Identification of Drug Targets in Helicobacter pylori by in silico ...

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79

Identification of Drug Targets in Helicobacter pylori by in silico Analysis: Possible Therapeutic Implications for Gastric cancer Deepthi Nammi1, Ravi C. P. K. Srimath-Tirumala-Peddinti2 and Nageswara Rao R. Neelapu3,* 1

Department of Bioinformatics, School of Life Sciences, GITAM Institute of Science, GITAM University, Rushikonda campus, Visakhapatnam–530045 (AP), India; 2Department of Bioinformatics, School of Life Sciences, GITAM Institute of Science, GITAM University, Rushikonda campus, Visakhapatnam–530045 (AP), India; 3Department of Bioinformatics, School of Life Sciences, GITAM Institute of Science, GITAM University, Rushikonda campus, Visakhapatnam–530045 (AP), India Abstract: Helicobacter pylori colonize stomach, inducing gastritis, ulcers and gastric cancer. Drugs are used to relieve pain, but not H. pylori infections. Hence, there is a need for discovery of drug targets and drugs for H. pylori. An objective of this current study is to identify drug targets for H. N.R.R. Neelapu pylori. RAST was used to compare genomes of 23 H. pylori strains with Homo sapiens sapiens, other Helicobacter species (H. acinonychis, H. hepaticus, H. mustalae) and among them, to identify 13471 unique genes. Bacterial genes which are non-homologous to humans and essential for pathogen are identified using BLASTp. Later, 29 potential drug targets were identified by subjecting these genes to property analysis. Eleven of the 29 drug targets are already experimentally validated, lending credence to our approach. These methods have enabled rapid identification of drug targets with possible therapeutic implications for gastric cancer.

Keywords: Drug targets, genome analysis, host-pathogen comparison, proteome comparison, strain-strain comparison, speciesspecies comparison. INTRODUCTION Gastric cancer is the most common cancer caused by Helicobacter pylori infection leading to the damage of gastric mucosa and gastric glands [1]. H. pylori is designated as a class 1 carcinogen by the WHO [2]. Treatment for H. pylori infection includes drugs to relieve pain and acidity, but not gastritis, peptic ulcers, and gastric cancer. Carcinogenic activity of H. pylori suggests the need for discovery of new drug targets and drugs for the prevention of H. pylori. Laboratory techniques are used to identify drug targets which can influence growth, colonization and virulence of H. pylori. Some of the laboratory techniques are signature tag mutagenesis [3], insertion mutagenesis [4], random insertion mutagenesis [5], deletion mutant analysis [6], null mutant analysis [7], mutant analysis [8] and structural studies [9]. Availability of the complete genome sequence of pathogen H. pylori, provided us the platform with an opportunity to mine the genome for harnessing the potential drug targets. Comparative genomic analysis between host and pathogen, strains, species, avirulent and virulent strains may reveal genes essential for pathogen, strain, species and virulence respectively providing us with a tremendous amount of information. This information can be harnessed for identifying potential drug targets for any given pathogen [10]. *Address correspondence to this author at the Department of Bioinformatics, School of Life Sciences, GITAM Institute of Science, GITAM University, Rushikonda, Visakhapatnam- 530045. AP, India; Tel: +91-891-2840464; Fax: +91-891-2790032; E-mail: [email protected]

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Comparison of pathogen and host genomes revealed potential drug targets in Staphylococcus aureus [11], H. pylori [10], Listeria monocytogenes [12], Leishmania infantum [13], L. major [14], Mycobacterium leprae [15], Pseudomonas aeruginosa [16] and Schistosoma mansomi [17]. Comparing the genomes of virulent and avirulent strains in Streptococcus pneumoniae, Pasteurella multocida, Mycobacterium tuberculosis, Stenotrophomas maltophilia, Pichinde arenavirus, duck enteritis virus revealed virulence factors [18], pathogenicity genes [19], genetic basis of virulence attenuation [20], strain specific pathogenic islands [21], and amino acid polymorphisms [22] that contribute to virulence. This information is a potential source to identify drug targets for a pathogens. Comparison among Leptospira interrogans strains revealed 88 drug targets [23]. Comparative genomic analysis of different species revealed essential genes [24-29]. Four new potential drug targets – trr1, rim8, kre2, erg6 were identified when genomes of eight fungal pathogen species were compared [24]. Reductive evolution in M. leprae [26], 1250 mycobacterial gene families [29] and drug targets essential for growth and survival [25] were revealed when the genomes of mycobacterial species were compared. Genome of H. pylori when compared with other family members of Enterobacteriaceae revealed similar components in the flagellar system [28] and differences in metabolism [27]. Genomics [30-35], proteomics [30, 31], metabolic pathway analysis [36] and reverse docking [37] are used to identify drug targets in H. pylori. Genome and proteome analysis in H. pylori strains HpB38, HpP12, HpG27, HpShi470, HpSJM180 identified 17 novel drug targets [30], whereas similar type of analysis identified 29 novel drug targets in © 2016 Bentham Science Publishers

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Nammi et al.

strain HpAG1 [31]. Essential gene analysis in H. pylori strain HpAG1, Hp26695 and J99 identified essential genes [38], outer membrane proteins and Mur A respectively as drug targets [34]. Metabolic pathway analysis in H. pylori identified lipopolysaccharide [36] and lysine biosynthesis pathway [34] as a source of potential drug targets. Reverse docking was used to identify drug target in H. pylori [37]. However, there are no specific reports to date, on comparing genomes of different H. pylori strains, and different Helicobacter species to identify drug targets in H. pylori. Therefore, the current paper deals with comparing genomes of host and pathogen; different H. pylori strains, and various Helicobacter species for screening drug targets and identification of novel drug targets using proteome analysis in 23strains of H. pylori. MATERIAL AND METHODS Sampling Genomes of 23 H. pylori strains (Table 1) are sampled based on availability of complete genome, strain history, Table 1.

pathogenicity report of the strains and geographical origin. In our study, identification of novel drug targets for H. pylori has been accomplished for the first time for all the 23 H. pylori strains by using an integrated approach of genome, proteome and metabolic pathway analysis followed by primary property analysis of the genes/proteins using computational resources. Drug Target Screening for Identification of Unique Molecules in H. pylori Comparative genome analysis was performed to screen the drug targets for pathogen H. pylori. Genome of 23 H. pylori strains (Table 1) were initially annotated and further reconstructed for metabolic pathways using RAST (Rapid Annotation Subsystem Technology) server [58]. Analysis was carried out by comparing genomes of pathogen H. pylori and host Homo sapiens sapiens using RAST to screen unique genes that are only present in pathogen and not present in the host. Another comparison of genomes was made between H. pylori and other Helicobacter species H. acinonychis, H. hepaticus, H. mustalae using RAST to

Statistics of genome for 23 H. pylori strains.

S. No

Strain Name

Genome Accession Number

Genome Length

Geographical Origin

Reference

1

HpF32

NC_017366

1,578,824 bp

East Asia

Furuta et al. [39]

2

HpF30

NC_017365

1,570,564 bp

East Asia

Furuta et al. [39]

3

Hp2017

NC_017374

1,548,238 bp

France

Avasthi et al. [40]

4

Hp2018

NC_017381

1,562,832 bp

France

Avasthi et al. [40]

5

Hp26695

NC_018939

1,667,867 bp

Europe

Manolov et al. [41]

6

Hp35A

NC_017360

1,566,655 bp

America

Muzny et al. [42]

7

Hp51

NC_017382

1,589,954 bp

East Asia

Kim et al. [43]

8

Hp52

NC_017354

1,568,826 bp

East Asia

Kim et al. [44]

9

HpCuz20

NC_017358

1,635,449 bp

Monte Carmelo near Cuzco, Peru.

Kersulyte et al. [45]

10

HpF16

NC_017368

1,575,399 bp

East Asia

Furuta et al. [39]

11

HpF57

NC_017367

1,609,006 bp

East Asia

Furuta et al. [39]

12

HpIndia 7

NC_017372

1,675,918 bp

India


13

HpSat464

NC_017359

1,560,342 bp

Satipo region, Peru


14

HpJ99

NC_000921

1,643,831 bp

Amerind strain

Merrell et al. [48]

15

HpB8

NC_014256

1,673,997 bp

Europe

Farnbacher et al. [49]

16

Hp908

NC_017357

1,549,666 bp

France

Devi et al. [50]

17

Hp83

NC_017375

1,617,426 bp

Greece

Muzny et al. [51]

18

HpSJM180

NC_014560

1,658,051 bp

Lima, Peru


19

HpAG1

NC_008086

1,596,366 bp

Europe

Oh et al. [53]

20

HpShi470

NC_010698

1,608,548 bp

Shimaa, Peru


21

HpG27

NC_011333

1,652,982 bp

Italy

Baltrus et al. [55]

22

HpP12

NC_011498

1,673,813 bp

Europe

Fischer et al. [56]

23

HpB38

NC_012973

1,576,758 bp

France, Europe

Thiberge et al. [57]

Novel Drug Targets for H. pylori


screen unique genes that are only present in pathogen and not present in other Helicobacter species. Genomes of 23 H. pylori strains were also compared among themselves using RAST to screen genes that are unique to each H. pylori strain. Genes which are unique to H. pylori in the above methods were filtered and catalogued (Fig. 1A). Drug Target Screening for Confirmation of Unique Molecules in H. pylori Bacterial genes which are non-homologous to humans are essential for pathogen. To identify these non-homologous genes in H. pylori strains homology at the level of sequence and structure was used as the parameter. BLASTp [59] which is based on principle of homology was used to confirm the uniqueness of the catalogued genes in H. pylori by comparing genes against Homo sapiens sapiens (Fig. 1B).

