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[144]
CHAPTER 5 Sequencing, Comparative Modelling and Docking
Analysis of Drug Target Genes
5.1 Introduction:
rug treatment is fundamental for managing and controlling TB, promoting
the cure of patients and breaking the chain of transmission. According to
World Health Organization, resistance to at least two drugs, namely,
Isoniazid (INH) and Rifampicin (RIF), causes multi-drug resistant tuberculosis
(MDR-TB). The drug resistance in M. tuberculosis is mostly caused by mutations in
specific regions in drug target genes and causes reduction in the sensitivity of M.
tuberculosis to anti-tuberculosis chemotherapy. The treatment of tuberculosis
involves DOTS, a multi-drug therapy approach.
Initially, when mono therapy with p-aminosalicylic acid or streptomycin was started
in 1940s, high treatment failure rates were observed. But, later on, this was controlled
by combination of two or more drugs (Pyle, 1947). Molecular genetic studies during
1970s showed that resistance to anti-TB drugs occurred due to naturally occurring
mutations in the genome of M. tuberculosis (David, 1970). Subsequent studies
demonstrated that within a population of M. tuberculosis there are mutants that arise
due to spontaneous point or deletion mutations (Zhang & Telenti, 2000). Mutations in
genes encoding drug targets or drug activating enzymes are responsible for resistance,
and such mutations have been found for all first-line drugs and some second line
drugs (Ramaswamy & Musser, 1998).
Mutants drug resistant to a particular drug occur approximately one in every 107 to
1010 cells (Parsons et al., 2004; Rattan et al., 1998). Therefore, mono drug therapy
results into rapid selection of drug resistant mutants, which dominate the lesions in
patients. Thus, combining two or more anti-tuberculosis drugs effectively reduces the
D
Chapter-5 Sequencing Comparative Modelling and Docking Analysis
of Drug Target Genes
2015
[145]
risk of selecting resistant mutants in a predominantly susceptible population of M.
tuberculosis. Therefore, by combining p-aminosalicylic acid and streptomycin during
1940s, the treatment failure rate was reduced to 9% (Canetti et al., 1963). Since then,
combined chemotherapy has remained the cornerstone for tuberculosis treatment. The
treatment of tuberculosis at public health level involves DOTS strategy through a
multi drug therapy approach. Multi drug resistant and extensively drug resistant TB
strains arise by sequential accumulation of resistant mutants to individual drugs until
the strain is resistant to drugs that define these forms of resistance.
M. tuberculosis use various mechanisms to avoid killing by drugs, including
mutations in genes that code for drug target proteins, a complex cell wall, which
blocks drug entry and membrane proteins that act as drug efflux pumps (Ramaswamy
& Musser, 1998). Earlier studies on molecular mechanisms of katG, encoding catalase
peroxidase and rpoB, encoding β-subunit of RNA polymerase gene emphasized them
as major targets, conferring resistance to M. tuberculosis against isoniazid and
rifampicin, respectively. A better understanding of mechanisms involved in drug
resistance of M. tuberculosis is essential for the development of new diagnostic tests
as well as new anti-TB drugs or identify new drug targets to treat MDR-TB/XDR-TB
patients. The systematic probing of these drug target genes through sequencing and
bioinformatics based approach in clinical isolates may offer great potential in terms of
identification of new drug targets and development of new anti-bacterial agents.
Various reports are available on molecular epidemiology and characterization of
multi-drug resistant tuberculosis isolates from Europe, America and other regions of
the world. The World Health Organization has identified India as a major hot spot for
Mycobacterium tuberculosis infection. From India, few reports on the prevalence of
drug resistant strain are available, especially from North India. The drug susceptibility
profile, molecular characterization of drug resistance genotypes or their prevalence in
the community has been explored by various research groups in North India (Gupta et
al., 2014; Singhal et al., 2014; Sharma & Madan, 2014; Sagar et al., 2013; Dave et al.,
2009; Siddiqi et al., 2002). But, report from tribal population, such as Sahariya, or
from North Central India on the characterization of drug resistance genotypes is still
lacking. Therefore, the present study was undertaken to characterize the mutations
prevalent in M. tuberculosis isolates from Sahariya tribe and their non-tribal
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of Drug Target Genes
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neighbours for two most important drug target genes, katG and rpoB. Further,
bioinformatics approach was used to forecast the structures of mutant katG and rpoB
proteins along with docking studies to elucidate the mechanism of drug resistance and
reveal the drug-drug target interactions at structural level.
