HELICOBACTER PYLORI INFECTION: A BIOINFORMATIC APPROACH

HELICOBACTER PYLORI INFECTION: A BIOINFORMATIC APPROACH

Ashwini Prasad ¹, Govindaraju Shruthi ¹, P. Sushma ¹, Anisha S. Jain ¹, D. Chandan ¹, M. N. Nagendra Prasad ², Shiva Prasad Kollur ³, Chandrashekar Srinivasa ⁴ and Chandan Shivamallu ^{* 1}

Department of Water & Health ¹, Faculty of Life Sciences, JSS Academy of Higher Education & Research, Sri Shivarathreeshwara Nagara, Mysore - 570015, Karnataka, India.

Department of Biotechnology ², JSS Science and Technology University, Sri Jayachamarajendra College of Engineering, Mysore - 570015, Karnataka, India.

School of Arts and Sciences, Amrita Vishwa Vidyapeetham, Mysuru Campus - 570026, Karnataka, India.

Department of Biotechnology ⁴, Davangere University, Davanagere - 577007, Karnataka, India.

ABSTRACT: Helicobacter pylori causative agent of acid peptic disease is a microaerophilic, spiral-shaped, gram-negative bacteria found in the gastric epithelium that may also lead to complications such as chronic gastritis, mucosa-associated lymphoid tissue (MALT) lymphoma, gastroduodenal ulcer and adenocarcinoma in stomach. Plantbioactives have always been potential therapeutics. Usage of modern bioinformatic tools plays a vital role in exploiting the potentials of alternative therapeutic molecules in managing diseases like peptic ulcers and their complications. The in-silico evaluation was carried out using molecular docking of the quorum sensing proteins of H. pylori with the ligand β-sitosterol obtained from the silver nanoparticles of Acorus calamus L. Among several given quorum sensing proteins the molecular interaction with the ligand β-sitosterol showed a high binding affinity with DnaA, PhnB, ToxB and Sip proteins. The results obtained from molecular interaction study revealed that the ligand β-sitosterol will be readily taken up by the organism, thereby facilitating easy inhibition or inactivation of quorum sensing molecules ToxB, DnaA, PhnB, and Sip, making it a novel therapeutic alternative to treat H. pylori infections.

In-silico Analysis, Molecular Docking, Molecular Interaction, Helicobacter pylori 

INTRODUCTION: The acid peptic disease is a condition due to frequent pathogenic actions involving excessive secretion of acid resulting in acid reflux, thereby damaging esophageal mucosa and laryngeal tissue. Further peptic ulcer condition is complicated by secretion of pepsin and gastric acid that damages mucosa and causes ulcers in the lower esophagus and duodenum region.

This acidity related disease hampers the lifestyle quality of individuals leading to rampant morbidity and mortality. Even in a developed country like the US, 40% of youth frequently complain of Heartburn, highlighting gastroesophageal reflux disorder (GERD) as one of the most common disorders ¹.

Presently, there are 3 lines of antibiotic therapies for the treatment of H. pylori-related complications viz., Concomitant & hybrid therapy, Bismuth quadruple & Levofloxin therapy, and Culture guided therapy ². The antibiotics of choice for treating H. pylori infections are metronidazole, tetracycline, clarithromycin, amoxicillin, fluoro-quinolones, tinidazole along with bismuth salts or proton pump inhibitors (PPIs). Resistance to particular antibiotics has made to use the combination of drugs in areas where there is more prevalence of resistance to a single antibiotic ³.

H. pyloriis an S-shaped, gram-negative bacteria with sheathed and polar flagella, which grow in microaerophilic conditions and is varied by its turns/spirals and size ⁴. H. pylori are most likely to spread from person to person through oral-oral, gastro-oral, or fecal-oral route. Other sources of transmission recorded in the literature also include contaminated water, iatrogenic modes, especially during endoscopy and rarely zoonotic transmission, too, though direct contact with the individuals remains the most prevalent spreading method ⁵. One of the most common bacteria that infect the world’s 50% of the population leading to peptic ulcer and adenocarcinoma is H. pylori ⁶. Acquisition of H. pylori infection is controlled by the persistence of infection and also infection rate loss ⁷.

