Bioactivity of crude extract and purified compounds against bacteria
6.3. Results and discussion
6.3.4. Antibacterial activity of Glabrin 1. Docking study
The current investigation aims at in silico analysis of the interaction between common bacterial proteins and Glabrin at the catalytic sites as predicted by CASTp (Table 6.3).
On performing the molecular docking of Glabrin with all target structures, the calculated binding energy falls within the range of 3-5 kcal/mol with a highest binding affinity of - 5.49 kcal/mol for 1ZOW and lowest binding affinity of -3.8 kcal/mol for 3BLM as shown in Table 6.7.
(C) (D)
(A) (B)
Chapter 6|134 Table 6.7 Binding energy of Glabrin and common targets for the antibacterial property.
Sr. No. Name of protein PDB ID Binding energy
(kcal/mol) S. aureus
1 Dihydropteroate synthase 1AD1 -4.16
2 β-ketoacyl-acyl carrier protein synthase III (KAS III or FabH)
1ZOW -5.49
3 β- lactamase 3BLM -3.8
4 Dihydrofolate reductase 3FRB -4.04
5 DNA gyrase subunit B 3G7B -4.06
E. coli enterotoxic
1 Dihydropteroate synthase 1AJ2 -4.43
2 β-ketoacyl-acyl carrier protein synthase II (KAS II or FabF)
1KAS -4.36
3 β- lactamase 1KE4 -4.32
4 Heat labile enterotoxin (LT) 1LTB -5.3
5 DNA topoisomerase 4 subunit A 1S16 -5.04
5 DNA gyrase subunit B 3G7B -4.43
Glabrin showed highest binding affinity of -5.3 kcal/mol when docked with heat labile enterotoxin (1LTB) of E. coli with five hydrogen bonds between Glabrin and TYR, ARG and THR residues of the protein (Table 6.8). The docked conformation between β-ketoacyl-acyl carrier protein synthase III (1ZOW) and Glabrin has three hydrogen bonds. Amino acid residues involved at the binding site are ALA12, ILE16, TYR11, GLU14, ASP279, and LYS283 which are similar to the isoniazid. All target proteins of S. aureus and E. coli showed the formation of one or more hydrogen bonds between the best active site of the protein (predicted by CASTp) and Glabrin. The least binding energy of -3.8 kcal/mol was observed when β-lactamase (3BLM) was docked with Glabrin along with the formation of three hydrogen bonds between the oxygen atom of Glabrin with residues PHE257, MET254, and TYR196. The intermolecular interactions between receptors and Glabrin are shown in Figure 6.8 and 6.9. Amino acids involved at docking site are given in Table 6.9. From the docked conformations we can deduce that the carboxyl group, hydroxyl group and nitrogen atom in Glabrin is biologically vital for forming hydrogen bond interactions with receptors.
Chapter 6|135 Table 6.8 Docking result of Glabrin and well known potent inhibitors with common bacterial proteins. (Docking study of selected proteins with well-known inhibitors is shown in Table 6.5).
Protein (IDs)
Binding energy (kcal/mol)
Inter molecular Energy (kcal/mol)
Inhibition constant
(µM)
Ligand efficiency (kcal/mol)
H-bonds
Residues involved in H-
Bonding
1AJ2 -4.43 -5.62 567.31 -0.37 3
GLN142:O;
LYS192:HN;
GLN142:O
1KAS -4.36 -5.56 633.14 -0.36 1 THR307:HG1
1KE4 -4.32 -5.51 681.18 -0.36 3
TYR256:HH;
PRO301:O;
PRO301:O
1LTB -5.3 -6.5 129.73 -0.44 5
TYR147:O;
ARG1:HE;
ARG1:HH21;
THR87:OG1;
THR87:HN
1S16 -5.04 -6.23 202.21 -0.42 4
GLU335:HN;
ARG336:HH21;
GLU335:OE1;
ARG336:HN
1AD1 -4.16 5.35 890.9 -0.35 4
ARG51:HH22;
ARG51:HE;
VAL48:O;
VAL48:O
1ZOW -5.49 -6.69 93.88 -0.46 3
ALA12:HN;
LYS283:HZ2;
ILE16:HN
3BLM -3.8 -4.99 1.64
(mM) -0.32 3
PHE257:OXT;
MET254::O;
TYR196:HH
3FRB -4.04 -5.23 1.1 (mM) -0.34 4
LEU24:HN;
LEU20:HN;
HIS23:HD1;
GLN19:HE22
3G7B -4.06 -5.26 1.05
(mM) -0.34 3
THR125:OG1;
LYS124:HZ2;
THR125:OG1
Chapter 6|136 Table 6.9 Amino acids involved at docking site between ligands and protein.
