This study (as shown in Table 1 and Figure 1), identifies the involvement of E. coli, C. jejuni, Streptococcus lutetiensis 033, Streptococcus infantarius, Campylobacter upsaliensis, Enterococcus ratti, Helicobacter sp. 'feline isolate, Helicobacter canadensis, Anaerobiospirillum sp. B0101, Anaerobiospirillum sp. 3J102 as well as uncultured bacterium (Prevotella) and unidentified bacterium (Lactobacillus). Ribosome is the site of protein synthesis in the cell, which based on the genetic information in deoxyribonucleic acids (DNA) that transcribes to ribonucleic acids (RNA). This study identifies difference in the secondary (2D) structure of consensus 16S rRNA produced by RNAalifold from ClustalO server and Aligner server multiple sequence alignments (MSA) (Figure 2 and 3). The optimal secondary structure (SS) from ClustalO MSA has a minimum free energy (MFE) of -764.31 (-383.21 plus -381.10 from covariance contributions) kcal/mol, free energy of the thermodynamic ensemble is -785.70 kcal/mol and frequency of the MFE structure in the ensemble is 0.00%, while that Aligner MSA has a MFE of -592.43 (-299.94 plus -292.49 from covariance contributions) kcal/mol, free energy of the thermodynamic ensemble is -607.10 kcal/mol and frequency of the MFE structure in the ensemble is 0.00%. These differences showed the unreliability of the secondary structure of RNA in determination of function.
Seventeen (17) antibiotic compounds that have mechanism which targeted 16S rRNA was used in this study to determine their possible binding to two key enzymes that involve in 16S rRNA and tRNA activities for protein synthesis in E. coli. The results of molecular docking of 17 antibiotics to pseudouridine synthase RsuA (1KSV) and methyltransferase KsgA (3TPZ) in Table 2 and Figure 4, indicate that the -cycline compounds possibly have strong affinities than the -mycin compounds. The free energies from molecular docking with two enzymes used in this study, reveal possible efficacy in the following order of: doxycycline > metacycline > streptomycin > rolitetracycline > tetracycline > tigecycline. Out of 17 antibiotics in this study, chlortetracycline and minocycline have high affinity for methyltransferase KsgA, kanamycin has almost equal affinity for both enzymes while the other 14 have high affinity for pseudouridine synthase RsuA.
The results of docking simulation showed that the binding of the best 3D structure model of 16S rRNA to 1KSV and 3TPZ with free energy of -367.52 kcal/mol and-371.55 kcal/mol respectively while the ligand (protein structure) root mean square deviation (RMSD) of 262.35Å and 212.23Å for 1KSV and 3TPZ respectively (Figure 5). The active site nucleotide residues of 16S rRNA for pseudouridine synthase RsuA (1KSV) are A58, A59, C70, U71, A72, A73, A74, G75, G77, G85, U86, G87, G88, G391, C392, C393, A567, G570, A571, G572, U756, C757, G758, A759, A760, G762, C763, G980, G981, U982, C1037, A1038, G1039, U1065, G1086, A1137, C1138, U1139, C1140; and active site nucleotide residues of 16S rRNA for methyltransferase KsgA (3TPZ) are C104, G105, A114, C115, C116, U173, U174, C190, U191, U192, C201, A202, A203, U217, A218, G223, A224, C225, C226, U227, C272, G273, A274, C819, U820, A821, G854, C868, A869, C886, G887, A888, C890, G891. Moreover, the active site nucleotide residues that may be directly interact with the active site amino acid residues of 1KSV are A59, G77, G981, U982, C1037, A1038, G1039, U1065, C1138, U1139, C1140, while that of 3TPZ are A114, C819, U820, A821, G854, G887, and A888.
