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Bioinformatics analysis and epitope screening of a potential vaccine antigen TolB from Acinetobacter baumannii outer membrane protein
Accepted Manuscript Bioinformatics analysis and epitope screening of a potential vaccine antigen TolB from Acinetobacter baumannii outer membrane protein
Xiaojie Song, Hua Zhang, Dongsheng Zhang, Weifeng Xie, Guanghui Zhao PII: DOI: Reference:
S1567-1348(18)30197-7 doi:10.1016/j.meegid.2018.04.019 MEEGID 3485
To appear in:
Infection, Genetics and Evolution
Received date: Revised date: Accepted date:
21 September 2017 9 April 2018 13 April 2018
Please cite this article as: Xiaojie Song, Hua Zhang, Dongsheng Zhang, Weifeng Xie, Guanghui Zhao , Bioinformatics analysis and epitope screening of a potential vaccine antigen TolB from Acinetobacter baumannii outer membrane protein. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Meegid(2018), doi:10.1016/j.meegid.2018.04.019
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ACCEPTED MANUSCRIPT
Bioinformatics analysis and epitope screening of a potential vaccine antigen TolB from Acinetobacter baumannii outer membrane protein #
#
Xiaojie Song 1 , Hua Zhang 2 , Dongsheng Zhang 3, Weifeng Xie4, Guanghui Zhao5
Department of Respiratory, Qilu Hospital of Shandong University, Qingdao,
Shandong Province 255036, People’s Republic of China
Department of Geriatric, Qingdao Municipal Hospital, Qingdao, Shandong Province
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2
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266011, People’s Republic of China
Department of Hepatobiliary Surgery, Qingdao Municipal Hospital, Qingdao,
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3
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1
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Shandong Province 266011, People’s Republic of China Department of ICU, Qingdao Municipal Hospital, Qingdao, Shandong Province
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4
266011, People’s Republic of China
Clinical Laboratory Medicine Center, Qilu Hospital of Shandong University, Jinan,
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5
*
Equal contribution
Corresponding author
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#
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Shandong Province 250012, People’s Republic of China
Correspondence Address: Guanghui Zhao, Ph.D Clinical Laboratory Medicine Center, Qilu Hospital of Shandong University, Jinan, Shandong Province 250012, PR China. Fax: +86 532 66850876. Email:[email protected]
ACCEPTED MANUSCRIPT Abstract The clinical isolation rate of multidrug-resistant or pan-resistant Acinetobacter baumannii (A. baumannii) is increasing, resulting that optional antibiotics are very limited in clinical practice. To deal with such a dilemma in treatment, the development of effective vaccines serves as a good strategy. Outer membrane proteins
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(Omp) often contain potential excellent vaccine antigens, and NCBI has published more than 300 Omp sequences of A. baumannii (including the duplicates). To
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accurately screen out the potential excellent antigen molecules from a large number of
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sequences, and avoid repetitive experimental processes is of great significance. In this study, we used the bioinformatics software to give extensive predictions of TolB
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protein. Results suggest it is a potential vaccine antigen. We then cloned the TolB gene fragments and confirmed it was highly conserved among the strains. Finally, we
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designed a good recombinant epitopes and conducted experimental verification. These findings provided grounds for animal immunology experiments in the future, and
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showed an orientation for the efficient development of A.baumannii vaccine.