81

Drug Target Identification A set of computational resources was used to analyse the characteristic features of the genes for identification of potential drug targets. GPCRpred [60], GPCRClass [61], Nrpred [62], Btxpred [63], Mitpred [64], SRTpred [65], VGIchan [66], Ntxpred [67] and ICM pred [68] are the computational resources (Table 2). Potential drug targets among the pool of catalogued genes were identified using the above list of servers (Fig. 1C). RESULTS Genome Wide in silico Analysis for Screening of Drug Targets in H. pylori Genome wide in silico analysis for screening of drug targets identified 13741 unique genes in 23 H. pylori strains.

Fig. (1). Integrated approach of A) drug target screening by genome and metabolic pathway analysis, B) Drug target confirmation by proteome analysis followed by C) drug target identification by primary property analysis of the genes/proteins using computational resources.

Table 2.

Computational Resources of Drug Discovery (CRDD) (http://crdd.osdd.net/) of Open Source Drug (http://www.osdd.net/) to identify the potentiality of the drug targets in Helicobacter pylori.

S. No

Server Name

Server Function

Reference

1

GPCR pred

Server is for predicting G-protein coupled receptors and for classifying them into families and sub-families

Bhasin and Raghava [60]

2

GPCRS class

Server is for predicting Amine Type of G-protein-coupled receptors based on dipeptide composition method. In this method type amine is predicted from dipeptide composition of proteins using SVM


3

Nrpred

Server is for classification of nuclear receptors on the basis of amino acid composition or dipeptide composition using SVM method. The overall prediction accuracy of amino acid composition and dipeptide composition based methods is 82.6% and 97.2%.


4

BTX pred

Server is for prediction of bacterial toxins and its function from primary amino acid sequence.

Saha and Raghava [63]

5

MIT pred

Server is for predicting mitochondrial proteins.

Kumar et al. [64]

6

SRT pred

Server classifies protein sequence as secretory or non-secretory proteins.

Garg and Raghava [65]

7

VGIchan

Server predicts voltage gated ion-channels and classifies them into sodium, potassium, calcium and chloride ion channels from primary amino acid sequences.

Saha et al. [66]

8

NTX pred

Server predicts neurotoxins and probable functions from primary amino acid sequences.


9

VICM pred

Server aids in broad functional classification of bacterial proteins into virulence factors, information molecule, cellular process and metabolism molecule.



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Table 3.

S.No

1 2 3

4 5

Nammi et al.

Total number of unique genes identified for each metabolic category in the 16 H. pylori strains by comparing genomes of H. pylori and Homo sapiens sapiens.

Metabolic Category

Amino Acids and Derivatives Carbohydrates Cell Division and Cell Cycle Cell Wall and Capsule Clustering-based subsystems

Hp

Hp

HpF

Hp

Hp

Hp

Hp

HpF

Hp

Hp

Hp J

Hp

HpCuz

HpSat

Hp

Hp

2017

2018

30

35A

51

F16

F32

57

52

908

99

India7

20

464

B8

83

51

51

50

53

53

53

51

51

52

51

50

52

51

52

52

49

36

36

37

37

36

37

36

37

36

36

37

37

36

37

37

23

3

3

3

3

3

3

3

3

3

3

3

3

3

3

3

3

43

43

43

42

43

43

43

48

43

43

43

43

46

45

43

45

63

63

82

83

83

83

66

67

66

63

66

67

66

67

67

75

41

41

48

38

50

47

41

41

35

41

41

41

41

41

41

45

33

33

32

31

31

31

32

31

30

28

31

30

31

31

32

34

26

26

26

26

26

27

26

26

26

26

26

26

26

26

26

26

Cofactors, Vitamins, 6

Prosthetic Groups, Pigments

7 8

DNA Metabolism Fatty Acids, Lipids, and Isoprenoids

9

Membrane Transport

22

22

22

23

22

22

22

22

22

22

22

22

22

22

22

23

10

Miscellaneous

0

14

13

14

14

14

0

0

0

0

0

0

0

0

0

0

11

Motility and Chemotaxis

38

38

38

38

38

38

38

38

38

38

37

38

38

38

38

40

3

3

2

2

2

2

3

3

3

3

3

3

3

3

3

7

12

Nucleosides and Nucleotides

13

Phosphorus Metabolism

2

2

3

3

3

3

2

2

2

2

2

2

2

2

2

1

14

Potassium metabolism

3

3

3

3

3

3

3

3

3

3

3

3

3

3

3

2

15

Protein Metabolism

82

82

83

83

83

83

82

84

82

83

82

82

83

82

82

75

16

RNA Metabolism

19

19

18

18

18

18

19

19

19

19

19

19

19

19

19

19

2

2

7

7

7

7

7

7

7

7

7

7

7

7

7

7

17

Regulation and Cell signalling

18

Respiration

28

28

28

28

28

28

28

28

28

28

28

28

28

28

28

28

19

Stress Response

15

15

15

15

15

15

15

15

15

15

15

15

15

15

15

16

20

Sulfur Metabolism

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

8

8

8

8

8

8

8

9

8

8

9

9

8

9

9

18

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

1

520

558

563

557

568

567

527

531

520

521

526

529

530

532

531

539

21

22

Virulence, Disease and Defense Iron acquisition and metabolism Total Number of Genes

Genome comparison between pathogen H. pylori and host Homo sapiens sapiens resulted in the identification of 9188 unique genes in 16 H. pylori strains (Table 3). Comparison of genomes between H. pylori and other Helicobacter species H. acinonychis, H. hepaticus, H. mustalae identified 4377 unique genes in 23 H. pylori strains (Table 4). Genome comparison among 23 H. pylori strains identified 176 unique

genes in H. pylori strains (Table 5). These molecules fall under 24 metabolic categories as shown in Tables 3-5. In Silico Proteome Analysis for Identification of Drug Targets in H. pylori Proteome analysis followed by gene property analysis of 13741 unique genes identified 29 potential drug targets