The results of ligand-protein dynamics simulation of methyltransferase KsgA (3TPZ) interaction with doxycycline and methacycline (Figure 6) showed that the interaction was stable over 1 ns simulation timestep. The RMSD of atomic positions did not significantly varied with radius of gyration (Rg) in a range of 19.9 to 20.4 Å for doxycycline and 20.2 to 20.8 Å for methacycline, over the simulation time. Difference was observed in the principal component analysis (PCA) for both ligand interaction with 3TPZ, which showed conformational variation in protein structure by amino acid residues due to ligand binding. Also, the difference was evident in the molecular mechanics Poisson–Boltzmann or Generalized Born surface area (MM-PB/GBSA) of the binding. SWAXS curves that were computed from PDB structures 1KSV and 3TPZ, have Guinier fit Rg of 22.8464 Å and 20.7851 Å respectively (Figure 7). Guinier fit Rg of 3TPZ was the same as an average Rg obtained by ligand-protein MD simulation.
Table 1
Cluster of diarrhoea microbes 16 rRNA sequences with pintail quality obtained by search on SILVA database.
SN | Accession number | Organism |
1 | KX162658.1.1443; KX162656.1.1450; KY750229.1.1207; Z83205.1.1525 | Escherichia coli |
2 | CP003025.16528.18064; CP003025.212088.213624; CP003025.335290.336826; CP003025.279149.280684; CP003025.271649.273185; CP003025.449268.450804 | Streptococcus lutetiensis 033 |
3 | CP013689.1.1531; CP013689.5810.7362; CP013689.225779.227331; CP013689.68577.70129; CP013689.284156.285708; CP013689.61083.62635; CP013689.398494.400046; CP013689.1808358.1809910; CP013689.1814190.1815742; | Streptococcus infantarius |
4 | CP007751.37529.39032; CP007751.392464.393967; CP007751.693858.695361 | Campylobacter jejuni subsp. jejuni D42a |
5 | CP007749.37394.38897; CP007749.392073.393576; CP007749.684989.686492 | Campylobacter jejuni subsp. jejuni M129 |
6 | AF497805.1.1428 | Campylobacter upsaliensis |
7 | AF326472.1.1523; AF539705.1.1503 | Enterococcus ratti |
8 | AF142062.1.1430 | Helicobacter sp. 'feline isolate |
9 | AF262037.1.1457 | Helicobacter canadensis |
10 | AF497808.1.1475 | Anaerobiospirillum sp. B0101 |
11 | AF497809.1.1453 | Anaerobiospirillum sp. 3J102 |
12 | JQ627624.1.1441 | Prevotella; uncultured bacterium |
13 | DI173490.1.1556 | Lactobacillus; unidentified |
Table 2
Binding energy score of the interaction between 16S rRNA-targeted antibiotics and two 16S rRNA enzymes, obtained from AutoDock Vina.
SN | Anti- 16S rRNA Compounds (Ligand) | DrugBank ID | Binding Free Energy (kcal/mol) |
16S rRNA pseudouridine synthase (PDB ID: 1KSV) | 16S rRNA methyltransferase KsgA (PDB ID: 3TPZ |
1 | Apramycin | DB04626 | -11.3 | -9.5 |
2 | Chlortetracycline | DB09093 | -9.9 | -11.4 |
3 | Doxycycline | DB00254 | -12.7 | -12.5 |
4 | Gentamicin | DB00798 | -9.9 | -7.5 |
5 | Kanamycin | DB01172 | -9.4 | -9.3 |
6 | Methacycline | DB00931 | -12.7 | -12.3 |
7 | Minocycline | DB01017 | -9.3 | -11.2 |
8 | Neomycin | DB00994 | -10.2 | -9.2 |
9 | Netilmicin | DB00955 | -9.7 | -8.4 |
10 | Omadacycline | DB12455 | -11.1 | -9.9 |
11 | Oxytetracycline | DB00595 | -11.7 | -11.2 |
12 | Paromomycin | DB01421 | -9.1 | -7.0 |
13 | Rolitetracycline | DB01301 | -12.3 | -10.5 |
14 | Streptomycin | DB01082 | -12.6 | -11.6 |
15 | Tetracycline | DB00759 | -12.2 | -11.3 |
16 | Tigecycline | DB00560 | -11.9 | -11.0 |
17 | Tobramycin | DB00684 | -9.0 | -7.3 |