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Keywords: Acinetobacter baumannii; TolB; Bioinformatics; T cell Epitope; B cell Epitope
ACCEPTED MANUSCRIPT 1. Introduction Acinetobacter baumannii is conditioned pathogens that can cause pneumonia, bloodstream infections, urinary tract infections, secondary meningitis, wound infections (Munoz-Price and Weinstein, 2008). The clinical isolation rate of multi-drug and pan-drug resistance strains is increasing, resulting that optional
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antibiotics are very limited in clinical practice. To deal with such a dilemma in treatment, the development of effective vaccines serves as a good strategy. The
multiple antigenic
vaccine, single subunit protein
vaccine,
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cell vaccines,
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development process of Acinetobacter baumannii Vaccine includes inactivated whole
polysaccharide vaccine and DNA vaccine. McConnell and Pachón (2010) and Harris
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et al. (2013) prepared inactivated whole cell vaccines using ATCC19606 strain and LAC-4 strain respectively. Through intramuscular injection, it can induce specific
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antibody production, alleviate tissue bacterial load and improve mouse survival. The results showed that inactivated whole cells had partial immunoprotection. However, it
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also has shortcomings, such as low immunogenicity, excessive invalid ingredients, potential virulence recovery. In the case of multiple antigenic component vaccines,
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McConnell et al. (2011) used the outer membrane complex to immunize mice and induce humoral and cellular immune responses. Huang et al. (2014) used a clinical multidrug-resistant strain to prepare outer membrane vesicles, which significantly
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increased the IgG and IgA in mice through intramuscular injection and intranasal perfusion. In the case of single protein component vaccines, Fattahian et al. (2011) immunized mice three times with recombinant biofilm-associated proteins, resulting with a humoral immune response, a significant increase in specific IgG antibody titers, and a significant decrease in the mortality of infected mice. Goel and Kapil (2001) immunized mice with iron regulators to obtain monoclonal antibodies. The results showed that antibody has the effect of antibiotics and regulators, which can effectively prevent iron absorption process. Luo et al. (2012) used recombinant outer membrane protein A to immunize the diabetic mouse model and induced humoral immune response. The 28-day survival rate of the infected mice was increased to 50%,
ACCEPTED MANUSCRIPT and the bacterial load of multiple tissues was significantly decreased. rOmpA antibody has an immune conditioning effect, which can enhanced phagocytosis of macrophages. Bentancor et al. (2012) immunized mice with trimeric autotransporters as candidate vaccines. The results showed that Ata antibody could reduce the adhesion of A.baumannii, enhance the phagocytosis of multinucleated neutrophils, mediate
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complement-dependent bactericidal effect and reduce tissue bacterial load. Russo et al. (2013) used the purified K1 capsular polysaccharide to immunize mice. The results
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showed that K1 antiserum could enhance phagocytosis mediated by PMNs and improve the clearance rate of bacteria. Although single protein component vaccine
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can induce a humoral or cellular immune response, it does not have sufficient protective power so it is difficult to apply to multiple serotypes of infection. DNA
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vaccine is a new type of vaccine. Compared with the traditional inactivated whole cell vaccine or single protein component vaccine, it showed superiority: firstly, DNA
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vaccine introduced into the host can continue to express specific protein antigen components, and then stimulate the host to produce a stro ng humoral immune and
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cellular immune response (Lever et al.,2017); Secondlly, the DNA vaccine is easy to construct recombinant multi- gene fragments, construction of recombinant; DNA
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vaccines with excellent epitopes can significantly improve the effectiveness against polyclonal infection (Wu et al.,2016). So far, NCBI has published more than 300 strains of acinetobacter baumannii
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outer membrane protein gene sequences (including repetitive sequences), involving how to accurately screen out potential excellent antigen molecules from a large number of sequences so as to further build its recombinant epitope vaccine to verify its immune protection effect, which is a very meaningful job. Based on that, bioinformatics software was applied to screen out potential antigen with good hydrophilicity, strong plasticity, high accessibility, high antigenicity index and numerous T cells and B cell epitopes. We finally screened the TolB protein, one of the components of the Tol-Pal system for follow-up studies.
ACCEPTED MANUSCRIPT 2. Materials and Methods 2.1.Basic physical and chemical properties prediction The gene sequences (Gene ID: 4917606) and amino acids (WP_011859984.1) sequences of TolB were obtained from the GenBank (https://www.ncbi.nlm.nih.gov).
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GENSCAN program was used to search the open reading frame (ORF) of TolB gene (http://genes.mit.edu/GENSCAN). The online server ProtParam (http://web.expasy.
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org/protparam/) was used to analyze basic physical and chemical properties of TolB
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protein, including molecular weight, theoretical isoelectric point, estimated half life in mammals, yeast, Escherichia coli, instability index, aliphatic index and grand average
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of hydropathicity. TMHMM server (http://www.cbs.dtu.dk/services/ TMHMM2.0/) and SignalP server (http://www.cbs.dtu.dk/services/SignalP/) were used to analyze its
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transmembrane structure and signal peptide, respectively. While NetNGlyc 1.0 server(http://www. cbs. dtu.dk/services/NetNGlyc) and NetPhos 3.1 server (http://ww w.cbs.dtu. dk/services/ NetPhos/) were used to analyze the post-translational
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2.2.Structural prediction
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modification sites of it, including N-glycosylation sites and phosphorylation sites.