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5.2 Results:
5.2.1 PCR Results for katG and rpoB:
All 27 multi-drug resistant isolates obtained in this study were sequenced. The PCR
products for katG and rpoB genes had targeted amplicons of 414 bp and 350 bp,
respectively (Fig. 5.1 & 5.2).
Fig. 5.1: Gel photograph showing amplicon of 414 bp for katG gene, M: 50 bp Molecular
Weight Marker
Fig. 5.2: Gel photograph showing amplicon of 350 bp for rpoB gene M: 50 bp Molecular
Weight Marker
5.2.2 Sequencing of Drug Target Genes:
The chromatograms of DNA sequence for both the genes i.e., katG and rpoB, were
generated from DNA analyser (ABI3730XL) using Sanger di-deoxy nucleotide
sequencing method that were used for further analysis (Fig. 5.3 A-D). The sequences
were confirmed for their identity using BLAST (www.ncbi.nlm.nih.gov/blast)
(Altschul et al., 1990) (Fig. 5.4 & 5.5). The variant alleles observed in the present
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study are submitted to NCBI with accession numbers: KR424777, KR424778,
KR424779 and KR424780.
Fig. 5.3 A-D: Chromatograms generated showing representative mutations
Fig. 5.4: Nucleotide Sequence Alignment of katG gene from representative isolates of
Mycobacterium tuberculosis from Sahariya tribe and non-tribe population. Nucletoide
changes are shown in the representative isolates with respect to reference gene M.
tuberculosis H37Rv at nucelotide position 1388 and 1586.
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Fig. 5.5: Nucleotide Sequence Alignment rpoB gene from representative isolates of
Mycobacterium tuberculosis from Sahariya tribe and non-tribe population. Nucletoide
change is shown in representative isolate with respect to reference gene M. tuberculosis
H37Rv at nucelotide position 1349
The nucleotide sequences were converted to putative amino acids using Expasy
Translate (http://web.expasy.org/cgi-bin/translate/dna_aa). Multiple sequence
alignments were performed using Clustal Omega
(http://www.ebi.ac.uk/Tools/msa/clustalo/) (Sievers et al., 2011).
5.2.3 Multiple Sequence Alignment:
In all 27 multi-drug resistant isolates, three types of mutations in katG gene were
observed. The most common mutation in all of the isolates was at codon 463
(Arginine 463 Leucine), the second was a combination of amino acid changes in two
isolates at codon 463 (Arginine 463 Leucine) and codon 529 (Asparagine 529
Threonine), and the third was the amino acid change in two isolates at codon 463
(Arginine 463 Leucine) and codon 529 (Asparagine 529 Histidine). The amino acid
sequence change, Arginine 463 Leucine, has been observed in other studies also. In
rpoB gene, only one type of mutation was observed at codon 531 (Serine 531
Leucine).
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For M. tuberculosis isolates from Sahariya tribe, the katG gene was observed to have
variation at codon 463 (Arginine 463 Leucine) in all the drug-resistant isolates
(100%), while the amino acid variations in two different combinations (Arginine 463
Leucine & Asparagine 529 Threonine and Arginine 463 Leucine & Asparagine 529
Histidine) was observed in about 10% of the isolates. For isolates from non-tribes, the
variation at codon 463 (Arginine 463 Leucine) was again observed in all the drug-
resistant isolates. For rpoB gene, out of 21 drug resistant isolates from Sahariya tribe,
81% isolates were observed to have variation at codon 531 (Serine 531 Leucine),
while the other 19% isolates had none. In non-tribes, out of 6 MDR-TB isolates, 50%
were observed to have the same mutation like that of isolates from Sahariya tribe
(Serine 531 Leucine), one isolate showed change for any amino acid at the same
position, whereas, rest of the two isolates (33%) did not show any mutation or
variation (Table 5.1).
