key: cord-0870728-geufw0mz authors: MubarakAli, Davoodbasha; Akshaya, Thirusangu; Sathya, Raghunathan; Irfan, Navabshan title: Study on the Interaction of Algal Peptides on Virulence Factors of Helicobacter pylori: In Silico Approach date: 2021-11-11 journal: Appl Biochem Biotechnol DOI: 10.1007/s12010-021-03716-4 sha: cfe16b3e6bffc0e76e51ed6f8774f664c5232cd5 doc_id: 870728 cord_uid: geufw0mz In the Asian region, Helicobacter pylori infects about 80% populations, which is most leading cause of peptic ulcers, and it is an asymptomatic infection. Studies reported that the particular bacteria carry specific virulence factors that leads to severe complications. These virulence factors can be used as a drug targets to inhibit their growth and pathogenicity. Chronic infection with H. pylori virulence factors are CagA, VacA and HtrA positive strains the risk factor of gastric cancer. In this study, we aimed to study the antagonistic interaction pattern between the potential eight algal peptides against the virulence factors of H. pylori through in silico analysis intended to treat peptic ulcer and prevent the further complications such as cancer. The proteins of virulent factors are docked using C-Docker algorithm and calculated the bind energy of the complexes. The results showed that the peptide derived from a green alga, Tetradesmus sp. are active against the three virulent factors such as cag-A, vac-A, and Htr-A with multiple hydrogen, vdW, electrostatic interactions, and mild π-hydrophobic bindings with the libdock energy score for CagA, VacA and HtrA are 175.625, 158.603 and 89.397 kcal/mol. These primes and the peptide lead to develop a better and potential inhibitors against H. pylori infection. H. pylori are more common in poorer nations because of the generally poor sanitation and hygiene conditions caused by poor socio-economic infrastructure. H. pylori are the main causative agent of peptic and gastric ulcers. It is a gram-negative, rod-shaped, microaerophilic, flagellated bacterium that causes stomach inflammation in more than half of the population; however, the affected individuals are asymptomatic. So this bacterium can enter our bodies and reside in the digestive tract. They cause sores called ulcers which may lead to cancer [1] . The World Health Organization (WHO) and the International Agency for Research on Cancer (IARC) have designated H. pylori as a class I carcinogen linked to the development of gastric cancer (GC) since 1994 [2] . Ulcers are the deep lesions penetrating through the entire thickness of the gastrointestinal tract that also lead to cancer because the stomach has a layer of mucus that is designed to protect from stomach acid. H. pylori attack this mucus lining and few part of the stomach exposed to acid together with the bacteria, and the acid can irritate the stomach causing ulcers or cancer There are different types of ulcers; most common are atrophy, metaplasia, and peptic and gastric ulcer, which appeared to damage the inner lining of the stomach. Furthermore, H. pylori infection is one of the most important risk factors for the development of stomach adenocarcinoma. Peptic ulcers develop due to an imbalance between aggressive factors and protective factors [3] . The virulence of the bacterial strains, environmental variables, and the genetic traits and lifestyles of the hosts all influence the clinical consequences of Helicobacter pylori infections. Certain foods or stress were considered to be the cause for peptic ulcer, however after the discovery of H. pylori [4] . Ulcers are not only the problems associated with H. pylori. Peptic ulcers can block the passage of food through the digestive tract, which causes vomiting and swelling from inflammation. Urease is the most important enzyme produced by the H. pylori, since it enables survival of the organism in a low pH environment and also aids colonization in the mucosal membrane of the stomach. The enzymes catalyze the degradation of urea to ammonia and carbon dioxide. Ammonia alkalizes the environment, leading to the neutralization of the acid fluid in the stomach, which allows bacterial survival. Urease was the first virulence factor of H. pylori used for diagnostic purposes in gastric pathology [5] . Medicinal plants and algae have achieved their therapeutic properties from their capability to produce renewable and various secondary metabolites which are known as phytochemical constituents. Algae used these phytochemicals as a protection