Insilico Subtractive Genomic Approach To Identify Essential

And Non-human Homolog Genes As Key Drug Targets In Bacillus

Anthracis CDC684 Strain M.Divya

1223309115M.Sc.

Bioinformatics

INTRODUCTION

• Genome Annotation/Genome Mining:Extraction, definition, ad interpretation of features on the genome sequence derived by integrating computational tools and biological knowledge.

• BackgroundBacillus anthracis, was the first bacterium shown to be the cause of a disease

• Disease– Cutaneous (affecting the skin)  – Inhalational (in the lungs)  – Gastrointestinal (in the digestive

tract)• Pathogenesis

– Protective antigen – Edema factor – Lethal factor

• Signs & Symptoms – Fever– Malaise

•Transmission – Modes of Transmission

•Anthrax As A Biological Weapon – Aerosol Release of Anthrax

Spores – Historical Events Of B.anthracis

Sverdlovsk, USSR—1979 United States—2001 World Health Organization (WHO)

•Counter Measures– Vaccines

•Current Anthrax Vaccine •Development of New Vaccines

– Anti-Toxin •PA-specific antibodies; •soluble receptor-based antitoxins; •polyvalent inhibitors (PV1); •dominant-negative of PA (DN-PA)

Antibiotics

OBJECTIVES1. To retrieve genome information of Bacillus

anthracis CDC684 from NCBI2. To select specific metabolisms out of all

metabolisms using subtractive genomic approach

3. To identify the ratio of essential genes and non-human homologous genes obtained in each metabolism

4. To retrieve the PDB structures of proteins derived from non-human homologous genes

5. To perform comparative analysis of PDB structures retrieved from CDC684 strain against Ames ancestor strain of Bacillus anthracis

6. To obtain complete protein profile of proteins for identify their probability to be used as drug targets

MATERIALS AND METHODS •Strain selection •Retrieving gene location information

• Protein sequence retrieval• Finding the essentiality of proteins • Identifying the non-human

homologous proteins • Finding the cellular localization • Obtaining PDB structures of non-

human homologous genes/proteins • Validation of obtained protein

structures

S.NO ATTRIBUTE TOOL

1 Sequence length, amino acid composition Prot Param

2 pI Prot Param

3 Molecular weight Prot Param

4 Hydropathicity(GRAVY), Prot Param

Hydrophobicity SOSUI

Aliphatic index Prot Param

Trans membrane helices SOSUI

5 Active sites Active site prediction server

6 Subcellular localization PSORTdb

7 Domain region,family ProDom

8 Cleavage sites with protease peptide mass expasy

9 Poat translational modifications ExPASy post translational tools

10 Functionalities,roles,importance SwissProt

11 Structure prediction

Secondary structure GOR-IV

Tertiary structure Swiss model, CPH model

12 Sequence identities with human and other species Hblast,blastP

13 Metabolic pathways KEGG

14 Gene location on chromosome NCBI Gene

15 Phylogenetic constraint analysis Phylogeny.fr

16 Structure alignment Dali

Complete protein profiling

RESULTS AND DISCUSSIONS•Retrieval of genome

information of Bacillus anthracis CDC 684 strain from NCBI

GENOME PXO1 PXO2

GENOME INFORMATION

RefSeq NC_012581 NC_012579 NC_012577

GenBank CP001215 CP001216 CP001214

Length 5,230,115 nt 181,773 nt 94,875 nt

GC content 35% 32% 33%

% coding 84% 75% 76%

Topology Circular Circular Circular

Molecule DNA DNA DNA

FEATURES

Genes 5717 208 117

Protein coding 5579 206 117

Structural RNAs 138 2 none

Pseudogenes None None None

Others 106 1 8

Contigs None None None

•Metabolisms selected for studies using subtractive genomic approach

S.no Gene Role Category # of Genes

% out of 5902

Genes

1 Amino acid biosynthesis 106 1.79 %

2 Biosynthesis of cofactors, prosthetic groups, and carriers

143 2.42 %

3 Cell envelope 489 8.28 %

4 Cellular processes 550 9.31 %

5 Central intermediary metabolism 66 1.11 %

6 Disrupted reading frame 36 0.60 %

7 DNA metabolism 145 2.45 %

8 Energy metabolism 299 5.06 %

11 Hypothetical proteins 803 13.6 %

22 Transport and binding proteins 579

• Ratio of number of essential genes and non human homologous genes in metabolisms Amino acid metabolism     -  4:6 Bio synthesis of cofactors, prosthetic groups & carriers - 21:10Cell envelope -11:38Cellular processes -7:6Central intermediary metabolism  -5:7Disrupted reading frame  -0:1DNA metabolism  -14:10Energy metabolism  -0:7Hypothetical proteins -Conserved -0:11Transport and binding proteins  -0:34

•Retrieval of 3-D structures for non human homologous proteins from PDB

S.NO Metabolism No. of PDB structures retrieved

1 Amino acid biosynthesis 3

2 Bio synthesis of cofactors, Prosthetic groups and carriers

6

3 Cell envelop 6

4 Cell processes 3

5 Central inter mediary metabolism 5

6 DNA metabolism 6

7 Transport and binding proteins 12

TOTAL 41

•Comparative analysis of non-human homologous PDB structures S.N

OMOLECULE NAME METABOLISM

1 Pdp Related Beta-Lactamase Cell envelope

2 Acetyl-Coenzyme A Synthetase Central intermediary metabolism

3 DNA Polymerase III Polc-Type Dna Metabolism

4 Homogentisate 1,2-Dioxygenase Energy metabolism

5 L-Lysine 2,3-Aminomutase Energy metabolism

6 2-Oxo Acid Dehyrogenase Alpha Subunit

Energy metabolism

7 Multidrug Resistance Protein D Transport and binding proteins

8 Glycerol-3-Phosphate Transporter Transport and binding proteins

9 ABC Transporter, ATP-Binding Protein

Transport and binding proteins

10 Amino Acid ABC Transporter Transport and binding proteins

11 Multi Antimicrobial Extrusion Protein (Na(+)/Drug Antiporter) MATE-Like MDR Efflux pump

Transport and binding proteins

•Complete protein profiling – By comparing both the protein profiles

DNA polymerase III epsilon unit is found to be more potential target.

– Comparison was done on the basis of following criteria 1. Length of the protein sequence2. Amino acid composition and pI3. Hydropathicity and hydrophobicity.4. Aliphatic index 5. Transmembrane helices6. Less sequence and structure similarity

• Identifying The Ligands For The Predicted Potential Drug Target

Ligand Binding Site Prediction Information

CONCLUSION • A total number of 6,042 genes are

located on the genome of Bacillus anthracis strain CDC684.

• Out of them 5,902 are protein coding.

• There are 392 proteins with their length less than 100 amino acids.

• Number of metabolisms considered for further analysis is 10 in number, containing 3,216 number of genes.– Out of them

•54 genes found to be essential and •135 genes found to be non-human homologs

• There are seven genes which are essential to the pathogen and are non-human homologous. These are considered to be more potential drug targets when compared with other targets.

FUTURE PROSPECTS• Out of 182 proteins only 41 PDB structures

are available. The remaining structures can be drawn by using homology modeling and other statistical, mathematical models.

• There are very few or no ligand molecules observed specific to bacillus anthracis potential drug targets. Several ligand molecules can be designed using Structure Based Drug Designing approach.

Thank You