Table 5.1: Molecular characteristics of katG and rpoB gene resistant
Mycobacterium tuberculosis isolates of Sahariya tribe and non-tribal population
Isolate No. Base Change Nucleotide
Position
Amino acid
Change
NCBI
Accession No.
katG Gene
GWL-82 CGG-CTG 1388 Arg463Leu KR424778
GWL-114 AAC-ACA 1586 Asp529Thr KR424779
GWL-250 AAC-CAC 1586 Asp529His KR424780
rpoB Gene
GWL-138 TCG-TTG 1349 Ser531Leu KR424777
GWL-360 TCG-TTG 1349 Ser531Leu NS
NS, Not submitted to NCBI
The multiple sequence alignment for all the 21 isolates belonging to Sahariya tribe
and 6 MDR-TB isolates from non-tribal population for both the drug target genes
revealed sequence variations in different isolates at amino acid level (Fig. 5.6 – 5.9).
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Fig. 5.6: Multiple Sequence Alignment of 21 MDR isolates of M. tuberculosis for
Sahariya tribal population. Changes in amino acids at two positions (Arginine 463
Lysine and Asparagine 529 Histidine as well as Asparagine 529 Threonine) are shown.
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Fig. 5.7: Multiple Sequence Alignment for rpoB gene from 21 MDR isolates of M.
tuberculosis for Sahariya tribal population showing change of amino acid (Serine 531
Leucine). Mutations were screened according to E. coli numbering system. The codon
531 position in E. coli and in respective isolates corresponds to 450 position in M.
tuberculosis.
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Fig. 5.8: Multiple Sequence Alignment for katG gene from 6 MDR isolates of M. tuberculosis
from non-tribal population showing change of amino acid (Arginine 463 Lysine).
Fig. 5.9: Multiple Sequence Alignment for rpoB gene of 6 MDR isolates of M.
tuberculosis for non-tribal population and change of amino acids (S531L). Mutations
were screened according to E. coli numbering system. The codon 531 position in E. coli
and in respective isolates corresponds to 450 position in M. tuberculosis.
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5.2.4 Template Selection:
Molecular Modelling of katG and rpoB mutant proteins was done by comparative
modelling using restraint based modelling implemented in Modeller 9.14 programme
(www.salilab.org/modeller). The sequences used to build up the proteins for katG and
rpoB mutants were taken after sequencing of drug target genes (i.e., katG and rpoB)
from clinical isolates of pulmonary tuberculosis patients and these nucleotide
sequences were converted to putative amino acids using Expasy Translate
(http://web.expasy.org/cgi-bin/translate/dna_aa). The amino acid sequences of mutant
katG and rpoB proteins in the clinical isolates were used for template selection for
protein modelling using Modeller 9.14 software and comparing the sequence
similarity with the proteins with PDB ID’s.
The template selection for 3 mutants (Arginine 463 Leucine, Asparagine 529
Threonine & Asparagine 529 Histidine) of katG protein and one mutant (Serine 531
Leucine) of rpoB protein in our clinical isolates was matched with the 1SJ2 ‘A’ and
1YNJ ‘C’ (PDB) as a template for katG and rpoB, respectively. The E value, percent
similarity and bit scores for the three katG mutants (Arginine 463 Leucine,
Asparagine 529 Threonine & Asparagine 529 Histidine) as well as one rpoB mutant
are shown in Table 5.2.
