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 ~  Abstract
 ~ Introduction
 ~  Materials and Me...
 ~  Amino Sequences ...
 ~ Results
 ~ Discussion
 ~  References
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  Table of Contents  
ORIGINAL ARTICLE
Year : 2017  |  Volume : 35  |  Issue : 4  |  Page : 522-528
 

The bioinformatics analyses reveal novel antigen epitopes in major outer membrane protein of Chlamydia trachomatis


1 State Key Laboratory Incubation Base of Xinjiang Major Diseases Research (2010DS890294) and Xinjiang Key Laboratory of Echinococcosis, First Affiliated Hospital of Xinjiang Medical University, Urumqi, Xinjiang 830011, P.R. China
2 State Key Laboratory Incubation Base of Xinjiang Major Diseases Research (2010DS890294) and Xinjiang Key Laboratory of Echinococcosis, First Affiliated Hospital of Xinjiang Medical University; Shenzhen Hospital of Southern Medical University, Urumqi, Xinjiang 830011, P.R. China
3 College of Basic Medicine of Xinjiang Medical University, Urumqi, Xinjiang 830011, P.R. China
4 Department of Microbiology and Immunologya and Pathology, University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA
5 State Key Laboratory Incubation Base of Xinjiang Major Diseases Research (2010DS890294) and Xinjiang Key Laboratory of Echinococcosis, First Affiliated Hospital of Xinjiang Medical University; College of Basic Medicine of Xinjiang Medical University, Urumqi, Xinjiang 830011, P.R. China

Date of Web Publication1-Feb-2018

Correspondence Address:
Ms. Amanguli· Yasheng
NO.137 South Road of carp mountain, Urumqi
P.R. China
Dr. Xiumin Ma
College of Basic Medicine of Xinjiang Medical University, Urumqi, Xinjiang 830011
P.R. China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmm.IJMM_17_251

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 ~ Abstract 

Purpose: The aim of this study was to predict the T-cell and B-cell epitopes in major outer membrane protein (MOMP) of Chlamydia trachomatis (CT) by using online software and also to analyse the secondary structure of MOMP through bioinformatics tools. Materials and Methods: The predictions of secondary structure of MOMP protein were carried out using SOPMA software, and the prediction of B-cell epitopes in MOMP protein was carried out using IEDB and LEPS software, while the T-cell epitopes were predicted by the software of IEBD and SYFPEITHI. The predictions from the software were combined with MOMP protein characteristics, including surface features, hydrophilicity, flexibility, accessibility and plasticity, to analyse the common epitope areas' response by T-cells and B-cells. Results: In the secondary structure of CT MOMP, the alpha-helices accounted for 41.62% of total amino acid, while the beta sheets and random coil accounted for 19.80% and 32.49%, respectively. Predictions combined with MOMP protein surface features, hydrophilicity, flexibility, accessibility and plasticity were further characterised, and three high-score B-cell epitope areas were found as located in 24–31, 307–311 and 318–327 amino acids of MOMP protein, respectively; in the meanwhile, three high-score T-cell epitope areas were found in 234–236, 323–329 and 338–343 amino acids of MOMP using major histocompatibility complex (MHC) class I HLA-A 0201 restrictive T-cell epitope analyser. Conclusion: We established the methods by using the biological information network technologies for looking the T-cell antigen epitopes and B-cell antigen epitopes in MOMP of CT, and three novel T-cell epitopes as well as three novel B-cell epitopes were identified in the current study. It provides important information for further studying the antigenicity of CT MOMP protein and also provides useful information for developing highly efficient subunit vaccines for CT.


Keywords: Antigen epitope, bioinformatics, Chlamydia trachomatis, major outer membrane protein, prokaryotic expression


How to cite this article:
ZHang T, Li H, Lan X, ZHang C, Yang Z, Cao W, Fen N, Liu Y, Yan Y, Yasheng A, Ma X. The bioinformatics analyses reveal novel antigen epitopes in major outer membrane protein of Chlamydia trachomatis. Indian J Med Microbiol 2017;35:522-8

How to cite this URL:
ZHang T, Li H, Lan X, ZHang C, Yang Z, Cao W, Fen N, Liu Y, Yan Y, Yasheng A, Ma X. The bioinformatics analyses reveal novel antigen epitopes in major outer membrane protein of Chlamydia trachomatis. Indian J Med Microbiol [serial online] 2017 [cited 2019 Aug 26];35:522-8. Available from: http://www.ijmm.org/text.asp?2017/35/4/522/224431

Tao ZHang, Huijun Li and Xi Lan have contributed equally to this work.