23

22

21

20

Stress Response

19

0/0/0

0/0/0

0/0/0

0/0/0

0/3/2

38/73/78

0/0/0

0/0/0

0/3/2

0/0/0

0/0/13

0/0/13

0/0/0

0/0/0

1/3/2

1/3/2

0/0/0

3/13/4

0/2/1

0/0/0

2/3/3

3/13/4

0/2/1

0/0/0

2/3/3

0/1/0

0/0//0

0/0/0

0/1/0

0/9/1

4/2/8

0/3/4

20/9/11

0/9/1

4/2/8

0/3/4

20/9/11

2/4/6

1/2/3

1/2/3

2/4/6

0/0/0

2/14/9

2/14/9

0/0/0

3/5/11

Hp2018

3/5/11

Hp2017

Total Number of Genes 38/73/78

aromatic compounds

Metabolism of

and metabolism

Iron acquisition

and Defense

Virulence, Disease

Metabolism

Sulfur

Respiration

Cell signalling

Regulation and

RNA Metabolism

Metabolism

Protein

metabolism

Potassium

Metabolism

Phosphorus

Nucleotides

Nucleosides and

Chemotaxis

Motility and

Miscellaneous

Transport

Membrane

and Isoprenoids

Fatty Acids, Lipids,

DNA Metabolism

Pigments

Prosthetic Groups,

Cofactors, Vitamins,

subsystems

Clustering-based

Cell Wall and Capsule

Cell Cycle

Cell Division and

Carbohydrates

Derivatives

Amino Acids and

Metabolic Category

43/77/84

0/0/0

0/0/0

0/3/2

0/0/0

0/0/0

1/1/14

0/2/0

1/3/2

3/13/4

0/2/1

0/0/0

2/3/3

0/1/0

0/0/0

1/8/2

4/2/8

1/4/5

20/9/11

3/4/7

1/2/3

0/0/0

2/14/10

4/6/12

HpF30

40/76/82

0/0/0

0/0/0

0/3/2

0/0/0

0/0/0

0/0/13

0/2/0

1/3/2

3/14/5

0/2/1

0/0/0

2/3/3

0/1/0

0/0/0

0/9/1

4/2/8

0/3/4

20/9/11

3/3/7

2/3/4

0/0/0

2/14/10

3/5/11

Hp26695

31/76/83

0/0/0

0/0/0

0/3/2

0/0/0

0/0/0

0/0/13

0/2/0

1/3/2

3/14/5

0/2/1

0/0/0

2/3/3

0/1/0

0/0/0

1/8/2

4/2/8

0/3/4

10/9/11

3/4/7

1/2/3

0/0/0

2/14/10

4/6/12

Hp35A

42/76/82

0/0/0

0/0/0

0/3/2

0/0/0

0/0/0

1/1/14

0/2/0

1/3/2

3/13/4

0/2/1

0/0/0

2/3/3

0/1/0

0/0/0

1/8/2

4/2/8

0/3/4

20/9/11

3/4/7

1/2/3

0/0/0

2/14/9

4/6/12

Hp51

43/76/83

0/0/0

0/0/0

0/3/2

0/0/0

0/0/0

1/1/14

0/2/0

1/3/2

3/13/4

0/2/3

0/0/0

2/3/3

0/1/0

0/0/0

1/8/2

4/2/8

1/3/4

20/9/11

3/4/7

1/2/3

0/0/0

2/14/10

4/6/12

HpF16

40/76/80

0/0/0

0/0/0

0/3/2

0/0/0

0/0/0

0/0/13

0/2/0

1/3/2

3/13/4

0/2/1

0/0/0

2/3/3

0/1/0

0/0/0

0/9/1

4/2/8

1/4/5

20/9/11

3/4/7

1/2/3

0/0/0

2/14/9

3/5/11

HpF32

41/76/83

0/0/0

0/0/0

1/3/3

0/0/0

0/0/0

0/0/13

0/2/0

1/3/2

3/15/5

0/2/1

0/0/0

2/3/3

0/1/0

0/0/0

1/8/2

4/2/8

0/3/4

20/9/11

3/4/7

1/2/3

0/0/0

2/14/10

3/5/11

HpF57

30/76/80

0/0/0

0/0/0

0/3/2

0/0/0

0/0/0

0/0/13

0/2/0

1/3/2

3/13/4

0/2/1

0/0/0

2/3/3

0/1/0

0/0/0

0/9/1

4/2/8

0/3/4

10/9/11

3/4/7

1/2/3

0/0/0

2/14/9

4/6/12

Hp52

38/74/77

0/0/0

0/0/0

0/3/2

0/0/0

0/0/0

0/0/13

0/2/0

1/3/2

3/13/4

0/2/1

0/0/0

2/3/3

0/1/0

0/0/0

0/9/1

4/2/8

0/2/3

20/9/11

2/4/6

1/2/3

0/0/0

2/14/9

3/5/11

Hp908

40/75/80

0/0/0

0/0/0

0/3/2

0/0/0

0/0/0

0/0/13

0/2/0

1/3/2

3/13/4

0/2/1

0/0/0

2/3/3

0/1/0

0/0/0

0/9/1

4/2/8

0/3/4

20/9/11

3/3/7

2/3/4

0/0/0

2/14/9

3/5/11

HpJ99

40/76/81

0/0/0

0/0/0

0/3/2

0/0/0

0/0/0

0/0/13

0/2/0

1/3/2

3/13/4

0/2/1

0/0/0

2/3/3

0/1/0

0/0/0

0/9/1

4/2/8

0/3/4

20/9/11

3/4/7

1/2/3

0/0/0

2/14/10

4/6/12

HpIndia7

39/78/79

0/0/0

0/0/0

0/3/2

0/0/0

0/0/0

0/0/13

0/2/0

1/3/2

3/14/4

0/2/1

0/0/0

2/3/3

0/1/0

0/0/0

0/9/1

4/2/8

0/3/4

20/9/11

3/4/7

1/4/3

0/0/0

2/14/9

3/5/11

HpCuz20

41/77/82

0/0/0

0/0/0

0/3/2

0/0/0

0/0/0

0/0/13

0/2/0

1/3/2

3/13/4

0/2/1

0/0/0

2/3/3

0/1/0

0/0/0

0/9/1

4/2/8

1/4/5

20/9/11

3/4/7

1/2/3

0/0/0

2/14/10

4/6/12

HpB8

35/74/77

0/0/0

0/0/0

0/3/2

0/0/0

0/1/0

1/1/14

0/2/0

3/2/3

1/13/3

0/1/1

0/0/0

0/6/5

0/1/0

0/0/0

2/8/2

0/3/4

1/5/6

17/6/8

4/3/9

1/2/2

0/0/0

0/12/7

3/4/10

Hp83

18/75/70

0/0/0

0/0/0

0/3/2

0/0/0

0/1/0

1/1/14

1/3/0

1/1/1

0/14/2

0/1/1

0/0/0

1/7/4

0/1/0

1/0/1

1/8/1

0/3/4

0/4/5

4/4/5

3/3/7

1/2/2

0/1/0

1/13/9

3/4/10

HpP12

37/73/78

0/0/0

0/0/0

0/4/2

0/0/0

0/0/0

0/0/13

0/2/0

1/3/2

2/12/3

0/2/1

0/0/0

2/3/3

0/1/0

0/0/0

0/9/1

4/2/8

1/4/5

18/7/9

3/4/7

1/2/3

0/1/0

2/14/10

3/5/11

HpSJM180

Total number of unique genes identified for each metabolic category in the 23 H. pylori strains by comparing genomes of H. pylori with species H. acinonychis, H. hepaticus, H. mustelae.

18

17

16

15

14

13

12

11

10

9

8

7

6

5

4

3

2

1

S.No

Table 4.

26/67/62

0/2/0

0/0/0

0/3/2

0/0/0

0/0/0

0/0/13

0/2/0

1/5/1

1/11/1

0/1/0

0/0/0

2/5/5

0/1/0

0/0/0

0/8/1

0/1/4

1/4/5

16/6/7

1/2/5

0/2/2

0/0/0

0/11/7

4/3/9

HpAG1

37/75/78

0/0/0

0/0/0

0/3/2

0/0/0

0/0/0

0/0/13

0/3/1

1/3/2

2/13/3

0/2/1

0/0/0

2/3/3

0/1/0

0/0/0

0/9/1

4/2/8

1/4/5

18/7/9

3/4/7

1/2/3

0/0/0

2/14/9

2/5/11

HpG27

37/73/78

0/0/0

0/0/0

0/3/2

0/0/0

0/0/0

0/0/13

0/2/0

1/3/2

2/12/3

0/2/1

0/0/0

2/3/3

0/1/0

0/0/0

0/9/1

4/2/8

1/4/5

18/7/9

3/4/7

1/2/3

0/0/0

2/14/10

3/5/11

HpShi470

43/77/83

0/0/0

0/0/0

1/3/3

0/0/0

0/0/0

0/0/13

0/2/0

1/3/2

3/13/4

0/2/1

0/0/0

2/3/3

0/1/0

0/0/0

1/8/2

4/2/8

1/4/5

20/9/11

3/4/7

1/3/3

0/0/0

2/14/9

4/6/12

HpSat464

34/6277

0/0/0

0/0/0

0/3/2

0/0/0

0/1/0

1/1/14

0/2/0

3/1/3

1/3/3

0/1/1

0/0/0

2/6/5

0/1/0

0/0/0

1/8/1

0/3/4

0/4/5

17/2/8

0/2/9

1/2/2

0/1/0

1/13/9

3/4/10

HpB38

Novel Drug Targets for H. pylori Current Cancer Drug Targets, 2016, Vol. 16, No. 1 83

