Analysis of Important H . pylori Outer Membrane Proteins by Detection of Common Sequences in Exposed Areas ; in Silico Study

1Department of Medical Biotechnology, School of Medicine, MashhadUniversity of Medical Sciences, Mashhad, Iran. 2Department of Microbiology, School of Medicine, Babol University of Medical Sciences, Babol, Iran. 3Department of Molecular and Cell Biology, Faculty of Basic Sciences, University of Mazandaran, Babolsar, Iran. 4Biotechnology Research Centre, Mashad University of Medical Sciences, Mashad, Iran.

After discovery of Helicobacter pylori pathogenesis approximately 30 years ago many scientists have shown that H. pylori strains cause various diseases including chronic gastritis, peptic ulcer, gastric adenocarcinoma and primary gastric B-cell lymphoma(J.V. Solnick and D. B. Schauer, 2001).
It has been proposed that H. pylori adhesins play important role for binding to the gastric mucosa and outcome of infection (D.Ilver et al., 1998, J. Mahdavi et al., 2002, N. Murata-Kamiya, 2011).Adherence of Helicobacter pylori and colonization to the gastric mucosa induces host immune and inflammatory responses.However, H. pylori escape from host immune response with different mechanisms (E.P. Reeves et al., 2008).
Currently, candidate receptors have been identified on the host gastric epithelium including oligosaccharides, glycolipids, glycoproteins and mucins.For instance , fucosylated oligosaccharide structure presented both in the H-  Zambon et al., 2003).
SabA is one of the major H. pyloriadhesins and the sialyl-Lewis x antigen is a binding site for SabA.The interaction of H. pylori with MUC1 in gastric mucus mediates by both SabA and BabA(S.K. Linden et al., 2009).Induction of interleukin-8(IL-8) secretion by two highly homologous OMPs, AlpA and AlpB, facilitate gastric injury and inflammation (H.Lu et al., 2007).
These two genes are capable of binding to the host cell laminin probably as adhesins.Additionally, other host related receptors have not been recognized yet (O. A. Senkovich et al., 2011).AlpA and AlpB mutants in C57BL/6 mice showed low colonization in gastric epithelial cells (H.Lu, J. Y. Wu, E. J. Beswick, T. Ohno, S. Odenbreit, R. Haas, V. E. Reyes, M. Kita, D. Y. Graham and Y. Yamaoka, 2007).Some of OMPs are associated with multiple intracellular signaling pathways and consecutive production of pro-inflammatory cytokines, such as interleukin-8 (IL-8), in epithelial cells and increased peptic ulcer disease (S.Censini et al., 1996, Y. Yamaoka et al., 2002).
In the present study we have focused on OMPs that play important role in the gastric disease.
The aim of this research is an extensive computational analysis for investigate OMPs in view of Secondary structures,tertiary structure, signal sequence,internal and external loops.Finding of homology modeling, prediction of accessible surface area amino acids among sequences of external loops and similarity of exposed amino acid area in these groups of proteins.The result of this study will be useful for detection adhesion motifs, reorganization of cell surface receptors and to get a better understanding of OMPs interaction with gastric epithelial cells and host inflammatory response.

Databases
Amino acids sequences of, BabA,BabB,AlpA ,AlpB,SabA and SabB were analyzed via recovering the genes from National Center for Biotechnology Institute and Uni-prot KB/Swiss-Prot.

Servers
The method has been used to predict secondary structure of the proteins wasbased on PHD and GOR 4 servers (H.M. Berman, J. Westbrook, Z. Feng, G. Gilliland, T. N. Bhat, H. Weissig, I. N. Shindyalov and P. E. Bourne, 2000, Aleksey A Porollo et al., 2004, B. Rost et al., 1994).The presence and location of signal peptide cleavage sites in amino acids sequences was predicted by SignalP 4.1 Server(H.Nielsen et al., 1997).
There are many freely accessible Web servers for protein structures prediction on the Internet.Our model can be built by Phyre servers, namely, structure prediction systems using such algorithm to provide models in PDB format and homology modeling(L. A. Kelley and M. J. Sternberg, 2009).
It is well known that a tertiary structure is mainly responsible for protein's function.Role of loops and turns in protein structure, folding, stability, and function is determined (J. S. Fetrow, 1995, P. T. Jones et al., 1986).Every OMP has a particular pattern of exposed amino acid sequences in external loops, which can be predicted by GETAREA and VADAR servers.Swiss PDB viewer and Discovery studio server were used for processing of data.Uniprot alignment was performed for sequence similarity OMPs detection.

RESULT
Sequences of the outer membrane proteins were retrieved from the data bases and analysis for detection of surface amino acids were   Uniprot alignment for measuring similarity between the OMPs showed that although there is a low similarity (7.6%) between BabA, BabB, AlpA, AlpB, SabA and SabB, the sever has found high similarities among BabA/ BabB (41.16%),AlpA/ AlpB (41.95%) and SabA/SabB (71.19%).
Although the most parts of similarities between proteins were not in the exposed regions, a number of conserved sequences were found in exposed external loops.The common regions were  Despite high sequence similarity between BabA and BabB, none of the similar areas were located on the exposed regions.The similarity between BabA, BabB, AlpA, AlpB, SabA and SabB were not in exposed amino acid sequences.