 ~ Introduction Top


Chlamydia trachomatis (CT) infection can cause human trachoma, and also Chlamydia is an important pathogen causing urogenital canal infection. In 1999, the World Health Organization estimated that over 92 millions of new urogenital canal CT infection patients were reported annually, and chlamydial infections seriously affected human health and became a heavy social financial burden.[1],[2] CTinfection can cause male urethritis, epididymitis, and female cervicite, endometritis, pelvic inflammation and salpingitis by sexual transmission, which lead to a series of diseases such as tubal infertility, ectopic pregnancy and spontaneous abortion and usually occur in mother - infant transmission, which give rise to neonatal inclusion conjunctivitis and pneumonia. Reproductive tract infection of CTis also a cofactor of cervical cancer caused by HBV [3],[4] and an important risk factor of HIV coinfection. Some autoimmune diseases such as reactive arthritis, Reiter syndrome and coronary heart disease are also closely related to CT infection.[5],[6]

CT infection has a conceal progress, so its clinical feature is slight even no symptom, and infection is recurrent and prolong, bring many difficulties of curing. Vaccination is the most effective and financial way to prevent and control CT infection. However, there is no commercially available vaccine yet. Previous studies showed that there have no desired protect effection when use CT thallus or target antigen to immune human and it will generate severe pathological immune response.[7],[8] Hence, immune dominant epitope by CT, especially constructed by the highly conserved T-cell and B-cell epitopes, would provide new solutions for the vaccine. Major outer membrane protein (MOMP) of CT exists in all serotypes. And it is exposed to the surface of chlamydia, occupied by more than 60% of total protein and it plays an important role in the process of mediate CT chlamydial adhere to the host cell. As an important immunogen, it can stimulate organs to generate both cellular and humoral immunities. In the current study, we introduced CTMOMP gene into pET42a-E.coilBL21DE3 prokaryotic expression system and induced the expression of MOMP; in addition, we did a bioinformatics analysis for MOMP protein, and three novel T-cell epitopes as well as three novel B-cell epitopes were found, which provide important information for further investigating the immunological characteristics of MOMP protein and for developing an effective vaccine for CTas well.


 ~ Materials and Methods Top


Chlamydia strain and chemical reagents

CT serovar D strain was gifted by Dr. Guangming Zhong at Department of Microbiology and Immunology of Life Science at the University of Texas Medical Research Centre Health Science Center at San Antonio. HeLa cell was purchased from standard strain conservation centre in the United States (ATCC, cat# CCL2). DNA extraction kit, polymerase chain reaction (PCR) reagents, PCR product recovery kit, DNA gel recovery kit, T-A cloning kits, plasmid extraction kit, restriction endonuclease, T4 DNA ligase, IPTG, DNA and protein reference standard and the Ni-NTA affinity chromatography materials were bought from TaKaRa Co., Prokaryotic expression vector pET42a and kanamycin resistance in the expression of host bacteria  Escherichia More Details coli BL21DE3 were bought from Novagen Co. Ltd.

Design and synthesis of primer

According to the Genbank (DNA sequence library in National Center for Biotechnology Information) to obtain CT MOMP length gene sequence and combined with DNAman software (Lynnon biosoft company in America) to design the following primers:

MOMPforward primer:

5′'-TATGGATCCTCACGAGCATCATTTGATGC-3′

MOMP reverse primer:

5′-GCGAAGCTTTCGTGACTTCATCTTGTTAA-3′

Primer was compounded at Invitmgen Company in Shanghai.

Analysis software

SOPMASever(affiliated to LBTI laboratory UMR 5305) was used to predict the secondary structure of CT MOMP; IEDB(a component of the National Institutes of Health in the Department of Health and Human Services.) and LEPS (China Medical University and National Taiwan Ocean University All Rights Reserved) were used to predict B-cell epitopes. SYFPEITHI (supported by the European Union) and IEDB were used to predict T-cell epitopes and online software three-dimensional (3D)Ligandsite(Structural Bioinformatics Group, Imperial College, London) and RasMol(Based on RasMol 2.6 by Roger Sayle Biomolecular Structures Group Glaxo Wellcome Research & DevelopmentStevenage, Hertfordshire, UK)were used to predict the 3D structure of MOMP.