22

of Genes

Total Number

0

0

0

0

2

0

2

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

1

0

1

0

0

0

1

0

1

0

0

0

0

0

0

0

0

Hp2018

0

Hp2017

4

0

0

0

0

1

0

0

1

0

0

0

0

0

0

0

1

0

0

0

0

1

0

HpF30

6

0

1

0

0

0

0

0

1

0

0

0

0

0

1

0

1

0

0

1

0

1

0

Hp26695

5

0

0

0

0

0

0

0

2

0

0

0

0

0

1

0

1

0

0

0

0

1

0

Hp35A

3

0

0

0

0

1

0

0

1

0

0

0

0

0

0

0

1

0

0

0

0

0

0

Hp51

3

0

0

0

0

1

0

0

0

0

0

0

0

0

0

0

1

0

0

0

0

1

0

HpF16

3

0

0

0

0

0

0

0

1

0

0

0

0

0

1

0

1

0

0

0

0

0

0

HpF32

5

0

0

0

0

0

0

0

3

0

0

0

0

0

0

0

1

0

0

0

0

1

0

HpF57

2

0

0

0

0

0

0

0

1

0

0

0

0

0

1

0

0

0

0

0

0

0

0

Hp52

4

0

0

0

0

0

0

0

2

0

0

0

0

0

1

0

1

0

0

0

0

0

0

Hp908

4

0

1

0

0

0

0

0

0

0

0

0

0

0

1

0

1

0

0

1

0

0

0

HpJ99

3

0

1

0

0

0

0

0

0

0

0

0

0

0

1

0

0

0

0

0

0

1

0

HpIndia7

4

0

0

0

0

0

0

0

1

0

0

0

0

0

1

0

1

0

0

1

0

0

0

HpCuz20

5

0

1

0

0

0

0

0

1

0

0

0

0

0

1

0

1

0

0

0

0

1

0

Hp B8

33

0

1

0

1

1

0

2

3

1

0

2

0

0

2

2

3

6

6

1

1

1

0

Hp83

43

0

2

0

2

1

1

3

3

1

0

4

0

1

1

2

2

7

9

1

1

1

1

HpP12

3

0

0

0

0

0

0

0

0

0

0

0

0

0

1

0

1

0

0

0

0

1

0

HpSJM180

4

0

0

0

0

0

0

0

1

0

0

0

0

0

1

0

1

0

0

0

0

1

0

HpAG1

4

0

0

0

0

0

1

0

2

0

0

0

0

0

1

0

0

0

0

0

0

0

0

HpG27

3

0

0

0

0

0

0

0

1

0

0

0

0

0

0

0

1

0

0

0

0

0

1

HpShi470

31

0

1

0

1

1

0

2

2

1

0

2

0

0

2

2

3

6

5

1

1

1

0

HpB38

6

0

1

0

0

0

0

0

0

0

0

0

0

0

0

0

1

0

0

3

0

1

0

HpSat464


and metabolism

Iron acquisition

and Defense

Virulence, Disease

Sulfur Metabolism

20

21

Respiration

Stress Response

19

Cell signalling

Regulation and

Metabolism

RNA

Metabolism

Protein

metabolism

Potassium

Metabolism

Phosphorus

Nucleotides

Nucleosides and

Chemotaxis

Motility and

Miscellaneous

Transport

Membrane

and Isoprenoids

Fatty Acids, Lipids,

DNA Metabolism

Pigments

Prosthetic Groups,

Cofactors, Vitamins,

subsystems

Clustering-based

Capsule

Cell Wall and

and Cell Cycle

Cell Division

Carbohydrates

Derivatives

Amino Acids and

Metabolic Category

Total number of unique genes identified for each metabolic category in the 23 H. pylori strains by comparing genomes of 23 H. pylori strains.

18

17

16

15

14

13

12

11

10

9

8

7

6

5

4

3

2

1

S.No

Table 5.

84 Nammi et al.


Table 6.


Drug targets identified in the 16 H. pylori strains by comparing genomes of H. pylori and Homo sapiens sapiens.

S. No

Drug Target

Metabolic Category

Strain

Gene ID

1

Menaquinone via futalosine step 1

Cofactors, Vitamins, Prosthetic Groups, Pigments

HpF30

GI:317178861


Hp2017

GI:325997655


Hp2018

GI:325996059


HpF32

GI:317180543


Hp51

GI:261837990


Hp52

GI:261839405


HpF57

GI:317182090

DNA Metabolism

HpF30

GI:317179582

DNA Metabolism

Hp52

GI:261839116

DNA Metabolism

Hp52

GI:261840193

Membrane Transport

Hp2017

GI:325997182

Membrane Transport

Hp2018

GI:325995586

Membrane Transport

HpF32

GI:317180105

Membrane Transport

Hp51

GI:261837744

Membrane Transport

Hp52

GI:261839155

Membrane Transport

Hp2017

GI:325997183

Membrane Transport

Hp51

GI:261837745

Membrane Transport

HpF30

GI:384899677

Membrane Transport

HpF32

GI:317181128

2

3

Type III restriction-modification system methylation subunit

Dipeptide transport system permease protein DppB

4

Dipeptide transport system permease protein DppC

5

Ferric siderophore transport system, biopolymer transport protein ExbB

Membrane Transport

Hp51

GI:261838720

Membrane Transport

Hp52

GI:261840120

6

Ribonuclease BN

RNA Metabolism

Hp2018

GI:325996663

7

Soluble lytic murein transglycosylase precursor


HpF30

GI:317178992


HpF32

GI:317180470

Type I restriction-modification system specificity subunit S

DNA Metabolism

HpF30

GI:317178850

DNA Metabolism

Hp52

GI:261839395

Membrane Transport

HpF30

GI:384899291

Membrane Transport

HpF32

GI:317180733

Membrane Transport

Hp51

GI:261837804


HpF30

GI:384899013


Hp35A

GI:315586640


HpF32

GI:317180434


Hp51

GI:261838080


Hp52

GI:447133031

Miscellaneous

Hp35A

GI:315587073

Miscellaneous

Hp2017

GI:325998043

Miscellaneous

Hp2018

GI:325996454

Miscellaneous

HpF32

GI:317180913

Miscellaneous

Hp51

GI:261838508

Miscellaneous

Hp52

GI:261839908

8

9

10

11

HoxN/HupN/NixA family nickel/cobalt transporter

Flagellar biosynthesis protein FliP

+

+

Na /H antiporter

85

86


Nammi et al.

Table 6. contd….

S. No

Drug Target

Metabolic Category

Strain

Gene ID

HpF32

GI:317180534

12

Molybdopterin-guanine dinucleotide biosynthesis protein MobA Cofactors, Vitamins, Prosthetic Groups, Pigments

13

Heavy metal RND efflux outer membrane protein of CzcC family

Cobalt-zinc-cadmium resistance

HpSat464

GI:308064104

14

(3R)-hydroxymyristoyl-[acyl carrier protein] dehydratase

Fatty Acids, Lipids, and Isoprenoids

Hp2017

GI:325998241

Fatty Acids, Lipids, and Isoprenoids

Hp2018

GI:325996652

in 23 H. pylori strains (Tables 6-8). Proteome analysis followed by gene property analysis of the 9188 unique genes filtered by comparing H. pylori and Homo sapiens sapiens identified 14 potential drug targets (Table 6). These molecules fall under 11 metabolic categories as shown in Table 6. Proteome analysis followed by gene property analysis of the 4377 unique genes filtered by comparing H. pylori and other Helicobacter species H. acinonychis, H. hepaticus, H. mustalae identified 9 potential drug targets (Table 7). These molecules fall under 7 metabolic categories as shown in Table 7. Proteome analysis followed by gene property analysis of the 176 unique genes filtered by comparing 23 H. pylori strains identified 9 potential drug targets (Table 8). These molecules fall under 8 metabolic categories as shown in Table 8. Critical Drug Targets for H. pylori Nine of the 29 predicted drug targets are critical for survival of H. pylori. Analysis showed that Menaquinone via futalosine step 1; Soluble lytic murein transglycosylase precursor; Membrane metalloprotease; Heavy metal RND efflux outer membrane protein, CzcC family; 3-polyprenyl4-hydroxybenzoate carboxy-lyase UbiX; Peptidyl-prolyl cistrans isomerase ppiD; Hydrolase (HAD superfamily); Potassium channel protein; and YafQ toxin protein might be the critical drug targets for survival of Helicobacter species (Table 9). Novel Drug Targets for H. pylori Sixteen of the 29 predicted drug targets are novel targets for H. pylori. Analysis showed that Molybdopterin-guanine dinucleotide biosynthesis protein MobA; 3-polyprenyl-4hydroxybenzoate carboxy-lyase UbiX; Type III restrictionmodification system methylation subunit; Type I restrictionmodification system, specificity subunit S; Ferric siderophore transport system, biopolymer transport protein ExbB; Ribonuclease BN; Soluble lytic murein transglycosylase precursor; D-alanine--D-alanine ligase B; Membrane metalloprotease; Hydrolase (HAD superfamily); Inner membrane protein YihY formerly thought to be RNase BN; SSU ribosomal protein S14p (S29e) ## Zinc-dependent; rRNA small subunit 7-methylguanosine (m7G) methyltransferase GidB; Heavy metal RND efflux outer membrane protein; and CzcC family, Membrane fusion protein of RND family multidrug efflux pump are the novel drug targets for species (Table 9). Experimentally Validated Drug Targets for H. pylori Eleven of the 29 predicted drug targets are experimentally validated. Analysis showed that Menaquinone via futalosine step 1; Type III restriction-modification system methylation