Table 5.2: E value, Percent Similarity and Bit score for katG as well as rpoB
mutant proteins used for the template selection
S. No. PDB IDs Bit Score Percent
Similarity
E Value
katG (Arginine 463 Leucine; Arginine 463 Leucine & Asparagine 529
Threonine; Arginine 463 Leucine & Asparagine 529 Histidine)
1. 1zbyA 416 37% 0.0 2. 1itkA 739 58% 0.0 3. 1mwvA 739 68% 0.0 4. 1sj2A 740 100% 0.0 5. 1u2kA 739 56% 0.0 6. 1ub2A 739 56% 0.0
rpoB (Serine 531 Leucine)
1. 1ynjC 1144 52% 0.0 2. 1twfB 1088 26% 0.0 3. 1iw7C 1144 52% 0.0
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5.2.5 Molecular Modelling:
The target alignment was constructed for all the mutant katG and rpoB proteins using
Modeller 9.14. Five different models were constructed from Modeller for each mutant
by using model single command (model-sinlge.py). Successful models were
generated, each with a Molpdf, GA341 and DOPE score. Molecular PDF (molpdf)
is the standard Modeller scoring function and is the sum of all restraints. DOPE or
Discrete Optimized Protein Energy is a statistical potential and is used to assess the
energy of the protein model generated through many iterations by MODELLER,
which produces homology models by the satisfaction of spatial restraints. DOPE is
based on an improved reference state that corresponds to non-interacting atoms in a
homogenous sphere with the radius dependent on a sample native structure; it thus,
accounts for the finite and spherical shape of the native structures. The DOPE method
is generally used to assess the quality of a structure model as a whole. Alternatively,
DOPE can generate a residue-by-residue energy profile for the input model, making it
possible for the user to spot the problematic region in the structure model. The DOPE
model score is designed for selecting the best structure from a collection of models
built by MODELLER. The molpdf and DOPE scores are not ‘absolute’ measures, in
the sense that they can only be used to rank models (molpdf is specific for a given set
of restraints and DOPE for a given target sequence). Other scores are transferrable.
For example GA341 score always range from 0.0 (worst) to 1.0 (native like);
however, GA341 is not as good as DOPE at distinguishing ‘good’ models from ‘bad’
models. A ‘good’ model will have a GA341 score near 1.0 (larger is better) but a
small DOPE core (DOPE scores, like molpdf, are “energies”, so small or negative) is
better. The models selected on the basis of lowest DOPE score for katG as well as
rpoB mutants are summarized in Table 5.3.
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Table 5.3: Values of Molpdf, DOPE score and GA341 score for the possible PDB
models built for various katG and rpoB mutants (The selection of the best model
is shown in red colour)
katG (Arginine 463 Leucine)
File Name Molpdf DOPE Score GA341 Score
TvLDH.B99990001.pdb 4206.37109 -81119.55469 1.00000
TvLDH.B99990002.pdb 3852.90234 -81904.48438 1.00000
TvLDH.B99990003.pdb 3803.60352 -82055.09375 1.00000
TvLDH.B99990004.pdb 4032.33667 -81863.45313 1.00000
TvLDH.B99990005.pdb 3729.07715 -81831.04688 1.00000
katG (Arginine 463 Leucine; Asparagine 529 Threonine)
File Name Molpdf DOPE Score GA341 Score
TvLDH.B99990001.pdb 3882.13965 -81832.34375 1.00000
TvLDH.B99990002.pdb 4150.41895 -81918.26563 1.00000
TvLDH.B99990003.pdb 3972.59326 -81919.35938 1.00000
TvLDH.B99990004.pdb 3877.48901 -81831.75000 1.00000
TvLDH.B99990005.pdb 3742.10889 -81991.32813 1.00000
katG (Arginine 463 Leucine; Asparagine 529 Histidine)
File Name Molpdf DOPE Score GA341 Score
TvLDH.B99990001.pdb 3853.39868 -81784.45313 1.00000
TvLDH.B99990002.pdb 3902.86548 -81664.55469 1.00000
TvLDH.B99990003.pdb 3939.23071 -81907.91406 1.00000
TvLDH.B99990004.pdb 4398.37598 -81288.10156 1.00000
TvLDH.B99990005.pdb 3781.92017 -82133.94531 1.00000
rpoB (Serine 531Leucine)
File Name Molpdf DOPE Score GA341 Score
TvLDH.B99990001.pdb 9319.8301 -102173.32031 1.00000
TvLDH.B99990002.pdb 9588.36035 -102747.38281 1.00000
TvLDH.B99990003.pdb 9302.90527 -100509.0.7813 1.00000
TvLDH.B99990004.pdb 9282.60742 -101986.33594 1.00000
TvLDH.B99990005.pdb 9207.24316 -100890.72656 1.00000
5.2.6 Modelled Structures:
The structures modelled for various katG and rpoB mutant proteins are shown in Figs.