Cell culture and infection

HeLa cells were subcultured by dulbecco's minimum essential medium(DMEM) medium which contained 10% foetal calf serum, then seeded them into 6-well cell culture plate, after the culture grows into monolayer cells, the culture fluid was discarded, washing once with DMEM media only, and then CT serovar D was inoculated for infection.

Clone and identification of Chlamydia trachomatis major outer membrane protein gene

Select the CT infection cell culture, split by proteinase K, and extract the CT genome DNA by phenol–chloroform method. Then, amplify the CT MOMP genes by PCR, the reaction system is 50 μl: DNA template 1 μl, 10 × buffer solution 5 μl; primer F and R 1 μl; 10 mM dNTP 4 μl; 25 mM MgCl2 4 μl; Taq polymerase 0.5 μl and add water to 50 μl. The parameter for PCR is 95 for 3 min, × 1; 94 for 30 s, 55 for 30 s, 72 for 1 min and × 30; 72 for 7 min. The product was detected by 2% agarose gel electrophoresis.

The construction of prokaryotic expression system of Chlamydia trachomatis major outer membrane protein

CT MOMP amplification fragment was purified by using DNA Fragment Purification Kit (BioDev-Tech) and was inserted into plasmid vector pMDl9-T using T-A cloning kit according to the manufacturer's instructions. The recombinant plasmid was amplified in E. coli DH5 and then extracted, according to blue and white spot screening and preliminary identification by double enzyme with Nde I and Xho I (TaKaRa). Alkali denaturation method was used to extract the objective recombinant plasmid pMDl9-T/CTMOMP, entrust Invitrogen company, determine the nucleotide sequence of insert fragments and compare with CTMOMP report gene sequences.

CT MOMP gene recombinant plasmid pMDl9-T/CTMOMP and expression vector with pET-42 a (Novagen) were have double enzyme cutted respectively, after recycling purpose fragment and used T4 DNA ligase (TaKaRa) to connect, and then transform into Expression of host bacteria:Ecoli BL21DE3(Novagen) form the prokaryotic expression system outbreak E. coli BL21DE3-pET-42a– CTMOMP and sequencing again.

Chlamydia trachomatis major outer membrane protein recombinant protein expression and purification

E. coli BL21DE3/CTMOMP vaccination in containing 50 (including g/ml kanamycin in LB broth, in 37°C 250 r/min rotation culturing for 2 h, then respectively to join tendency for 0.5/mmol/L IPTG to continue the culturing for 4–6 h, induced expression of recombinant protein rCtMOMP. Using SDS-PAGE and Bio-Rad gel image analysis system to measured rCTMOMP expression, then used Ni-NTA rCTMOMP affinity chromatography purification to purify rCtMOMP, and by protein kit (Beyotime) determine its concentration.


 ~ Amino Sequences Of Chlamydia Trachomatis Major Outer Membrane Protein Top


Validation of gene sequence analysis was eventually carried out and the corresponding amino sequences were deducted by using DNAman software. Multiple sequences were detected according to the CT MOMP gene sequence provided by GenBank.

Prediction of secondary structure of protein

The prediction for the secondary structure of CT MOMP protein was made using online software SOPMA Sever.

(http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl? page=/NPSA/npsa_sopma.%20 html).

Prediction of Chlamydia trachomatis major outer membrane protein antigen epitopes

B-cell epitope prediction software

The prediction on hydrophilicity, antigenicity and flexibility of B-cell epitope was made by using online prediction software IEDB (http://tools. immuneepitope. org/tools/bcell/iedb_input) and LEPS (http://leps.cs.ntou.edu.tw/index.php).

T-cell epitope prediction software

The prediction on MHC-I type HLA-A 0201 of restrictive T-cell epitope was made by using online resources SYFPEITHI (http://www.syfpeithi. De) and IEDB (http://tools.immuneepitope.org/tools/bcell/iedb_input).

Prediction of tertiary structure of Chlamydia trachomatis major outer membrane protein

The prediction of tertiary structure of CT MOMP was made by using online server 3DLigandsite (http://www.sbg.bio.ic.ac.uk/~3dligandsite/), combined with RasMol software to analyse and reveal different models of demonstration, such as cartoon, structure and group.