Two online services, SWISS-MODEL (http: // swissmodel. Expasy. Org /) and
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SOPMA (http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/ npsa_ sopma. html), were employed to predict secondary structure and 3D structure of TolB, the protein structure acquired was marked with Vector NTI software. 2.3.Bacterial strains and mice In order to investigate the conservation of TolB protein between different strains, we randomly selected five clinical isolates, named Ab1, Ab2, Ab3, Ab4 and Ab5 respectively, and one standard strain ATCC 17978. Six-week-old female BALB/c mice were purchased from the Shandong University Laboratory Animal Center. All the mice were maintained under specific-pathogen- free conditions. All the animal
ACCEPTED MANUSCRIPT experiments were approved by the Animal Ethics Committee of Shandong University Qilu Hospital. 2.4.DNA extraction and PCR Bacterial DNA was extracted from the six A. baumannii strains through DNA
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Extraction Kit. A pair of primers were designed according to the TolB gene of A. baumannii ATCC 17978. Restriction enzyme recognition sequence and protective
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base were inserted into the 5ˊ-end of both the forward and the reverse primers
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(Forward primer: 5ˊ- CGGGGTACCGTGCCTTTTAATAACGACAATGGA -3ˊ , Reverse primer 5ˊ - CCCAAGCTTTTATTTAGGTGCCCAGGCTGG -3ˊ , Kpn I
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and HindIII recognition sites were introduced and underlined). The TolB gene of the other five clinical isolates were also amplified by this primer. Specifically, the primer
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concentration was diluted to 10 μM with sterile ddH2 O, a 50μl PCR reaction system was prepared in a 0.5 ml tube: Forward Primer 1μl, Reverse Primer 1μl, PCR Mix (TransGen, China) 25μl, DNA template 2μl, ddH2 O 21μl. Then run the PCR program
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of initial denaturation at 94℃for 5 min, then undergo a proceeding of 35 reaction cycles as denaturation at 94℃for 30 s, anneal at 57℃ for 30 s, polymerization at 72℃
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for1 min, followed by a final polymerization at 72℃ for 10 min. After amplification, the PCR products were identified by 1% agarose gel electrophoresis.
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2.5.TolB gene cloning and identification PCR products were cloned into the pEASY-T1 simple vector (TransGen Biotech, China) to generate a recombinant cloning plasmid. After preparation of DH5α competent cells, 50μl recombinant plasmid was incubated with it on ice for 20 min, heat shock at 42℃ for 30s, then immediately was put on ice for 2min. The reaction system was added to 250 μl of LB liquid medium and incubated at 200 rpm, 37℃for 1h, and then transferred to the LA plate. A single white colony was picked and verified by PCR, positive colonies were inoculated into 3 ml LK medium and shaken overnight at 180 rpm, 37℃. The recombinant cloned plasmid pEASY-TolB was
ACCEPTED MANUSCRIPT extracted from the cultures, and then identified by double digestion using Kpn I and HindIII. The digested product was sequenced to obtain the gene sequence. 2.6.Sequencing and alignment of gene sequence The PCR products of A. baumannii ATCC17978 and the other five clinical
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isolates were sequenced and compared for homology. Briefly, nucleotide BLAST of NCBI was used to analyze the TolB gene conserve among different strains. A highly
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conserved gene sequence will help the potential vaccine antigen to maintain
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suitability for polyclonal strains.
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2.7.Epitope screening
The protein sequence of A. baumannii ATCC17978 was selected as analysis object.
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IEDB server（http://tools. immuneepitope.org/main/ html/tcell _tools.html）was used to analyze potential B cell epitopes on TolB protein and predicted items are mainly related to flexibility, antigenicity, accessibility and epitope, meanwhile, two online
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servers ABCpred and BepiPred were used to predict peptide segment of B cell epitopes. T cell epitopes were also predicted by IEDB server, including potential
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CD4+ T cell epitopes binding to MHC class Ⅱ molecules and CD8+ T cell epitopes binding to MHC classⅠwere screened out.