5.10 & 5.11, respectively. The wild type structure of katG protein is shown in Fig.
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5.10 A, whereas, the crystal structure for rpoB protein in the database is not available.
In the present study, the three dimensional structure for the amino acid sequence of
rpoB M. tuberculosis H37Rv was obtained from NCBI database (Accession No.
NP_215181.1) and the query sequence were searched against PDB to find out protein
structure of related family in PDB database (www.rscb.org) (Natraj et al., 2008). The
modelled mutant protein structures were submitted to I-TASSER online server for
protein energy minimization. The root mean square deviation values obtained for the
three mutants katG, Arg463Leu (0.899), Arg463Leu; Asp529Thr (1.959),
Arg463Leu; Asp529His (0.807) as well as one rpoB Ser531Leu (2.763) mutant
structures were observed higher than wild type katG (0.703) and rpoB (1.167) protein
structures, respectively.
Fig. 5.10 A-D: Structures of wild type as well as mutant katG proteins
A: Wild type katG protein; B: Mutant kat G (R463L)
C: Mutant katG (R463L, N529T); D: Mutant katG (R463L, N529H)
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Fig. 5.11 A-B: Structures of wild type and mutants rpoB proteins.
A: Wild type rpoB protein; B: Mutant rpoB (S531L)
5.2.7 Ramachandran Plot for various katG and rpoB mutants:
The 3D structures of various mutants for katG (R463L, R463L & N529T, R463L &
N529H) and rpoB (S531L) obtained from homology modelling were submitted to
RAMPAGE (www. http://mordred.bioc.cam.ac.uk/~rapper/rampage.php) for analysis
(Fig. 5.12 & 5.13). The Φ and Ψ distributions for non-glycine non-proline residues in
mutant structures for katG and rpoB proteins are summarized in Table 5.4.
Fig. 5.12 A-C: Ramachandran plots generated for various katG mutant proteins.
A: R463 L; B: R463L, N529T; C: R463L, N529H
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Fig. 5.13: Ramachandran plot generated for rpoB mutant (S531L)
Table 5.4: Ramachandran Plot calculations for katG and rpoB mutants
Mutants Number of
residues in
favoured region
N (%)
Number of
residues in
allowed region
N (%)
Number of
residues in
outlier region
N (%)
katG Mutant (R463L) 709 (96.1%) 22 (3.0%) 7 (0.9%) katG Mutant (R463L &
N529T) 709 (96.1%) 21 (2.8%) 8 (1.1%)
katG Mutant (R463L & N529H)
708 (95.9%) 20 (2.7%) 10 (1.4%)
rpoB Mutant (S531L) 1080 (92.3%) 70 (6.0%) 20 (1.7%)
5.2.8 Docking:
The root mean square deviation values (RMSD) obtained after protein energy
minimization in I-TASSER online server (http://zhanglab.ccmb.med.umich.edu) for
wild type and mutant structures of katG and rpoB proteins are shown in Table 5.5.
The docked complexes were analysed and the interactions between drug and wild type
as well as mutant proteins were visualized through PyMOL. The best conformations
were measured on the basis of hydrogen bond interactions along with bonding
distance.
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5.2.8.1 Docking of katG wild type as well as mutant proteins with Isoniazid:
The computational analysis of docking score and interaction energy between katG
wild type as well as mutant modelled proteins of Mycobacterium tuberculosis and
isoniazid were carried out through AutoDock4.0. The docking results showed that
isoniazid binds to wild type katG protein at one of its active site residue HIS108 with
one hydrogen bond formation, whereas, the drug binds to different positions for two
of its mutant forms, viz., Arg463Leu and Arg463Leu & Asp529Thr, with formation of
one hydrogen bond at two different residues, LYS274 and HIS270, respectively (Fig.
5.14 A-C). The drug binds to third mutant (Arg463Leu & Asp529His, a semi
conservative substitution) at TYR229 and VAL230 residues forming 2 hydrogen
bonds (Fig. 5.14 D). The residues HIS108 and VAL230 are also one of the proven
active site residues for isoniazid interaction (Tally et al., 2008).