subunit; Type I restriction-modification system, specificity subunit S; Dipeptide transport system permease protein DppB; Dipeptide transport system permease protein DppC; HoxN/HupN/NixA family nickel/cobalt transporter; Oligopeptide transport system permease protein OppC; FliP gene; Na+/H+ antiporter; Potassium channel protein; and DNA binding protein HU are the validated drug targets for H. pylori (Table 9). DISCUSSION In silico methods helped in identifying novel drug targets in addition to the existing H. pylori drug target's pool. Mining the genome of pathogen identified nearly 29 potential drug targets for H. pylori. These novel drug targets fall under the following categories of functions such as Cofactors, Vitamins, Prosthetic Groups, and Pigments; DNA Metabolism; Membrane Transport; RNA Metabolism; Cell Wall and Capsule; Motility and Chemotaxis; Miscellanous; Virulence; Respiration; Fatty acids, lipids and isoprenoids; Protein metabolism; Potassium metabolism; and Regulation and cell signalling. Drug Targets Influencing Cofactors, Vitamins, Prosthetic Groups, Pigments of the Pathogen Menaquinone via futalosine step 1; Molybdopteringuanine dinucleotide biosynthesis protein MobA; and 3-polyprenyl-4-hydroxybenzoate carboxy-lyase UbiX are the drug targets influencing cofactors, vitamins, prosthetic groups, pigments of the pathogen. Menaquinone via futalosine step 1 gene is identified as a drug target for H. pylori. Menaquinone is an important component of the electron transfer pathway. An alternative pathway is present in the human commensal intestinal bacteria H. pylori and Campylobacter jejuni. Disruption of Menaquinone via futalosine pathway had shown inhibition of bacteriostatic growth [69]. Therefore, designing an inhibitor for Menaquinone via futalosine step 1 would affect the growth of H. pylori. 3-polyprenyl-4-hydroxybenzoate carboxy-lyase UbiX gene is identified as a drug target for H. pylori. 3-polyprenyl4-hydroxybenzoate carboxy-lyase UbiX catalyzes biosynthesis of ubiquinone during logarithmic growth. ubi mutants accumulate hydroxybenzoates and show defect in both the aerobic and anaerobic ubiquinone biosynthesis pathways [70]. Ubiquinone deficiency produces pleiotropic phenotypes decreasing the rate of survival of H. pylori. Therefore, designing an inhibitor for 3-polyprenyl-4-hydroxybenzoate carboxy-lyase UbiX would affect the growth of H. pylori. Molybdopterin-guanine dinucleotide biosynthesis protein MobA is identified as a drug target for H. pylori. Modified


Table 7.

S. No 1

2


87

Drug targets identified in the 23 H. pylori strains by comparing genomes of H. pylori with species H. acinonychis, H. hepaticus, H. mustelae. Drug Target

Metabolic Category

Soluble lytic murein Cell Wall and Capsule transglycosylase precursor

Membrane metalloprotease

Clustering-based subsystems

Species Compared

Strain

Gene ID

H. hepaticus, H. Mustalae

Hp35A

GI:384896059

H. acinonychis, H. hepaticus, H. Mustalae

Hp51

GI:387782356


Hp52

GI:384887698


Hp908

GI:384891026


HpB8

GI:298736342


HpCuz20

GI:384892678


HpF16

GI:385217413

H. acinonychis, H. hepaticus, H. mustalae

HpF32

GI:385215941


HpF57

GI:385249163


HpG27

GI:208434567

H. acinonychis, H. hepaticus

HpAG1

GI:108563055


HpIndia7

GI:385220532

H. mustalae

HpJ99

GI:15611657


HpP12

GI:210134852

H. mustalae

HpB38

GI:254779368


Hp83

GI:385225346

H. mustalae

HpSJM180

GI:308184433


HpShi470

GI:188527507


Hp26695

GI:15645269

H. hepaticus

Hp2017

GI:385223699


HpF30

GI:384899049

H. hepaticus

Hp2018

GI:384899049

H. mustalae

HpSat464

GI:384894310


Hp51

GI:387782423

H. hepaticus, H. mustalae

Hp35A

GI:384896124


Hp52

GI:384887766


Hp908

GI:384890951


HpB8

GI:298736266


HpCuz20

GI:384892609


HpF16

GI:385217543


HpF32

GI:385215818


HpF57

GI:385249095


HpG27

GI:208434495


HpAG1

GI:108562979


HpIndia7

GI:385220641


HpSJM180

GI:308184358


HpShi470

GI:188527578

H. hepaticus

Hp2017

GI:385223625


HpF30

GI:384899117

H. hepaticus

Hp2018

GI:384899117

H. mustalae

HpSat464

GI:384894381

88


Nammi et al.

Table 7. contd….

S. No 3

4

Drug Target

Metabolic Category

3-polyprenyl-4Cofactors, Vitamins, hydroxybenzoate carboxy- Prosthetic Groups, lyase UbiX Pigments

Menaquinone via futalosine Cofactors, Vitamins, step 1 Prosthetic Groups, Pigments

Species Compared

Strain

Gene ID


Hp35A

GI:384895247


Hp51

GI:387782991

H. acinonychis, H. mustalae

Hp52

GI:384888322


Hp908

GI:384891822


HpB8

GI:298735943


HpCuz20

GI:384893443


HpF16

GI:385218132


HpF32

GI:385216630


HpF57

GI:385249886


HpG27

GI:208435344


HpAG1

GI:108563862


HpIndia7

GI:385221343


HpJ99

GI:15612434


HpSJM180

GI:308185245


HpShi470

GI:188528246

H. hepaticus

Hp2017

GI:384891822


HpF30

GI:384899709


Hp26695

GI:15646085

H. mustalae

HpSat464

GI:384894981


Hp35A

GI:384895940


Hp51

GI:387782231


Hp52

GI:384887575


Hp908

GI:384891160


HpB8

GI:298736482


HpCuz20

GI:384892802


HpF16

GI:385217341


HpF32

GI:385216014


HpF57

GI:385249295


HpG27

GI:208434690


HpAG1

GI:108563188


HpIndia7

GI:385220404


HpJ99

GI:15611782


HpSJM180

GI:308184563


HpShi470

GI:188527377


Hp26695

GI:15645397


HpF30

GI:384898918

H. mustalae

HpSat464

GI:384894182



89

Table 7. contd….

S. No

Drug Target

Metabolic Category

Species Compared

Strain

Gene ID

5

Peptidyl-prolyl cis-trans isomerase ppiD

Protein Metabolism


Hp35A

GI:384896300


Hp51

GI:387782586


Hp52

GI:384887930


Hp908

GI:384891356


HpB8

GI:298736064


HpCuz20

GI:384892993


HpF16

GI:385217719


HpF32

GI:385215652


HpF57

GI:385249481


HpG27

GI:208434248


HpAG1

GI:108563383


HpIndia7

GI:385220824


HpJ99

GI:15611978


HpSJM180

GI:308184755


HpShi470

GI:188527796


Hp26695

GI:15645592


HpF30

GI:384898730


HpSat464

GI:384894549

H. hepaticus

Hp2017

GI:385224032


Hp35A

GI:384896600


Hp51

GI:387782887


Hp52

GI:384888212


Hp908

GI:384891689


HpB8

GI:298735648


HpCuz20

GI:384893315


HpF16

GI:385218017


HpF32

GI:385216520


HpF57

GI:385249642


HpG27

GI:208435224


HpAG1

GI:108563697


HpIndia7

GI:385221202


HpJ99

GI:15612312


Hp12

GI:210135484


HpB38

GI:254779869


Hp83

GI:385225981


HpSJM180

GI:308185079


HpShi470

GI:188528115


Hp26695

GI:15645940


HpF30

GI:384898439


HpSat464

GI:384894850

H. hepaticus

Hp2017

GI:385232226

6

Heavy metal RND efflux outer membrane protein, CzcC family

Virulence, Disease and Defense

90


Nammi et al.

Table 7. contd….

S. No

Drug Target

Metabolic Category

Species Compared

Strain

Gene ID

7

Hydrolase (HAD superfamily)



HpP12

GI:210134963


HpB38

GI:254779264


Hp83

GI:385225291


Hp12

GI:210134693


HpB38

GI:254779488


Hp83

GI:385225571


HpP12

GI:210134650

8

9

Table 8.

Potassium channel protein

YafQ toxin protein

Potassium metabolism


Strain specific drug targets identified in the 23 H. pylori strains by comparing genomes of 23 H. pylori strains.

S.No.

Drug Target

Metabolic Category

Gene ID

Strain

1

D-alanine--D-alanine ligase B


GI:15645358

Hp26695

2

LSU ribosomal protein L36p

Protein Metabolism

GI:15645910

Hp26695

3

Membrane fusion protein of RND family multidrug efflux pump

Virulence, Disease and Defense

GI:15645231

Hp26695

4

Oligopeptide transport system permease protein OppC

Membrane Transport

GI:387781939

Hp35A

5

Inner membrane protein YihY, formerly thought to be RNase BN


GI:254779925

HpB38

6

DNA-binding protein HU ## epsilon-proteobacterial type

DNA Metabolism

GI:254779192

HpB38

7

SSU ribosomal protein S14p (S29e) ## Zinc-dependent

Miscellaneous

GI:108563676

HpAG1

8

rRNA small subunit 7-methylguanosine (m7G) methyltransferase GidB


GI:210134579

HpP12

9

YafQ toxin protein


GI:210134650

HpP12

nucleobases that mimic the normal DNA or RNA bases are traditionally referred to as base analogs. A particularly interesting group of mutagenic base analogs is comprised of N-hydroxylated derivatives of purines and pyrimidines. Examples of these agents include 6- N-hydroxylaminopurine (HAP), 2-amino-6-hydroxyaminopurine (AHAP) and N4hydroxycytidine. HAP is a highly effective mutagen for bacteria, yeast, and mammalian cells. Molybdopterin guanine dinucleotide is encoded by genes mobAB, moaABCDE, moeAB and mogA. MobA catalyzes the synthesis of molybdopterin guanine dinucleotide (MGD) from molybdopterin (MPT) and GTP. Inactivation of the mobA gene blocks the addition of GMP to MPT. Mutations either block the synthesis of precursor Z or MPT or activation and insertion of the molybdenum ion into the MPT leading to HAP-hypersensitivity. In E. coli and Salmonella, hypersensitivity to N-hydroxylated compounds, including hydroxylamine (HA), was found to be associated with a defect in the synthesis of the molybdenum cofactor (MoCo) [71]. If, H. pylori were not able to assimilate Mo then it would affect the growth of the organism. Thus, designing an effective inhibitor would affect the growth of the organism.