mechanism against pathogen, and those algae are specific protein fragments, and this protein is an essential component required to repair cells, and also algal peptides have less toxicity and more antibacterial properties [6, 7] . Numerous species of algae are reported to be rich in proteins, carbohydrates, and bioactive compounds. Bioactive compounds isolated from algal peptides are showing anti-cancer activity and preliminary anti-COVID-19 [8, 9] . In the previous research Cbf-K16, a cathelicidin-like antimicrobial peptide, demonstrated wide antibacterial action which is followed Cbf-K16 was found to have effective antibacterial and anti-inflammatory properties, as well as down regulating the expression of adhesion-and cytotoxin-related genes in drug-resistant H. pylori, making it a promising anti-infective therapy option [10] . Sun et al. have recently reported that anti-adhesive peptide which is derived from wheat germ protein is effectively inhibited H. pylori adhesion to gastric epithelial cells [11] . The aim of this study was to investigate the antagonistic interaction pattern between the potential 8 algal peptides against the virulence factors of H. pylori through in silico analysis intended to treat peptic ulcer and prevent the further complication such as cancer. Literature studies reported about the H. pylori and also its virulence genes. Commonly there are several genes that have been implicated in disease related to H. pylori. Studies show that several virulence factors are thought to be important once contact with the host cell epithelium. The study reported showed that 21 virulence genes of H. pylori are the reason behind the ulcerogenesis, and specifically three gene markers CagA, VacA, and HtrA significantly play the role ( Table 1) . The potent antibacterial, antitumor, and antimicrobial algal peptide sequences (Table 2) were collected. Among the 12 peptides, 8 peptides were selected for the docking studies. In molecular mechanics simulations, an essential step is first to assign a force field to calculate the potential energy of input molecule. The Discovery Studio client uses a CHARMm-style residue topology file (RTF) to implement force field calculation. Additionally, each of the supported force fields may include some residue patches to allow variations of the residues. To make stable molecules, energy minimization is performed on structures before docking analysis; it relaxes the conformation and removes the steric overlap that produces bad contacts [24] . In the minimization algorithm, max steps are 2000, RMS gradient is 0.01, and the energy change is 0.0. In advanced, partial charge estimation is Momany-Rone fixed in the parameter window. Minimized molecules were subjected to the libdock protocol. Libdock is an algorithm for docking small molecules into an active receptor site. Initially, a hotspot map is calculated for the receptor active site which contains polar and a polar group. This hotspot map is subsequently used to rigidly align the ligand conformation to form favorable interactions [25] . In the protocol window, virulence factors are submitted in the input receptor column, and in the ligand column, all 8 minimized peptides are uploaded. The binding spot is chosen based on the receptor cavity in the coordinates of − 5.05467 (X), 60.4518 (Y), and − 24.6123 (Z) with the radius of 10.9 Å. Other docking preferences, conformation method, minimization algorithm, and advanced options are mentioned in Table 3 . Anticancer and antioxidant activities [12] 2 NIPP-1(pro-gly-trp-asn-gln-trp-phe-leu) NIPP-2(val-gluval-leu-pro-pro-ala-glu-leu) Inhibited ethanol-induced cytotoxicity in HepG2/CYP2E1 cells [13] 3 Gly-Met-Asn-Asn-Leu-Thr-Pro-Leu-Glu-Gln Fractions on human umbilical vein endothelial cells (HUVECs) [14] 4 LDAVNR, MMLDF Anti-inflammatory activity [15] 5 Ile-Ala-Glu, Phe-Ala-Leu, Ala-Glu-Leu, Ile-Ala-Pro-Gly, and Val-Ala-Phe Anti-oxidant activity, anti-hypertensive activity, antimicrobial activity, anti-diabetics activity, and anti-obesity activity [16] 6 ELWKTF Antioxidant peptides [17] 7 WPRGYFL, GPDRPKFLGPF, WYGPDRPKFL, SDWDRF Tetradesmus obliquus ACE inhibitory activity [18] 8 PHA, PHP, PHS Anti-oxidant, anti-hyaluronidase, anti-collagenase, antiinflammatory activity [19] 9 FGMPLDR, MELVLR ACE inhibitory peptides [20] 10 GVPMPNK, RNPFVFAPTLLTVAAR, LRSELAAWSR Anti-diabetic peptides [21] 11 AIVFQAQH Anti-osteopenic activity [22] 12 PIZ, FEIHCC ACE inhibitory peptides [23] The three main