 ~ Results Top


Cloning of Chlamydia trachomatis major outer membrane protein gene

Using primers to amplify the CT MOMP gene, the length of target fragment was consistent with our predicted(1160 bp) [Figure 1].
Figure 1: Chlamydia trachomatis major outer membrane protein gene amplification product. (1) Chlamydia trachomatis major outer membrane protein amplification band and (2) DNA marker

Click here to view


rCT major outer membrane protein expression and purification

Under IIPTG induction, prokaryotic expression system of e.c. was constructed with our fabrication: E. coli BL21DE3-pET-42a– CTMOMP express rCTMOMP effectively, the recombinant protein is mainly exist in the form of soluble, SDS-PAGE detection shows in the location of the relative molecular mass of about 43,000 has a protein bands in purified rCTMOMP.[Figure 2].
Figure 2: Chlamydia trachomatis major outer membrane protein genetic recombination express product. (1) Protein marker, (2) empty plasmid and (3) rCT major outer membrane protein

Click here to view


Amino sequences coded by Chlamydia trachomatis major outer membrane protein

Corresponding amino sequence to be translated was predicted using online software DNAman and 394 amino acid residues were coded:

MKKLLKSVLVFAALSSASSLQALPVGNPAEPSL MIDGILWEGFGGDPCDPCTTWCDAISMRMGY YGDFVFDRVLQTDVNKEFQMGAKPTTATGNA AAPSTCTARENPAYGRHMQDAEMFTNAAYM ALNIWDRFDVFCTLGATSGYLKGNSASFNLV GLFGDNENHATVSDSKLVPNMSLDQSVVELYTDTTF AWSAGARAALWECGCATLGASFQYAQSKPKVEELNV LCNAAEFTINKPKGYVGQEFPLDLKAGTDGVTGTKD ASIDYHEWQASLALSYRLNMFTPYIGVKWSRASFD ADTIRIAQPKSA

TTVFDVTTLNPTIAGAGDVKASA EGQLGDT MQIVSLQLNKMKSRKSCGIAVGTTIVDADK YAVTVETRLIDERAAHVNAQFRF.

Results of prediction of antigen protein secondary structure of Chlamydia trachomatis major outer membrane protein

Prediction of secondary structure of CT MOMP antigen protein was made by using online software SOPMA. Results show that α-helical structure accounted for 41.62% of total amino acid and β-sheets and random coil account for 19.80% and 32.49%, respectively. Distribution of different structures of CT MOMP antigen protein is shown in [Figure 3].
Figure 3: Secondary structure of major outer membrane protein

Click here to view


Results of B-cell antigen epitope prediction of Chlamydia trachomatis major outer membrane protein

We made prediction on combined hydrophilicity, antigenicity and flexibility of CT MOMP by using online software IEDB and LEPS (http://leps.cs.ntou.edu.tw/index. Php). High-valued regions were assumed as potential B-cell antigen epitopes. Plasticity analyse three score higher B-cell epitope areas which were located in 24–31, 307–311 and 318–327 amino acid sequences. According to the predicted result, high-valued amino acid sequences were identified [Figure 4] and [Figure 5].
Figure 4: (a) Prediction of hydrophilicity, (b) prediction of flexibility, (c) prediction of beta-turns, (d) prediction of exposed surface, (e) prediction of polarity

Click here to view
Figure 5: The parameters of IEDB online prediction. (a) β-angle prediction. (b) Surface accessibility index prediction. (c) Flexible area prediction. (d) Antigenicity index prediction. (e) Hydrophilia index prediction. (f) Linear epitope prediction

Click here to view


Results of T-cell antigen epitope prediction of Chlamydia trachomatis major outer membrane protein

In order to obtain most accurate results, online prediction software SYFPEITHI (http://www.syfpeithi. de) and IEDB (http://tools. immune epitope. org/tools/bcell/iedb_input) were used to predict MHC-I type HLA-A 0201 restrictive T-antigen epitopes and 13 high-valued epitopes of them were selected. General analysis results are shown in [Table 1].
Table 1: The major histocompatibility complex-I human leucocyte antigens-A 0201 restrictive T-cell epitopes of major outer membrane protein predicted by SYFPEITHI and IEDB software

Click here to view


Combining the above two results, four potential T-cell antigen epitopes, which were located in 234–236, 323–329 and 338–343 amino acid sequences, were finally identified.