Drug Targets Influencing DNA Metabolism of the Pathogen Type III restriction-modification system methylation subunit, Type I restriction-modification system specificity subunit S and DNA binding protein HU are the drug targets influencing DNA metabolism of the pathogen. Type I restriction-modification system specificity subunit S and type III restriction-modification system methylation subunit of restriction–modification (R-M) systems are identified as drug targets for H. pylori. H. pylori are naturally competent and prone to take DNA from the environment [72]; and bacteriophages also infect H. pylori [73]. Missense and frameshift mutations can accumulate and inactivate genes when bacteriophages/free DNA/plasmids enter into other cells. Evidence is there that sometimes even both endonuclease and methylase genes of R-M systems have been turned off. However, H. pylori in a population have a very good defensive system, where R-M systems protect the genome of H. pylori from accumulated mutations when bacteriophages/free DNA/plasmids enter into cells. Mutant strains lacking this display a pleiotropic phenotype, including increased mutability, hyper recombination, and increased sensitivity to DNA-damaging agents. Therefore,


Table 9.


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Summary of the 29 drug targets identified in 23 H. pylori strains.

S.No.

Drug Target

Method 11

Method 22

1

Menaquinone via futalosine step 1

+

+

2

Type III restriction-modification system methylation subunit

+

+

3

Dipeptide transport system permease protein DppB

+

+

4

Dipeptide transport system permease protein DppC

+

+

5

Ferric siderophore transport system, biopolymer transport protein ExbB

+

+

6

Ribonuclease BN

+

+

7

Soluble lytic murein transglycosylase precursor

+

8

Type I restriction-modification system, specificity subunit S

+

+

9

HoxN/HupN/NixA family nickel/cobalt transporter

+

+

10

Flagellar biosynthesis protein FliP

+

+ +

+

+

Method 33

Validated4

Novel5

+

+

+

11

Na /H antiporter

+

12

Molybdopterin-guanine dinucleotide biosynthesis protein MobA

+

13

Heavy metal RND efflux outer membrane protein, CzcC family

+

14

NADH-ubiquinone oxidoreductase chain J

+

+

15

(3R)-hydroxymyristoyl-[acyl carrier protein] dehydratase

+

+

16

Membrane metalloprotease

+

17

3-polyprenyl-4-hydroxybenzoate carboxy-lyase UbiX

+

+

18

Peptidyl-prolyl cis-trans isomerase ppiD

+

+

19

Hydrolase (HAD superfamily

+

+

20

Potassium channel protein

+

21

YafQ toxin protein

+

22

+ +

+

+

+ +

+

D-alanine--D-alanine ligase B

+

+

23

LSU ribosomal protein L36p

+

+

24

Membrane fusion protein of RND family multidrug efflux pump

+

+

25

Oligopeptide transport system permease protein OppC

+

26

Inner membrane protein YihY, formerly thought to be RNase BN

+

27

DNA-binding protein HU ## epsilon-proteobacterial type

+

28

SSU ribosomal protein S14p (S29e) ## Zinc-dependent

+

+

29

rRNA small subunit 7-methylguanosine (m7G) methyltransferase GidB

+

+

1

+ + +

2

Drug targets identified in the 16 H. pylori strains by comparing genomes of pathogen H. pylori and Host Homo sapiens sapiens. Drug targets identified in the 23 H. pylori strains by comparing genomes of pathogen H. pylori with species H. acinonychis, H. hepaticus, H. mustelae. 3Strain specific drug targets identified in the 23 H. pylori strains by comparing genomes of 23 different H. pylori strains. 4Experimentally validated drug targets either by genetically or biochemically. 5Novel drug targets identified in this study.

designing an inhibitor for Type I restriction-modification system specificity subunit S and type III restrictionmodification system methylation subunit decreases the rate of survival of H. pylori due to gross changes occurring in the genetic material. DNA binding protein HU was identified as a drug target for H. pylori. The major function of the bacterial cell envelope, besides protecting the cell from the environment,

is to permit and control the exchange and communication between the environment in which the bacteria lives and the interior of the cell. It was observed that the cells lacking the protein HU were abnormally sensitive to common features of H. pylori like acid and oxidative stress. Alterations in composition of H. pylori outer membrane was demonstrated when histone like protein HU is not expressed [74]. Non colonization of the hup mutant strain in a mouse infection

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model under oxidative stress and colonization of the strain when hup mutant strain was complemented with the functional hup gene in the presence of oxidative stress, is the evidence for the role of Histone like DNA binding protein HU. Therefore, blocking Histone like DNA binding protein HU might make organism susceptible to oxidative and acid stress. Drug Targets Influencing Membrane Transport of the Pathogen Dipeptide transport system permease protein DppB; Dipeptide transport system permease protein DppC; Ferric siderophore transport system biopolymer transport protein ExbB; HoxN/HupN/NixA family nickel/cobalt transporter; and Oligopeptide transport system permease protein OppC are the drug targets influencing membrane transport of the pathogen. Dipeptide transport system permease protein DppBC and oligopeptide transport system permease protein OppC are identified as drug targets for H. pylori. Dipeptide DppABCDF and oligopeptide oppABCD genes belong to a class of ABCtype transporter. Dipeptide transport system permease protein DppBC and oligopeptide transport system permease protein OppC are responsible for transporting dipeptides and oligopeptides respectively. Dipeptide and oligopeptide system mutants in H. pylori lacked the ability to use certain dipeptides, hexapeptides, and nonapeptides due to compromisation of either substrate binding domain or permease domains [75]. Therefore, designing an inhibitor to dipeptide transport system permease protein DppBC and oligopeptide transport system permease protein OppC affect the growth and survival of H. pylori. Ferric siderophore transport system biopolymer transport protein ExbB is identified as a drug target for H. pylori. All bacterial pathogens have developed highly sophisticated iron assimilation systems in response to iron-limiting conditions encountered in environment and host’s body fluids. Production of siderophores, small nonproteinaceous molecules with extremely high affinity for iron (III), is one of the most successful and widely utilized strategies for iron assimilation [76]. Common components of both siderophore-dependent and host iron-binding protein-dependent iron acquisition systems are receptor proteins involved in binding of siderophores and interacting with the host iron-binding proteins. These large outer membrane proteins are responsible for the transport of iron or iron-containing compounds through the impermeable outer membrane [77]. Ferric siderophore transport system ton B periplasmic binding protein; Ferric siderophore transport system Exb B biopolymer transport protein; and Haemin uptake system ATP binding protein in H. pylori are responsible for the transport of iron or iron-containing compounds through the impermeable outer membrane. Sequence analysis in E. coli, Haemophilus influenzae, Neisseria meningitides and Pseudomonas putida provided information on existence of mechansim that utilizes Ton-independent heme. Knockout mutant and complementation studies in Neisseria meningitides established this fact [78]. Designing an effective inhibitor to the existing multiple proteins for the utilization of hemecontaining compound's effects the survival of H. pylori in their natural habitat, human mucosal surfaces.

Nammi et al.