Fig. 5.14 A-D: Docking of isoniazid with wild type and mutant katG proteins A: Docking
of isoniazid with wild type katG protein B: Docking with mutant katG (R463L) C:
Docking with mutant katG (R463L & N529T) D: Docking with mutant katG (R463L &
N529T).
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5.2.8.2 Docking of rpoB wild type as well as mutant proteins with Rifampicin:
The docking of rifampicin with wild type and mutant proteins also showed variation
in binding energies, formation of hydrogen bonds and their distances. Interaction of
rifampicin with wild type rpoB protein revealed formation of 1 hydrogen bond at
LYS799 residue along with the formation of 1 oxygen bond at THR829 residue, a
hypothesized active site for rifampicin binding, since no crystal structure is available
for rpoB protein till date. Whereas, its interaction with mutant protein (one of the
most common mutation in 81bp region, RRDR) resulted in the formation of 1
hydrogen bond at ILE522 residue with bond distance of 2.23 Å, smaller than bond
distance at THR829 residue 2.46 Å in the wild type (Fig. 5.15 A & B). The binding
energies were also observed varying.
Fig. 5.15 A-B: Docking of rifampicin with wild type and mutant rpoB proteins A: Docking
of rifampcin with wild type rpoB protein B: Docking with mutant rpoB (S531L)
The binding energies as well as hydrogen bond distances calculated for wild type and
mutant proteins of katG and rpoB genes are shown in Table 5.5.
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Table 5.5: Molecular interactions of wild type and mutant structures of katG
and rpoB proteins
Structure
Number of
Hydrogen
Bonds
Interactive
Residues
H-Bond
Distance
Binding
Affinity
(Kcal/mol)
RMSD
Value
katG Wild Type 1 HIS108:HE2 1.79 Å -4.05 0.703
katG Mutant
KR 424778 (R463L) 1 LYS274: HN 2.0 Å -4.84 0.899
katG Mutant
KR 424779
(R463L, N529T)
2 HIS270:HE2 1.9 Å -4.09 1.959
katG Mutant
KR 424780
(R463L, N529H)
2 TYR229:HN
VAL230:HN
2.0 Å
1.9 Å -4.21 0.807
rpoB Wild Type 1 LYS799: HZ
THR829: O
2.19 Å
2.46 Å -9.09 1.167
rpoB Mutant
KR 424777 (S531L) 1 ILE522:HN 2.23 Å -9.59 2.763
HN: Hydrogen Nitrogen; O: Oxygen
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5.3 Discussion:
The molecular basis of drug resistance in multi-drug resistant tuberculosis isolates is
well studied (Gupta et al., 2014; Singhal et al., 2014; Sharma & Madan, 2014; Sagar
et al., 2013; Dave et al., 2009; Siddiqi et al., 2002; Ramaswamy & Musser, 1998;
Rattan et al., 1998). Recently, Bhat et al (2015) reported the situation of MDR-TB in
Sahariya tribe from Gwalior region. But, till date, no data is available on the
mutations in M. tuberculosis isolates responsible for causing drug resistance in
Sahariya tribe and their non-tribal neighbours. To the best of our knowledge this is the
first ever investigation on sequencing of drug target genes and drug-drug target
interactions in mycobacterial isolates from Sahariya tribe and their non-tribal
populations from North Central India. Mycobacterium tuberculosis uses various
mechanisms, including mutations in genes that code for drug target genes and become
resistant to particular drugs.
In present study, the sequencing of katG gene revealed common mutation Arg463Leu
in all the isolates of Sahariya tirbe and non-tribes, whereas mutation at codon 529
(Asp529Thr & Asp529His) was observed only in two isolates of Sahariya tribe. In
rpoB gene, the most common mutation Ser531Leu was observed in the 81 bp
Rifampicin Resistance Determining Region (RRDR) in Sahariya tribe as well as non-
tribe isolates. A bioinformatics approach was used to forecast the structure of mutant
katG and rpoB proteins along with docking studies to elucidate the mechanism of
drug resistance. The present investigation on drug-drug target interactions of wild
type and mutant structures of katG protein, revealed variation in their interactive
residues, is being HIS108 in wild type katG protein, already proved interactive site for
INH (Tally et al., 2008) and LYS274, HIS270 and TYR229 & VAL230 in katG
mutant structures. The docking analysis of wild type and mutant structure of rpoB
protein affirmed LYS799 and THR829 binding residues for wild type protein and
ILE522 for mutant protein.