Protein Ligands Nearby amino acid residues
1AJ2 Glabrin GLN142, GLN143, ASN143, PRO145, ALA151, GLY189, PHE190, GLY191, LYS192
Sulfamethoxazole ASN22, GLU60, SER61, THR62, ARG63, ASP96 1KAS Glabrin THR270, SER271, PRO272, HIS303, THR307,
ALA309, PHE400
Isoniazid ASP265, HIS268, MET269, THR270, GLY399 1KE4 Glabrin ALA252, GLN253, SER254, TYR256, PRO301
Clavulanic acid LEU251, SER254, PRO303, ALA304 1LTB
Glabrin ARG1, ALA86, THR87, TYR 122, TYR142, TYR147, ARG148
4-Nitrophenyl α-D-
galactopyranoside GLN30, GLU79, THR80, ARG145 1S16 Glabrin GLN275, THR333, GLU335, ARG336
Norfloxacin ARG31, HIS34, GLN37, MET274, THR333, GLU335 1AD1
Glabrin ASN10, VAL48, ARG51, ILE57, ASP83, ASN102, GLN104, ARG238
Sulfamethoxazole ILE8, ILE9, LEU10, ASN11, ASP83, ARG51, ASP83, GLN104
1ZOW Glabrin ALA12, ILE16, GLU14, ASP279, LYS283
Isoniazid ALA12, ILE16, ASP18, TYR266, LYS265, LYS283, 3BLM Glabrin PRO193, TYR196, LYS 251, MET254, LYS255,
GLU256, PHE257
Clavulanic acid LYS240, ASN242, LYS234
Chapter 6|137 Figure 6.8 Predicted docked conformation of Glabrin against common S. aureus proteins.
(A) Dihydropteroate synthase; (B) β-ketoacyl-acyl carrier protein synthase III or KAS III or FabH; (C) β- lactamase; (D) Dihydrofolate reductase and (E) DNA gyrase subunit B.
Figure 6.9 Predicted docked conformation of Glabrin against common E. coli proteins.
(A) Dihydropteroate synthase; (B) β-ketoacyl-acyl carrier protein synthase II (KAS II or FabF);
(C) β- lactamase; (D) Heat-labile enterotoxin (LT) and (E) DNA topoisomerase 4 subunit A.
(A) (B)
(D)
(C)
(E)
(A) (B)
(D)
(C)
(E)
Chapter 6|138 6.3.4.2. Cell leakage
The cytoplasmic membrane of bacteria acts as a barrier between cytoplasm and extracellular medium by maintaining the chemiostatic balance which is necessary for the survival and metabolism of the cell. The leakage of low molecular weight cytoplasmic constituents often leads to the leakage of larger cellular contents either due to further damage of the membrane or gradual breakdown of proteins and nucleic acids by autolytic enzymes of the cell (Johnston et al., 2003). It was observed that the amount of low molecular weight metabolites increased with increasing time of exposure due to the continuous release of cellular materials through the compromised cell membrane of treated bacterial strains as compared to controls (Figure 6.10). The leakage of cellular material could be an indication of a disturbance and disorganization of the cytoplasmic membrane integrity when the concentration of antimicrobials was bacteriostatic. At high antimicrobial dose, it would cause lethality to microbial cells. The results shows that Glabrin effectively induces the cell damage in both Gram-positive as well as Gram- negative bacteria, although the effect is more pronounced against SA. Finally, cell lysis occurs due to pores formation or disruption of cell membrane causing loss of metabolic activity and ultimately cellular leakage resulting to the cell death. Similar observations of cell membrane damage and subsequent leakage caused due to bioactive compounds was reported earlier (Cushnie and Lamb, 2005).
Chapter 6|139 Figure 6.10 Cell leakage analysis.
The absorbance of the cell materials at 260 and 280 nm released after treatment with Glabrin at 0, 4, 8 and 16 h incubation period, where SA- A, B, and ETEC- C, D. The data are expressed as mean ± standard error.
6.3.4.3. FESEM study
The bacteria ETEC and SA were examined by FESEM to observe morphological changes caused by treatment with Glabrin (Figure 6.11). FESEM images of untreated ETEC and SA exhibited intact, well-defined cellular contents and were normal in size (Figure 6.11 A and C) whereas Glabrin is more likely to have direct contact with the cell membrane through damaged cell wall after treatment of compound (Figure 6.11 B and D). These findings indicate that Glabrin cause lysis of SA and ETEC by weakening peptidoglycan layers thereby rupturing cell wall and leading to pores formation. These
(A) (B)
(C) (D)
Chapter 6|140 damaging effects inturn trigger the leakage of ions and cellular materials ultimately causing the cell death (Cowen, 1999).
Figure 6.11 Field emission scanning electron micrographs of S. aureus (A and B) and E. coli enterotoxic (C and D). A and C untreated bacterial cells; B and D cells treated with Glabrin.