DISCUSSION
The gastric pathogen,H.pylori, is one of the bacteria that lead to development of chronic gastritis, gastric or duodenal ulcer, gastric cancer and MALT-lymphoma(M.J. Blaser, 1997).H. pylori induce strong immune responses in host cells.Moreover, the infection was not eradicated in most cases and patients may subject to severe gastroduodenal disease and gastric malignancy.Adaptation of H. pylori to their host immune system cause persistence of the bacteria for years and decades in their niches(N.Murata-Kamiya, 2011).Despite multiple treatments in H.pyloriassociated disease,there is a high incidence in developing countries(M.Oleastro and A. Menard, 2013).The remarkable progress in recent years has resulted from taking advantage of bioinformatics tools with the structure and function of proteins and application of this information for protein engineering, modeling and manufacturing of vaccines .Outermembrane proteinscouldpossiblybeone ofthemost important proteins in H. pylori.Furthermore,OMPs act as adhesins and involve in gastric colonization(B.Kalali et al., 2014).It was found thatOMPs of bacteriaare appropriatecandidates for vaccine production(Ravinder Singh et al., 2014).In the current study, we have systematically analyzed exposed amino acid sequences for detectingadhesion motifs and possiblereceptors as well.Structural similarities between proteins are a suitable predictionfor finding a functional similarity among the proteins.Our study has focused on similarity of exposed amino acid area in several H.pyloriOMPs.Our results have shown that there was ahighsimilarity betweenAlpA and AlpB(41.95%),SabA/SabB(71.1%)andBabA / BabB(41.16%).Although the mentioned similarities are previously reported by a number of research groups, our study has concentrated on the common exposed amino acid sequences.Furthermore, finding a common amino acid sequence of the proteins which share a high similarity in their exposed amino acid sequences will likely provide us a possible target site for new drugs or vaccines.Moreover, detection of these sequences is very useful for finding complementary receptors on surface of gastric epithelial cell and specific targeting of cell surface receptors.
BabA play a role in CagAtranslocation by theH.pylori TFSSand lead to inflammationand intestinal metaplasia(N.Ishijima et al., 2011, S. M. Sheu et al., 2012).Although, BabA interacts with a Lewis b on the human gastric MUC5AC, the biding role of the other proteins in HOP (helicobacter outer membrane proteins) group (BabB, BabC, and HP0227) has not been identified so far(D.Ilver, A. Arnqvist, J. Ogren, I. M. Frick, D. Kersulyte, E. T. Incecik, D. E. Berg, A. Covacci, L. Engstrand and T. Boren, 1998).Sequence similarity between BabA/ B genes was 41.16% and the proteins are highly similar in their N-and C-terminal regions but none of thesimilar areas has located on the exposed amino acid region.
Two greatly homologous OMPs, AlpA and AlpB located in the alpAB operon.AlpA/B causes interleukin-8(IL-8) secretion and expansion of the inflammatory responses (S.Odenbreit et al., 2002, S. Odenbreit et al., 1996, S. Odenbreit et al., 1999).Even though, the pair proteins will bind to host laminins, their corresponding receptors for these proteins remains unknown up to now (O. A. Senkovich, J. Yin, V. Ekshyyan, C. Conant, J. Traylor, P. Adegboyega, D. J. McGee, R. E. Rhoads, S. Slepenkov and T. L. Testerman, 2011) .Various types of bioinformatic tools were used to analyses sequence similarity and detection of exposed amino acid sequences.As a result, afew regions of exposed amino acid sequences were detected as common regions which were 170-171, 34-37 in AlpA and 186-187,54-57 in AlpB, respectively.The biological importance of AlpA and AlpB in adherence to host cells and tissues was reported in several studies and Exposed amino acid sequences of OMPs family play important role in binding to gastric tissue(S.Odenbreit, G. Faller and R. Haas, 2002, S. Odenbreit, M. Till and R. Haas, 1996, S. Odenbreit, M. Till, D. Hofreuter, G. Faller and R. Haas, 1999).Moreover, these two proteins are expressed in all H.pylori strains at steady rates whereas other H.pylori OMPs produce at variable rates which emphasis a critical function of AlpA and AlpB in the bacteria (S. C. Baik et al., 2004).
SabAof H. pylori can bind to various receptors such as sialyl-dimeric-Lex, sialylated structures on the surface of erythrocytes ,salivary mucin MUC7, MUC5B and sialylated moieties on the extracellular matrix protein laminin(J.Mahdavi Our finding has shown that thesimilarity rate between two proteins SabA/ SabBwas 71.19% but there was a short amino acid sequence in the exposed area which was common between two proteins (86-89/56-58).
Detection of Exposed amino acid sequences in OMPs is very helpful for characterization of the receptors.Adherenceto the gastric epithelium with specific receptors is a crucial step in beginning of infection.In addition, it can be considered for better understanding of the pathways of H. pylori colonization.In this way, we can recognize possible receptor on gastric epithelial cells.In fact, detection of these receptors probably helps us for control of H. pylori infection and to understand fundamental mechanisms of H. pylori colonization in chronic infection.Additionally,Adhesion motifs of the OMPsare a stimulator for host cell immune responseand induce pro-inflammatory intracellular signaling.Therefore, these proteins can be used as a purpose for vaccine expansion and protein docking study.Indeed,further studies shouldbe performedinvaccine design and potential targets in future.

CONCLUSION
Exposed amino acid of OMPs is the main sequences which are related to interaction with stomach epithelial cells.Despite extensive studies over several H. pyloriadhesions, some of their receptors are still unknown.
A number of common regions,between SabA/SabB and AlpA/AlpB, located on the exposed aminoacid areas.In spite of high similarity in BabA/B Homologous, none of the similar areas is located on the exposed amino acid regions.
This study has provided accessible surface areaamino acid sequences, which will be helpful for detection of possible receptors on gastricepithelial cells andto design novel targets to prohibit progress of disease that leads to severe pathologic outcomes.