Results of united epitope prediction of T-cell and B-cell antigen

Using multiple sequence alignment from DNAMAN [Please provide manufacturer details such as company, city, state, and country name.]software to compared with T- and B-epitopes, it shows highly overlapped sequence 323-327 of T-cell and B-cell combined epitopes [Table 2].
Table 2: United epitope prediction of T-cell and B-cell antigens

Click here to view


Demonstration and analyses of Chlamydia trachomatis major outer membrane protein tertiary structure

Segments of CT MOMP code amino acid were submitted to the server 3DLigandsite (http://www.sbg.bio.ic.ac.uk), making prediction and analysis on protein 3D structure [Figure 6]. We applied different demonstration models, such as structure and group [Figure 7]a and [Figure 7]b, to show specific position of each amino acid on the tertiary structure of CT MOMP by using RasMol and 3DLigandsite analysis software. There is a huge similarity between the blue area shown in the structure model and the flexible area predicted by secondary structure. Group structure discovered that blue area further assembles even basically distributed to the surface of the structure, indicating that it is most likely to be the combined epitope of antigen and antibody.
Figure 6: Three-dimensional structure mimic diagram of major outer membrane protein analysed by three-dimensionalLigandsite

Click here to view
Figure 7: Model exhibition of major outer membrane protein three-dimensional structure. (a) Structure model exhibition, (b) anterior view of group model exhibition

Click here to view



 ~ Discussion Top


CTis an important pathogen which causes human urogenital system inflammation, especially female tubal infection and inflammatory response.[9],[10],[11] Antibiotic therapy is the main treatment to CT infection; however, lots of chlamydial infections were unsymptomatic, and it already caused disease outcomes to human before it was identified. Preventing CT infection, especially vaccination, is still the effective way to control chlamydial infection in human.

Epitope, also known as antigenic determinant, is a special chemical group that can determine the specific antigen in antigen molecules. It is the basic unit that TCR/BCR specific binding to antibody, which was finding in foot-and-mouth disease virus (FMDV) at first.[12],[13] In immune response, the epitopes recognised by TCR and BCR are different, so the epitopes were differentiated into B-cell epitopes and T-cell epitopes. B-cell epitopes were located on the surface of antigen molecules, which can induce B-cells, generating specific antibody during humoral immune response; while T-cell epitopes are the linear peptides that specific antigen disposed by APC and then presented to TCR through via MHC molecules, then induce body leaded to cellular immune response. Epitope vaccine is made by choose the epitope amino acid sequence which has strongest antigenicity on the antigen.[14],[15] With the development of biological technology, epitope vaccines have become the hot spot of molecule vaccine research.[16]

MOMP is the outer membrane protein of all serotypes of CT, it not only can prevents the fusion of phagosome and lysosome, which benefits chlamydia reproduction in phagocytosis and damages the host cell, but also serves as an important immunogen that can stimulate body to generate humoral and cellular immunity.[17],[18] MOMP is the key protein in the immune system which can defence the CT infection, and it has higher specificity and immunogenicity.MOMP have existed abundant B-cell and T-cell epitopes and protective immunity antigen.[19],[20] So, the vaccine's research and development always use MOMP as the target antigen. This research used bioinformatics technology to predict the B-cell epitopes of CT MOMP and found that α-helix structure were occupied 41.62% of total amino acids, β-sheets were occupied 19.80% and random coil occupied 32.49% of total amino acids. It has more possibility to form B-cell antigen epitope and has stronger immunogenicity. In the process of predicted T-cell epitopes, we find that the predict of MHC-I antigen's epitope have higher veracity. This research predicted that MOMP antigen protein HLA-A0201 MHC-I nine peptide restricted epitope through two software named SYFPEITHI and IEDB, and found there are 3 zones have high score: amino acid sequence 234–236, 323–329 and 338–343, these sequences are easier to form T-cell epitopes.

In this research, we predicted the secondary structure of CTMOMP antigen protein and T-cell and B-cell epitopes, and found three novel B-cell epitopes and three novel T-cell epitopes, which provide important information for further investigating the immunological characteristics of MOMP protein, and to develop an effective vaccine for controlling CTinfection.

Financial support and sponsorship

This work was supported by Scientific Research Project of Science Department of Xinjiang Autonomous Region (2015211C035).

Conflicts of interest

There are no conflicts of interest.



 
 ~ References Top

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World Health Organization. Global Prevalence and Incidence of Selected Curable Sexually Transmitted Infections: Overviews and Estimates. Geneva, Switzerland: World Health Organization; 2001. Available from: http://www.who.int/hiv/pub/sti/who_hiv_aids_2001.02.pdf. [Last accessed on 2013 Apr 19].  Back to cited text no. 1
    
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
 
 
    Tables

  [Table 1], [Table 2]



 

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