HoxN/HupN/NixA family nickel/cobalt transporter gene is identified as a drug target for H. pylori. Nickel/cobalt transporters are required for import of Ni (II) and Co (II) through nickel-cobalt transporters into bacterial cell. The incorporation of nickel into the metal center of nickeldependent enzymes like ureases and its expression is essential for H. pylori colonization. Therefore, nickelcontaining enzyme urease is an essential colonization and pathogenesis factor of the gastric pathogen H. pylori, as it allows the bacterium to survive the acidic conditions in the gastric mucosa. Mutagenesis of nixA in H. pylori results in reduced nickel transport and urease activity [79]. Therefore, inactivation of HoxN/HupN/NixA family nickel/cobalt transporter gene in H. pylori reduces nickel transport, urease activity effecting colonization of organisms. Drug Targets Influencing RNA Metabolism of the Pathogen Ribonuclease BN is identified as a drug target for H. pylori. Ribonuclease, BN, lacking RNase H and RNase D activity was identified in E. coli and it is different from other exoribonucleases known till date in E. coli. RNase BN is a substrate specific with specificity towards C-C-A sequence in tRNA than other types of tRNA and substrate specificity was proved both in vitro and in vivo. Mutants of these proteins affect the processing of t RNA’s and ultimately synthesis of protein [80]. Therefore, an effective inhibitor for Ribonuclease BN can block the function of protein synthesis. Drug Targets Influencing Cell Wall and Capsule of the Pathogen Soluble lytic murein transglycosylase precursor and Dalanine--D-alanine ligase B are the drug targets influencing cell wall and capsule of the pathogen. Soluble lytic murein transglycosylase precursor is identified as drug target for H. pylori. Murein (peptidoglycan) hydrolases are essential for growth of bacteria. The cell is surrounded by murein an exoskeleton of the bacterial murein sacculus. Sacculus provides mechanical stability to bacteria, by cross-linking polymer murein to reinforce cell wall, and in addition maintains cell shape. The growth of bacteria is dependent on murein sacculus enlargement and division. Murein sacculus bag-shaped structure that completely surrounds the bacterium is needed for two processes during growth: firstly, for enlargement of the murein net and, secondly, for the splitting of the septum to allow separation of the daughter cells, i.e., murein-degrading enzyme may play a role in recycling of muropeptides during cell elongation and/or cell division. Lytic murein transglycosylase mutants’ analysis demonstrated alterations in murein sacculus structure indicating the role of lytic transglycosylases in murein sacculus maturation. Murein synthesis inhibitors, irrespective of their specific site of action, cause bacteriolysis [81]. Therefore, an effective inhibitor for soluble lytic murein transglycosylase precursor can block the function of murein recycling leading lysis of H. pylori cells. D-alanine--D-alanine ligase B is identified as drug target for H. pylori. Peptidoglycan, the backbone of this structure, contains the D-amino acids D-alanine, D-glutamate, and diaminopimelate, which may contribute to its stability


against proteolytic degradation. D-Alanine is one of the central molecules of the cross-linking step of peptidoglycan assembly. There are three enzymes involved in the D-alanine branch of peptidoglycan biosynthesis: the pyridoxal phosphate-dependent D-alanine racemase (Alr), the ATPdependent D-alanine:D-alanine ligase (Ddl), and the ATPdependent D-alanine:D-alanine-adding enzyme (MurF). Inhibiting enzymes involved in the D-Ala pathway of peptidoglycan biosynthesis inhibits cross-linking leading to extensively weaker cell walls and cell death [82]. Therefore, enzymes involved in the D-Ala pathway of peptidoglycan biosynthesis are attractive targets for novel antibiotic design. Quercetin is the lead molecule possessing antibacterial activity against Ddl (HpDdl) enzyme of H. pylori. The molecular docking analysis demonstrated that quercetin and HpDdl showed better interaction, affinity and absorption [83]. Drug Targets Influencing Motility and Chemotaxis of the Pathogen Flagellar biosynthesis protein FliP was identified as a drug target for H. pylori. Motility-associated gene flip codes for a flagellar basal body component involved in flagellar assembly and its regulation to colonize gastric mucosa. Inactivation of fliP genes shuts down assembly of flagellar assembly followed by motility loss in H. pylori. Mutants of nonmotile H. pylori did not colonize animal models. So, loss of flagellar synthesis reduces motility function, thereby affecting colonization and thus the survival rate of organism [84]. Therefore, inactivation of flagellar biosynthesis protein FliP gene in H. pylori reduces motility and effect colonization of organisms. Drug Targets Influencing Miscellaneous Metabolism of the Pathogen Na+/H+ antiporter; Membrane metalloprotease; Hydrolase (HAD superfamily); Inner membrane protein YihY formerly thought to be RNase BN; SSU ribosomal protein S14p (S29e) ## Zinc-dependent; and rRNA small subunit 7-methylguanosine (m7G) methyltransferase GidB are the drug targets influencing miscellaneous metabolism of the pathogen. Na+/H+ antiporter was identified as a drug target for H. pylori. Na+/H+ antiporters are integral membrane proteins present virtually in all cell types from bacteria to higher eukaryotes. In bacteria, Na+/H+ antiporters function in the opposite direction, using the proton motive force to extrude Na or Li when present at toxic concentrations in the cytoplasm or to maintain a lower cytoplasmic pH in an alkaline environment [85]. Defective HpNhaA mutants when grown in alkaline pH range showed severe growth retardation due to the confirmational changes demonstrated by lower antiporter activity when compared to the wild type. Complementation of the HpNhaA mutants reestablished antiporter activity. Thus, if Na+/H+ antiporter is inactivated, then it can act as a suitable drug target for H. pylori [86]. Metalloprotease was identified as a drug target for H. pylori. H. pylori produce a metalloprotease of approximately 200 kDa, associated with the outer membrane fraction of the bacterium. The surface expression of this metalloprotease activity raises the possibility that this enzyme may be


93

involved in the proteolysis of a variety of host proteins in vivo and thereby contributes to gastric pathology. It has been shown, that protease-deficient mutants are less virulent than wildtype strains [87]. Therefore, an effective inhibitor for membrane metalloprotease can affect survival of H. pylori. The haloacid dehalogenase (HAD) superfamily includes a variety of enzymes that catalyze the cleavage of substrate C-Cl, P-C, and P-OP bonds via nucleophilic substitution pathways. All members possess the alpha/beta core domain, and many also possess a small cap domain. The active site of the core domain is formed by four loops (corresponding to sequence motifs 1-4). Position substrate, cofactor-binding residues as well as the catalytic groups mediate the "core" chemistry. The cap domain is responsible for the diversification of chemistry within the family. A tight betaturn in the helix-loop-helix motif of the cap domain contains a stringently conserved Gly (within sequence motif 5), flanked by residues whose side chains contribute to the catalytic site formed at the domain-domain interface. To define the role of the conserved Gly in the structure and function of the cap domain loop of the HAD superfamily members phosphonoacetaldehyde hydrolase and betaphosphoglucomutase, the Gly was mutated to Pro, Val, or Ala. The catalytic activity was severely reduced in each mutant [88]. Therefore, an effective inhibitor for haloacid dehalogenase (HAD) superfamily can block the function of many important molecules which are important for survival of H. pylori. Inner membrane protein YihY formerly thought to be RNase BN now known as elaC gene and renamed as rbn is identified as a drug target for H. pylori. In eukaryotes, archaea and in some eubacteria removal of 3' precursor sequences during maturation of tRNA is catalyzed by an endoribonuclease, termed RNase Z. In contrast, in Escherichia coli, a variety of exoribonucleases carry out final 3' maturation. Yet, E. coli retains an RNase Z homologue, ElaC, displaying structural and catalytic properties identical to those ascribed to RNase BN. The properties of RNase BN are off exoribonucleases against tRNA substrates containing nucleotide substitutions within the -C-C-A sequence and relatively inactive against other types of RNAs. This substrate specificity in in vitro is consistent with a processing function in in vivo. Mutants of these proteins would affect the processing of transfer RNA’s and ultimately protein synthesis [89]. Therefore, an effective inhibitor for Ribonuclease BN can block the function of protein synthesis. SSU ribosomal protein S14p (S29e) ## Zinc-dependent is identified as a drug target for H. pylori. S14 is one of the proteins from the small ribosomal subunit. In E. coli, S14 is required for the assembly of 30S particles and also responsible for determining the conformation of 16S rRNA at the A site. It belongs to a family of ribosomal proteins that include bacteria, algae, etc. Mutants of these proteins would affect the association of ribosomal units, and ultimately protein synthesis [90]. Therefore, an effective inhibitor for SSU ribosomal protein S14p can block the function of protein synthesis. rRNA small subunit 7-methylguanosine (m7G) methyltransferase GidB is identified as a drug target for H.