reasons for the selection of these particular virulence markers are as follows: first, CagA (cytotoxin associated gene) is a cancer-promoting gene. It causes cell proliferation and cell elongation. Second, VacA (Vacuolating cytotoxin gene) is a multifunctional toxin, and it causes cell vacuolation. The last is HtrA-suppression of HtrA proteolytic activity is sufficient to kill H. pylori. These three proteins were collected from their genes CagA, VacA, and HtrA, respectively, and downloaded from protein databases based on their residues length, classification, and resolution values. Energy minimization step for ligands and proteins before docking. Some proteins have less energy value with only in its minimized. Molecular mechanics mainly depend upon threeparameter force field, parameter sets, and minimizing algorithms. The potential energy of the molecules in molecular mechanics is calculated by using force field concepts. A force field is a set of functions and constants used to describe the potential energy of a molecule. The parameter set includes atomic mass, Vander Waal's radii, bond length, bond angle, the dihedral angle which defines a reference point, and force constants allowing for the calculation of potential energy caused due to the inclusion of attractive or repulsive interactions between atoms. Algorithms to calculate new geometrical positions are so-called minimizers or optimizers [26] . Force field applied three virulence factors, and the 8 peptides were minimized by the smart minimizer (steepest descent 1000 steps + conjugated gradient 1000 steps The docking algorithm run between the vacA virulence factor and 8 peptides results illustrated that, totally 222 conformers generated, 81 poses docked and 2 molecules failed to dock. The failing of the legends due to the clashes of active site amino acids with the amino acids of peptide. Figure 3 shows that the highest binding molecule Tetradesmus (3) formed 17 hydrogen bonds and 3 attractive charges that made the molecule bind with highest energy of 158.603 k cal/mol compared to other molecules. Only two ligands docked and 35 conformers generated in the CagA libdock with peptides. Tetradesmus (3) and (2) only formed bonding with the CagA. Specifically, Tetradesmus (3) formed around 8 H-bonds, one π-alkyl, and π-cationic interaction that made the molecule fit proper orientation inside the binding pocket of CagA. The binding energy was found to be 175.625 kcal/mol (Table 7) . In silico molecular modeling studies will lead to generation of a potent molecule in less time with reduction in the usage of chemicals and animals and also reduce the expenses. Owing to its invasiveness and pathogenicity, numerous treatment and control methods were employed to target the H. pylori [27, 28] . Hence, to develop the vaccine against the pathogen is the significant task and at the same time the discovery and development of antibiotic among the most powerful and successful achievements of modern science and technology for the control of infectious diseases. Prolonged usage of broad spectrum antibiotics leads to the emergence of drug resistance. Apart from that, the algae have achieved their therapeutic properties from their capability to produce renewable and various secondary metabolites which are known as phytochemical constituents, and these phytochemicals were used as a protection mechanism against pathogen. There is a tremendous need for novel antimicrobial agents from different microbes in general and microalgae in particular provides important sources of chemical compounds and secondary metabolites which have many therapeutic applications. In this study, microalgal peptide derived from Tetradesmus sp. exhibited as antibacterial and inhibitor of H. pylori. It almost acts on all the three selected virulence factors, VacA, CagA, and HtrA through antagonizing effect. These peptides could be lead molecules to treat the H. pylori-induced diseases as well as to develop a vaccine. Helicobacter pylori virulence dupA gene: Risk factor or protective factor Helicobacter pylori and gastric cancer: A state of the art review Peptic ulcer disease. The Lancet Helicobacter pylori infection and peptic ulcers Antioxidant peptide purified from enzymatic hydrolysates of Isochrysis Zhanjiangensis and its protective effect against ethanol induced oxidative stress of HepG2 cells Perforated peptic ulcer disease: A review of history and treatment. 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