Plant mediated synthesis of nanobactericides is a protocol which exploits plant or their products to synthesize the desired class of nanobactericides ⁸. In a plant-mediated synthesis of nanobactericides, there are two different types of extracellular synthesis, which involves the extraction of plant products in the form of aqueous extract and using it to synthesize nanobactericides ⁹. Hence the present study makes a primary attempt to prosper the fact to develop novel nanobactericides against the treatment of H. pylori biofilm formation. The developed nanobactericides are synthesized using a facile route by exploring the herbal plants, which are reported to have a high therapeutic index. One of the advantages of developing plant-based nanobactericides is that it may protect the host immune system and can minimize the risks of drug resistance to prevent latent infections. As the pathogens can inhabit various host niches and the conventional medicines often fail or damage the host self-defense, which may lead to a compromised immune system. Hence, the use of nanobactericides can be highly advantageous.

Quorum sensing in H. pylori is an evident phenomenon. Still, it lacks in understanding the detailed mechanism of various proteins involved in quorum sensing and requires in-depth and vast research in the field. Currently, only a few quorum sensing genes have been annotated, which includes LuxS and ToxB. These are the only two quorum sensing proteins which have been structurally annotated and the sequence of which is currently available in Protein Data Bank and GenBank.

The other known quorum sensing proteins Table 1 requires structural annotation and elucidation of the mechanism of action, which has been carried out in the current study through in-silico annotation and analysis using structural elucidation and molecular interaction studies and is the main focus of the current research work.

TABLE 1: QUORUM SENSING PROTEINS OF H. PYLORI

*Note: Proteins obtained from cross-reference of KEGG and GenBank.

LuxS is a well-known quorum sensing molecule involved in the production of autoinducer II, which is found to be involved in the formation of lesions, which results in the easy access of the pathogen into the host cell. Thereby further spreading the infection to the nearby cells and tissues, leading to host tissue necrosis. The inhibition of LuxS prevents the ability of the organism to invade host tissue/cells through cytolysis ¹⁰.

ToxB, GTP cyclohydrolase is an essential protein involved in the acclimatization of H. pylori in acidic conditions. This protein prevents the degradation of the bacterial cell wall in the host intestine and helps in the growth and homeostasis of the bacteria. The particular protein lays the foundation for the establishment of biofilm in the host, thereby directly influencing the propagation of infection and invasion into the host cell ¹¹.

Quorum sensing pathway of H. pylori has been annotated using in-silico tools such as KEGG (Kyoto Encyclopedia for Genes and Genomics) wherein, LuxS, DnaA, PhnA, PhnB, FlaB, ToxB and Sip were found to be the key quorum sensing molecule involved in biofilm formation in H. pylori Fig. 1.

FIG. 1: EXHIBITING THE QUOROM SENSING OF H. PYLORI INVOLVING QUORUM SENSING MOLECULES LUXS, PHNA, PHNB, FLAB, TOXB AND SIP OF H. PYLORI (IN GREEN COLOR). (IMAGE COURTESY- KEGG PATHWAY DATABASE: HPY02024)

MATERIALS AND METHODS:

Structural Annotation of Protein of H. pylori: Structure of LuxS and ToxB proteins were procured from Protein Data Bank (PDB) and were found to be fit for further in-silico analysis without the requirement of any further modifications. The structures of DnaA, PhnA, PhnB, FliC, and Sip were unannotated and required ab-initio structure building. The sequences of these five proteins were obtained from the GenBank database of H. pylori proteome and were subject to structure prediction using the Raptor-X tool ¹¹.

Binding Site Prediction of Proteins: The binding sites of LuxS and ToxB proteins were analyzed and obtained through the ligand explorer tool. The amino acid residues of the binding sites of structurally annotated proteins DnaA, PhnA, PhnB, FliC, and Sip were predicted using B Spread tool of Yang Zhang Lab ^{12, 14}.

