chapter 5 sequencing, comparative modelling and docking...

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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

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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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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)


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)


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)


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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chapter 5 sequencing, comparative modelling and docking...

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