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2.8.Epitope recombination
In order to achieve the best immunogenicity, predicted epitopes are reorganized by series connection, the conjugate was a flexible peptide GGGG. All the arrangement schemes were analyzed and compared by DNAstar software, The optimal combination of recombinant epitopes selected by DNAstar software. The ideal combination is that all epitopes in the recombinants remain relatively independent, no new epitope is formed, no epitope is drifted, and antigenicity index of each epitope are high.
ACCEPTED MANUSCRIPT 2.9. Experimental verification of B cell epitopes B cell epitopes of TolB were detected by ELISA. Briefly, Blood was collected from the eyelids of BALB/c mice that had been infected with A.baumannii ATCC 17978 and then the serum was separated by centrifugation. The B-cell epitopes (epitopes 1 to 4) were coated in 96-well plates (10μg/ml) and PBS was used as a
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control well, then add 50μl mouse serum to each well. After washing, continue to add goat anti- mouse IgG horseradish peroxidase labeled secondary antibody (100μl/well).
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After washing the plate, 100 μl of TMB chromogenic reagent was added to each well.
450 nm using a microplate reader.
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2.10. Experimental verification of T cell epitopes
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The reaction was terminated after 30 minutes at 37℃. The OD value was detected at
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Lymphocyte proliferation assays were used to identify TolB protein CD8+ T cell epitopes and CD4+ T cell epitopes. The BALB/c mice infected with A.baumannii ATCC 17978 selected and the spleen was removed in a sterile environment. The
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spleen was grinded into cell suspension with a 200- mesh copper mesh, and the red blood cells in the spleen were removed using red blood cell lysate. The lymphocyte
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separation solution was added to separate the lymphocytes and resuspended in RMPI-1640 cell culture medium containing 10% fetal bovine serum. The cell suspension was added into a 96-well plate (100 μl/well) and incubated at 37℃, 5%
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CO2 for 24 h. The predicted CD8+T cell epitope (epitope 5) and CD4+ T cell epitope (epitope 6) were added into the plate and stimulated with 1 μg/well ConA as a positive control. After 24 hours, CCK-8 reagent (10 μl/well) was added and the culture was continued for 4h. The OD value at 450 nm was detected by a microplate reader. 3. Results 3.1.Physical and chemical characteristics of TolB
ACCEPTED MANUSCRIPT The TolB protein has 381 amino acids with a molecular weight of 41.8289 kDa, the theoretical isoelectric point was 6.34, the percentage of basic amino acid residue (Arg + Lys) was 64%, and the percentage of acidic amino acid residue (Asp+Glu) was 61%. The half- lives (in vivo) in mammals, yeasts and Escherichia coli were 30h, 20h and 10h respectively, the instability index was 42.98%, the fat solubility index was
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84.29%, the amphiphilic index was -0.072. Protein post-translational modification (PTM) plays a critical role in cellular control mechanism, mainly including
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phosphorylation, glycosylation and acylation. We found 25 hits on the TgIMP1
and 2 sumoylation site (Table 1).
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3.2.TolB protein secondary structure prediction
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sequence: 3 N-glycosylation sites, 4 O-glycosylation sites, 47 Phosphorylation sites
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We used three online services PRABI, CFSSP and Jpred to predict the secondary structure of TolB. The results showed that Alpha helix accounted for 5.25% to 25.98%, with the average 14.74%, Extended strand accounted for 23.26% to 54.59%,
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with the average 31.89%, Random coil accounted for 16.27% to 65.62%, with the average 49.02%, Beta turn accounted for 0 to 14.44%, with the average 2.38%.
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Extended strand and random coil took up a large proportion. These secondary structures are conducive to the formation of antigenic epitopes (Table S1).
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3.3.Modeling of three-dimensional structure We used an on- line service SWISS-MODEL to predict the 3D structures of TolB. The modeling and 3D structure of TolB was based on 1crz.1.A, which encoding Escherichia coli TolB protein. This sequence has a 39.05% identity with TolB (Fig.1A-B). There are many β-turns and random coils on TolB protein. Furthermore, we labeled the previously selected epitopes on the 3D model using Vector NTI software (Fig.2A-B). The results showed that TolB is a barrel- like structure and the surface exposed a lot of flexible areas, which is suitable for the formation of antigenic epitopes.