Ligand Preparation: The 3-dimensional structure of the ligand β-sitosterol was prepared in two steps, in the first step; the 2-dimensional structure of β-sitosterol was drawn in Chemdraw (v.8.0) software and was saved as .cdx file. In the second step, 2 dimensional structure of β-sitosterol was converted to a 3-dimensional structure by the addition of 3D coordinates to the structure and was made explicit, using Openbabel software ¹⁵.

Molecular Docking: The molecular interaction studies of the prepared ligand with all the seven proteins were performed using rigid docking studies using Autodock suite (v.4.2.6) using genetic algorithm setting to check the interaction of β-sitosterol with the proteins where the grid was set for the binding site, and the protein macromolecule was set as a rigid molecule, and the various possible confirmations of ligand were generated¹⁶.

Visualization: Upon completion of molecular docking, the various confirmations of a ligand in association to respective proteins were visualized to analyze various interactions of ligands with the binding site residues of the protein using UCSF Chimera visualization tool, based on the type of interaction, a number of interactions and docking score, the best-fit orientation of the ligand was selected ¹⁶.

RESULTS AND DISCUSSION:

In-silico Analysis:

Structural Annotation of Proteins of H. pylori: The structure of LuxS and ToxB obtained from Protein Data Bank did not require further refinement other than removal of the bound ligand, structures of LuxS and ToxB devoid of a ligand is represented in Fig. 2 and Fig. 4 respectively. The structures of DnaA, PhnA, PhnB, FliC, and Sip obtained from RaptorX were validated through a Ramachandran plot. The validated structures which obeyed structure-activity relationship were selected and the structures of DnaA, PhnA, PhnB, FliC, and Sip are represented in Fig. 9 - Fig. 13 respectively.

FIG. 2: A: STRUCTURE OF LUXS (PDB ID:1J6X) DEVOID OF COFACTOR ZN, REPRESENTED IN RIBBON PRESET; B: STRUCTURE OF TOXB (PDB ID:4RL4) DEVOID OF LIGAND PPV, REPRESENTED IN RIBBON PRESET; C: STRUCTURE OF DNAA REPRESENTED IN RIBBON PRESET; D: STRUCTURE OF PHNA REPRESENTED IN RIBBON PRESET

FIG. 3: A: STRUCTURE OF PHNB REPRESENTED IN RIBBON PRESET; B: STRUCTURE OF FLIC REPRESENTED IN RIBBON PRESET; C: STRUCTURE OF SIP REPRESENTED IN RIBBON PRESET

Validation of Protein Structures: The structures of DnaA, PhnA, PhnB, FliC, and Sip procured from RaptorX were validated through Ramachandran plot analysis using Phenix (v.1.13) and are depicted in Fig. 4 - Fig. 8. Only structures which had at least 95 percent of the residues in the favorable region and around 2 percent of the residues in the allowed regions were considered to be fit for molecular interaction studies.

FIG. 4: RAMACHANDRAN PLOT PREDICTION FOR STRUCTURE OF DNAA; A: GENERAL PLOT; B: GLYCINE RESIDUES OF DNAA STRUCTURE; C: PROLINE RESIDUES OF DNAA STRUCTURE; D: PRE-PROLINE RESIDUES OF DNAA STRUCTURE

FIG. 5: RAMACHANDRAN PLOT PREDICTION FOR STRUCTURE OF PHNA; A: GENERAL PLOT; B: GLYCINE RESIDUES OF PHNA STRUCTURE; C: PROLINE RESIDUES OF PHNA STRUCTURE; D: PRE-PROLINE RESIDUES OF PHNA STRUCTURE

FIG. 6: RAMACHANDRAN PLOT PREDICTION FOR STRUCTURE OF PHNB; A: GENERAL PLOT; B: GLYCINE RESIDUES OF PHNB STRUCTURE; C: PROLINE RESIDUES OF PHNB STRUCTURE; D: PRE-PROLINE RESIDUES OF PHNB STRUCTURE

FIG. 7: RAMACHANDRAN PLOT PREDICTION FOR STRUCTURE OF FLIC; A: GENERAL PLOT; B: GLYCINE RESIDUES OF FLIC STRUCTURE; C: PROLINE RESIDUES OF FLIC STRUCTURE; D: PRE-PROLINE RESIDUES OF FLIC STRUCTURE