94


pylori. GidB gene encoding a conserved 7-methylguanosine (m (7) G) methyltransferase is specific for G527 located in 530 loop of the 16S rRNA. 7-methylguanosine (m7G) methyltransferase (gidB) exhibits glucose-inhibited division (GidB) revealing a methyltransferase fold. Mutations in the gene encoding a 7-methylguanosine (m7G) methyltransferase (gidB) results in the loss of a conserved 7-methylguanosine modification in 16S rRNA conferring low-level streptomycin (STR) resistance in Mycobacterium tuberculosis [91]. Therefore, inhibitor for 7-methylguanosine (m7G) methyltransferase (gidB) blocks the 7-methylguanosine modification in 16S rRNA making it more susceptible to antibiotic streptomycin (STR). Drug Targets Influencing Virulence of the Pathogen Heavy metal RND efflux outer membrane protein of CzcC family and membrane fusion protein of RND family multidrug efflux pump are the drug targets influencing virulence of the pathogen. Heavy metal RND efflux outer membrane protein of CzcC family is identified as a drug target for H. pylori. Cobalt, Zinc and Cadmium are required in trace amounts for bacterial growth. Homeostasis of Cobalt, Zinc and Cadmium are tightly regulated, because they are toxic when present in excess. Excess of Cobalt, Zinc and Cadmium levels when detected by the pathogen induce the genes such as Heavy metal RND efflux outer membrane protein of CzcC. Active efflux of metal ions is controlled by the gene contributing to Co/Zn/Cd resistance [92]. Interactions between Cobalt, Zinc and Cadmium resistance proteins was revealed and established in the H. pylori. Knockout mutant studies of CzcA and CzcB suggested a metal efflux pump operating in H. pylori and were not able to colonize the stomach. Blocking the function of the gene would lead to excess flow of trace elements across the membrane into pathogen thus becoming toxic and finally killing the pathogen H. pylori. Therefore, inactivation of Heavy metal RND efflux outer membrane protein, CzcC, renders the bacteria hypersusceptible to the excess flow of trace elements killing H. pylori. Membrane fusion protein of RND family multidrug efflux pump is the drug target identified for H. pylori. The multiple drug resistance (MDR) phenotype is often associated in bacteria with efflux pumps in the cytoplasmic membrane. Collectively, these proteins belong to five families of transporters that include the ATP-binding cassette (ABC), major facilitator superfamilies (MFS), multidrug and toxic compound extrusion (MATE), resistance nodulation division (RND) and the small multidrug resistance (SMR) families [93]. A remarkable feature in some of these systems is the wide range of substrates that are recognized by a single pump protein. The role of RestrictionNodulation-Division (RND) multiple drug efflux systems has been established in several pathogens like E. coli, H. influenzae, N. gonorrhoeae, P. aeruginosa and H. pylori. RND efflux systems are widespread among gram-negative bacteria. H. pylori contain three putative RestrictionNodulation-Division (RND) efflux systems, named hefABC, hefDEF, and hefGHI. The RND family efflux systems are dependent on proton motive force. Such systems are responsible for resistance to a wide variety of structurally

Nammi et al.

and chemically unrelated antibiotics and other antimicrobial compounds. Upregulation of these systems leads to multiple antibiotic resistance, whereas inactivation of RND systems renders the bacteria hypersusceptible to a wide variety of antimicrobials [94]. Therefore, inactivation of membrane fusion protein of RND family multidrug efflux pump renders the bacteria hypersusceptible to antimicrobials effecting survival of organism [94]. Drug Targets Influencing Respiration of the Pathogen NADH-ubiquinone oxidoreductase chain J is identified as a drug target for H. pylori. The NADH:ubiquinone oxidoreductase (Complex I), provides the input to the respiratory chain from the NAD-linked dehydrogenases of the citric acid cycle. The complex couples the oxidation of NADH and the reduction of ubiquinone, to the generation of a proton gradient which is then used for ATP synthesis. The complex occurs in the mitochondria of eukaryotes, plasma membranes of purple photosynthetic bacteria and the closely related respiratory bacteria. Inhibitors affect the electrontransfer step from the high-potential iron-sulphur cluster to ubiquinone acting directly at the ubiquinone-catalytic site which is related in complex I and glucose dehydrogenase [95]. Inhibitors designed to bind to NADH-ubiquinone oxidoreductase chain J competitively inhibit the protein from functioning which results in chemical asphyxiation of cells. Drug Targets Influencing Fatty Acids, Lipids and Isoprenoids of the Pathogen (3R)-hydroxymyristoyl-[acyl carrier protein] dehydratase is identified as a drug target for H. pylori. (3R)hydroxymyristoyl-[acyl carrier protein] dehydratase belongs to the thioester dehydratase family, FabZ subfamily involving in unsaturated fatty acid biosynthesis. This enzyme catalyzes the dehydration of both short and long chain betahydroxyacyl-ACPs. There is a relation to fabA gene and suppression of mutations in lipid A biosynthesis. Mutations in E. coli (3R)-hydroxymyristoyl-[acyl carrier protein] dehydratase effect biosynthesis and growth [96]. Therefore, an effective inhibitor for (3R)-hydroxymyristoyl-[acyl carrier protein] dehydratase loses the ability to suppress cell division/lipid A biosynthesis gene, envA and thus effect the survival of organism. Drug Targets Influencing Protein Metabolism of the Pathogen Peptidyl-prolyl cis-trans isomerase ppiD and LSU ribosomal protein L36p are the drug targets influencing the protein metabolism of the pathogen. Peptidyl-prolyl cis-trans isomerase ppiD was identified as a drug target for H. pylori. Periplasmic peptidyl-prolyl isomerases play an important role in the starvation-stress response (SSR) of Salmonella enterica serovar Typhimurium by functioning in folding of cell envelope proteins. Peptidyl-prolyl cis-trans isomerase ppiD encode a peptidyl-prolyl isomerase that catalyze the rate-limiting cis-trans isomerization of Xaa-Pro peptide bonds, and are important in the proper folding of outer membrane proteins located in the cell envelope. Bacteria can resist starved cells to high temperature, acidic pH, and the antimicrobial peptide. In conclusion, the presence of these


periplasmic PPIases contributes to the survival, and possibly recovery, of Salmonella cells exposed to nutrient-poor conditions and other types of environmental stress. Knockout mutants and disruption studies demonstrated lack of resistance to thermal, acid, and antimicrobial peptide stress [97]. Therefore, an effective inhibitor for Peptidyl-prolyl cistrans isomerase ppiD can block the function of proper folding of outer membrane proteins and thus the survival of the organism in high temperature and acidic pH. LSU ribosomal protein L36p of H. pylori which is essential for protein biosynthesis is identified as for drug target. Therefore, blocking the protein would become a target for therapeutic intervention. The 50 S ribosome subunit consists of 35 different molecules: two rRNA's and 33 proteins. Stable association of RNA and proteins is based on cross linking of proteins, interactions in ribosomes and RNAs and also the association of the ribosome. Mutants of these proteins affect the association of ribosomal units and ultimately protein synthesis [90]. Therefore, an effective inhibitor for LSU ribosomal protein L 36p can block the function of protein synthesis. Drug Targets Influencing Potassium Metabolism of the Pathogen Potassium channel protein is identified as a drug target for H. pylori. H. pylori contains a two-transmembrane RCK (regulation of K+ conductance) gene coding for a putative K + channel (HpKchA) which is essential for colonization of the murine stomach. Mutant ΔhpKchA of H. pylori demonstrated a sturdy growth defect, when compared to wild type at low K+ concentration. When compensated with additional KCl, ΔhpKchA proved the biological function of K+ channel, as a K+ uptake system. Further, it was proved that colonization of the gastric murine mucosa is dependent on HpKchA [98]. Therefore, an effective inhibitor for HpKchA can block the K+ uptake effecting colonization and subsequent survival of H. pylori. Drug Targets Influencing Regulation and Cell Signalling of the Pathogen YafQ toxin protein is identified as a drug target for H. pylori. Toxin–Antitoxin (TA) systems (suicide modules) are identified on chromosomes and plasmids of bacteria to induce post-segregational killing. During stress TA toxins target replication and translation to inhibit cell growth, which is reversible. When stress prolongs cell death occurs. TA antitoxin prevents toxin activity during environmental stress [99]. Locus dinJ–yafQ, was identified in H. pylori. YafQ (endoribonuclease) cleaves mRNA at a particular frame and sequence, blocking elongation of translation inducing reversible cell growth arrest. This temporary arrest of growth is vital to repair DNA. Coexpression or coinduction of dinJ–yafQ genes in a module or independently restored normal growth, confirming that DinJ antitoxin is the cognate of toxin YafQ [100]. Therefore, an effective inhibitor of YafQ toxin blocks the short-term growth arrest that is essential to repair DNA affecting the rate of survival of H. pylori due to gross changes in the genetic material.


95

CONCLUSION Comparative genomics of H. pylori and Homosapien sapiens; H. pylori and other Helicobacter species (H. acinonychis, H. hepaticus, H. mustalae); and 23 H. pylori strains identified 29 bacterial genes which are nonhomologous to humans and are essential for pathogen. Nine of the 29 drug targets were found to be critical for the species and fifteen were found to be important for survival of species in the host. Eleven of the 29 predicted drug targets are already experimentally validated lending credence to our approach. Soluble lytic murein transglycosylase precursor, 3-polyprenyl-4-hydroxybenzoate carboxy-lyase UbiX, Hydrolase (HAD superfamily), rRNA small subunit 7-methylguanosine(m7G)methyltransferase GidB and Peptidyl-prolyl cis-trans isomerase ppiD are the novel drug targets for species. These novel drug targets may have possible therapeutic implications for gastric cancer. CONFLICT OF INTEREST The authors confirm that this article content has no conflict of interest. ACKNOWLEDGEMENTS DN, STPRCPK and NNRR are thankful to the GITAM University, Visakhapatnam, India for providing the facility and support. NNRR and DN are thankful to University Grants Commission, New Delhi for the project funding [UGC Project F.No.42-636/2013 (SR) letter dated 25-032013]. DN is thankful for the Project Fellowship sponsored by UGC, New Delhi, India. The authors also thankful to Prof. I. Bhaskar Reddy and Dr Malla Rama Rao for constant support throughout the research work. We profusely thank Dr Ch. Surekha, GITAM University, Visakhapatnam, India for critical comments and reviewing of the manuscript. REFERENCES [1] [2]

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Accepted: May 29, 2015