can save costs and time because it can predict drug structure through mathematical equations, visualization in three dimensions, and can evaluate interactions between compounds and targets before synthesizing these structures into drugs [10][11][12].Based on this background, this study aims to analyze the interactions of several compounds thought to be present in Streptomyces sp.GMY02 strain against FimA and Mfa1 proteins in P. gingivalis fimbriae in silico.

Ligand Preparation
The ligand chosen in this study was metronidazole as a positive control and compounds suspected to be present in Streptomyces sp.strain GMY02.The candidate compounds produced by Streptomyces sp.GMY02 were predicted by using genome mining tool, AntiSMASH version 7 (available online in https://antismash.secondarymetabolites.org)(antiSMASH bacterial version) with NCBI accession number of whole genome sequence CP077658.PubChem as SMILES files converted to PDB format using NovoPro (https://www.novoprolabs.com/tools/smiles2pdb), then converted to mol2 format with UCSF Chimera software (version 1.16).The ligands tested and their structures can be seen in Table 1.
Table 1.List of compounds to be docked with FimA and Mfa1 fimbriae and their structures

Protein Preparation
Crystal structure of FimA protein in P. gingivalis fimbriae (PDB ID: 6JZK) and Mfa1 (PDB ID: 5NF2), taken from the Protein Data Bank (PDB) (http://www.rcsb.org/pdb)as PDB files.The crystal structure of the FimA protein has a resolution of 2.10 Å, while that of the Mfa1 protein has a resolution of 1.73 Å.The crystal structure of the FimA and Mfa1 proteins can be seen in Figure 1.Protein preparation was carried out by removing all unique ligands, all atoms, water molecules, and ions to obtain native receptor proteins, then polar hydrogen and charges were added, The protein preparation procedure was carried out using Chimera (version 1.16).

Molecular Docking
Protein and ligand preparation, energy minimization, and molecular docking were performed using UCSF Chimera (version 1.16) and AutoDock (version 1.5.7)software.
The molecular docking simulation method was validated using the root-square deviation (RMSD) calculations by redocking the native ligand.The best conformation of the docked native ligand was retrieved and superimposed with the native protein prior to docking, and RMSD was calculated.An acceptable RMSD value should be less than 2.0 Å [13].Docking was performed with a grid box that includes all receptor structures with dimensions of 50×50×50 Å and centered at 1.287, -3.755, and 92.921 respectively the X, Y, and Z coordinates for the FimA protein, and dimensions of 50x50x70 Å and centered on 29.787, 3.436, and 84.082 respectively the X, Y, and Z coordinates for the Mfa1 protein, and the Genetic Algorithm (GA) Run number of 50.

Data Visualization
Visualization of the interaction results of docking of the best ligand conformation which has the highest binding affinity of the compounds suspected to be present in Streptomyces sp.strain GMY02 with protein, using LigPlot plus.In addition, with LigPlot Plus it can also be seen the amino acid residues around the interactions that occur, the hydrogen bonds, and the hydrophobicity.
3 Results and Discussion

Docking Analysis
The results of the docking of compounds suspected to be present in Streptomyces sp.strain GMY02 with FimA protein in P. gingivalis fimbriae is shown in Table 2. Table 2 shows all the compounds tested have a higher binding energy compared to SRT whose value was -2.66 kcal/mol and metronidazole (positive control) which has a binding energy of -3.66 kcal/mol.The highest binding energy of the compounds suspected to be found in Streptomyces sp. the GMY02 strains with FimA protein are reductasporine (-10.04 kcal/mol), hopene (-6.27 kcal/mol), and sangivamycin (-5.46 kcal/mol) and RMSD is 1,99.The results of the docking of compounds suspected to be present in Streptomyces sp.strain GMY02 with Mfa1 protein in the fimbriae of P.gingivalis is shown in Table 3. Table 3 shows all the compounds tested, except sangivamycin and ectoine, which have a higher binding energy compared to ACT which is -2.84 kcal/mol and metronidazole (positive control) which has a binding energy of -3.68 kcal/mol.The highest binding energy of the compounds suspected to be found in Streptomyces sp. the GMY02 strain with Mfa1 protein are reductasporine (-9.14 kcal/mol), hopene (-6.76 kcal/mol), and alkylresorcinol: propylresorcinol (-5.86 kcal/mol).RMSD is 0,15.
Two compounds that have the highest binding energy with the FimA and Mfa1 proteins (reductasporine and hopene) were further analyzed to determine their interactions with the active sites of the FimA and Mfa1 target proteins.Figure 2 illustrates the interaction display of the best ligand conformation binding results of reductasporine and hopene with FimA and Mfa1 proteins using LigPlot plus software.Fig. 2. Interaction of metronidazole and the best ligand conformation binding results from reductasporine and hopene with FimA and Mfa1 proteins using LigPlot plus software Reductasporine contains a novel tryptophan dimer (TD) core structure.Reductasporine contains an indolocarbazole pyrrolinium core structure which may be a key new bioactivity profile.This new core structure gives reductasporin a bioactivity profile that is different from other TD core structures.TD has biological activity as an antibacterial and antifungal.Several tryptophan derived from marine alkaloids show strong and promising antimicrobial activity, whether against bacteria, fungi, or viruses [14,15].
Hopene is an important precursor for synthesizing bioactive hopanoids.Hopanoids are a group of pentacyclic triterpenoids consisting of a hopene skeleton and side chain sequences.Hopanoids which are natural products play an important role in stabilizing the structure of bacterial membranes such as the effect of sterols on eukaryotes.Hopanoids interact with glycolipids on the bacterial outer membrane to form a highly ordered bilayer in a manner similar to the interaction of sterols with sphingolipids on eukaryotic plasma membranes [16,17].
Reductasporine and hopene are 2 compounds that are thought to be present in Streptomyces sp.GMY02 strain had the highest binding energy with P. gingivalis fimbriae, even higher than metronidazole.The binding energy generated by the ligand when it binds to the target protein site can trigger a specific biological response, the more negative the binding score, the higher the effect on the activity of the target protein, it can be indicate that the compounds suspected to be present in Streptomyces sp.strain GMY02 have the potential to destruct P. gingivalis fimbriae.
This work suggests the application of the mentioned ligands or compounds to control P. gingivalis biofilm found in the oral cavity.By approving their potency in silico, it is necessary to carry out in vitro experiment to BIO Web of Conferences 75, 02004 (2023) https://doi.org/10.1051/bioconf/20237502004BioMIC 2023 confirm the computational predictions that have been made.

Conclusion
Based on the data obtained from this study, it can be concluded that Streptomyces sp.strain GMY02 have the potential to destruct the P. gingivalis fimbriae.

Table 2 .
List of docking results for compounds suspected to be present in Streptomyces sp.strain GMY02 with FimA fimbria

Table 3 .
List of docking results for compounds suspected to be present in Streptomyces sp.strain GMY02 with Mfa1 fimbriae