Studies in North Indian population have documented the mutation in katG at codon
Arg463Leu (Dave et al., 2009; Siddiqi et al., 2002). However, other variations, like
Thr275Ala, Arg409Ala and Asp695Ala along with change at Arg463Leu were
observed in isoniazid resistant clinical isolates from South Africa, Switzerland and
USA (Pretorius et al., 1995). Large number of studies have shown a most common
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mutation at Ser315Thr position (Gupta et al., 2014; Samad et al., 2014; Poudel et al.,
2012; Yuan et al., 2012; Ajbani et al., 2011; Aslan et al., 2008). In South India, a
novel mutation at Gly297Pro was observed along with Ser315Thr change (Ravibalan,
2014). Variable percentage of mutations in rpoB gene has been reported by different
groups. The locus Ser531Leu is suggested to confer resistance to rifampicin at high
frequency, ranging from 30% (Siddiqi et al., 2002), 44.2% (Yuan et al., 2012), 52%
(Khan et al., 2013), 58.7% (Poudel et al., 2012), 64% (Aslan et al., 2008), 72%
(Ravibalan, 2014), 97% (Ajbani et al., 2011), 98% (Wang et al., 2013), to 100% of
the isolates (Dave et al., 2009). In rpoB gene, mutations in some other loci, such as
His526Tyr and Asp516Val, have been reported (Sharma & Madan, 2014; Ravibalan,
2014; Khan et al., 2013; Aslan et al., 2008; Somoskovi et al., 2006; Hwang et al.,
2003; Siddiqi et al., 2002, Bodmer et al., 1995). It was observed that mutations at
Arg463Leu and Ser531Leu in katG and rpoB genes, respectively, of Sahariya tribe
and non-tribes are not different from those studied in various parts of the country.
Thus, the control of MDR-TB is critical, while the level of MDR-TB is increasing,
expanding into clones (Kritski et al., 1997). This stresses the need of early detection
of MDR-TB to control tuberculosis.
Studies on the structural basis of drug resistance for isoniazid and rifampicin in MDR-
TB isolates with various mutations have revealed decreased stability and flexibility of
proteins at isoniazid and rifampicin binding sites, which lead to impaired action
(Kumar & Jena, 2014; Samad et al., 2014; Ubyaan et al., 2012; Ramasubban et al.,
2011; Purohit et al., 2011). Through docking studies it was observed that the mutant
protein with positive binding energy fails to cause rifampicin inhibition and thus,
provides resistance (Kumar & Jena, 2014). The docking studies on mutant proteins of
both genes revealed that these mutations have the potential to alter the interaction of
their respective drugs to their active residues. The present study indicates that these
mutations make the protein unstable and interactive sites rigid. It is suggested that in
wild type protein the binding sites are flexible and favours drug binding (Ramasubban
et al., 2011). Thus, mutations observed in MDR-TB isolates in the present study
might be causing impaired activity of the active binding sites.
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5.4 Conclusion:
The data obtained in the present study may be considered as a representation of
molecular characteristics of MDR-TB isolates in Sahariya tribe and non-tribes. The
study also explores the possible functional variation in isoniazid and rifampicin
resistance with respect to the mutations identified. Already reported mutations,
Arg463Leu and Ser531Leu for katG and rpoB genes, respectively, were observed in
most of the isolates. Only two novel mutations, Asp529Thr and Asp529His for katG
gene, were identified in only two different isolates of Sahariya tribe. The knowledge
of structural mechanism of drug resistance may pave the way for designing of new
drug targets to tackle the situation of multi-drug resistant tuberculosis in near future.
Thus, the application of molecular methods to detect drug resistance in tuberculosis is
important and rapid. As a result, suitable therapy can be initiated to break the
transmission of MDR-TB in this population.
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