FIG. 8: RAMACHANDRAN PLOT PREDICTION FOR STRUCTURE OF SIP; A: GENERAL PLOT; B: GLYCINE RESIDUES OF SIP STRUCTURE; C: PROLINE RESIDUES OF SIP STRUCTURE; D: PRE-PROLINE RESIDUES OF SIP STRUCTURE

Binding Site Prediction of Structurally Annotated Proteins: A congruence binding site obtained from various algorithms of Bspread was considered for molecular interaction studies of DnaA, PhnA, PhnB, FliC, and Sip. Each protein showed involvement of at least six residues information of the binding site. The binding sites of respective proteins have been depicted in Fig. 9 - Fig. 13.

Molecular Interaction Studies: The molecular interaction studies performed through molecular docking of β-sitosterol against the quorum sensing proteins LuxS, ToxB, DnaA, PhnA, PhnB, FliC, and Sip, exhibited exceptionally high binding affinity and molecular interaction with proteins ToxB, DnaA, PhnB, and Sip, whereas, β-sitosterol did not show significant binding and interactions with LuxS, PhnA, and FliC. The annotation of molecular interaction of β-sitosterol with ToxB, DnaA, PhnB, and Sip visualized using UCSF Chimera is depicted in Fig. 14 -Fig. 23.

Interaction of β-sitosterol with ToxB: β-sitosterol was found to bind ToxB at a binding site specified above along with additional amino acid residues surrounding the binding site of ToxB, overall of β-sitosterol was found to interact with Val49, Arg50, Leu51, His52, Ile87, Leu137, Met145, Thr149, Asn150, Asn151, Met154, and Leu 169.

Interaction of β-sitosterol with DnaA: β-sitosterol was found to bind DnaA at a binding site specified above along with additional amino acid residues surrounding the binding site of DnaA, overall of β-sitosterol was found to interact with TYR114, THR152, GLY153, LYS156, THR157, HIS158, ILE281, and ILE309 as shown from Fig. 24 - Fig. 30.

FIG. 30: EXHIBITING Β-SITOSTEROL INTEGRATED IN BINDING POCKET OF DNAA DEPICTED IN HYDROPHOBICITY SURFACE WITH GOLDEN HUE, WHERE BINDING POCKET OF DNAA IS ALSO DEPICTED IN SOLID HYDROPHOBICITY SURFACE MODE

Interaction of β-sitosterol with PhnB: β-sitosterol was found to bind PhnB at a binding site specified above along with additional amino acid residues surrounding the binding site of PhnB, overall of β-sitosterol was found to interact with ASN8, ASN32, ILE54, GLY59, SER63, SER64, LEU67, ILE71, GLY86, LEU87, and ALA89 as shown in the Fig. 31 - Fig. 37.

Interaction of β-sitosterol with Sip: β-sitosterol was found to bind Sip at a binding site specified above along with amino acid residues surrounding the binding site of Sip, overall of β-sitosterol was found to interact with THR119, ASN120, GLU121, TYR136, ASN189 and PHE203 as shown in the Fig. 38 – Fig. 44.

CONCLUSION: Molecular interaction studies reveal that the ability of Acarus calamus in inhibiting biofilm formation in H. pylori might be due to the inhibitory effect of phytobio-active component, β-sitosterol, against quorum sensing molecules- ToxB, DnaA, PhnB, and Sip, making it a novel therapeutic alternative to treat H. pylori infections.

ACKNOWLEDGEMENT: Authors acknowledge the Head of the Institute, JSS Academy of Higher Education, Mysuru, for the facilities. C. S. greatly acknowledges the funding support from DST DST-SERB (YSS/2015/001135/LS (Ver-I).

CONFLICTS OF INTEREST: The authors declare no conflict of interest.

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    How to cite this article:

    Prasad A, Shruthi G, Sushma P, Jain AS, Chandan D, Prasad MNN, Kollur SP, Chandrashekar S and Shivamallu C: Helicobacter pylori infection: a bioinformatic approach. Int J Pharm Sci & Res 2020; 11(11): 5469-83. doi: 10.13040/IJPSR.0975-8232.11(11).5469-83.

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