ACCEPTED MANUSCRIPT 3.4.Amplification of TolB gene The TolB gene was amplified from the genome of A.baumannii ATCC 17978 and five clinical isolates by PCR. The gene fragment of about 1145bp was obtained by 1% agarose gel electrophoresis, which was consistent with the expectation (Fig.3).
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3.5.Alignment of TolB gene sequence
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The 4 gene fragments were sequenced and aligned with the software, including ATCC17978, Ab1, Ab2 and Ab3, the TolB proteins from these strains share 96.2%
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similarity (Fig.4). The results showed that TolB gene was highly conserved among
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different strains. 3.6.Prediction of Linear-B cell epitopes on TolB
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Online service IEDB was used to analyze the flexibility, antigenicity, accessibility and epitope of TolB protein (Fig.5). In order to improve the reliability of the
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prediction results, we intercept the positive value higher than the threshold set by the software itself. Based on the prediction results of epitope, and considering the good
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flexibility, strong accessibility and high antigenicity index, we finally selected 7 peptides as the most promising epitopes (Fig.6).
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3.7.Prediction of T cell epitopes on TolB We used online service IEDB to predict the T cell epitopes on TolB. The results are given in units of IC50nM for combinatorial library and SMM_align. Therefore a lower number indicates higher affinity. This high affinity is necessary for potential T cell epitopes. The predicted output showed that there were many potential T cell epitopes on TolB protein, including CD4+ T cell epitopes binding to MHC class Ⅱ molecules and CD8+ T cell epitopes binding to MHC classⅠ(Table S2). 3.8.Screening of antigen epitopes
ACCEPTED MANUSCRIPT Six epitopes, including four B cell epitopes, one CD4+ T cell epitope and one CD8+ T cell cell epitope, were screened by IEDB server, ABCpred and BepiPred softwares (Table 2). Further, these epitopes are labeled on the predicted 3D structure and found that they all exist on the protein surface.
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3.9.Recombination of antigen epitopes These six antigenic epitopes can form a total of Seven hundred and twenty
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combinations schemes, through the DNAstar Protean analysis, and finally found this
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arrangement: epitope2-epitope5-epitope4-epitope6-epitope1-epitope3, was the best combination scheme (Fig.7). The polypeptide sequence of the designed recombinant
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epitope vaccine was: TDGEQPKGGGGMLFTASMNGGGGDTEARYTPGGGGRIA YVLR NPATPAERGGGGGSPQIGGGGLDESPSFS. Corresponding
nucleotide
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sequence was: CAGATGGTGAACAACCAAAAGGTGGCGGTGGCATGCTATTT ACAGCATCGATGAATGGTGGCGGTGGC GATACTGAAGCGCGTTACACACCAGG TGGCGGTGGCCGTATCGCTTATGTATTACGTAATCCTGCCACTCCTGCTGA
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ACGTGGTGGCGGTGGC GGCTCGCCTCAAATTGGTGGCGGTGGCTTAGATG
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AATCACCAAGTTTCTCA.
3.10. Verification of antigenic epitopes
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Antibody recognition experiments showed that the serum of mice infected with A.baumannii ATCC 17978 could specifically recognize the four B cell epitopes, and the specificity of epitope 1 is the strongest, as shown in Figure 8A. Lymphocyte proliferation assay results showed that both epitopes 5 and 6 were able to stimulate lymphocyte proliferation, and epitope 6 stimulated a higher level of lymphocyte proliferation, as shown in Figure 8B. More importantly, the recombinant epitopes performed better than the single epitopes in both experiments. Therefore, the antigen epitopes screened by bioinformatics software was proved to be effective. 4. Discussion
ACCEPTED MANUSCRIPT The Tol-Pal complex is composed of five core proteins: TolQ, TolR, TolA, TolB and Pal. The most classic function of the Tol-Pal system is in maintaining the integrity of the cell envelope of E. coli (Godlewska et al.,2009). In addition, the Tol-Pal system is potentially involved in cell division. It was reported that the disruption of any of the genes within the tol-pal cluster resulted in a variety of
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phenotypic changes in E. coli (Bernadac et al.,1998), Caulobacter crescentus (Yeh et al.,2010), Erwinia chrysanthemi (Dubuisson et al.,2005), and Pseudomonas putida
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(Llamas et al.,2000). These changes included as follows: a reduction in the kinetics of bacterial growth, changes in motility, decreased virulence. Turner et al. (2015) found
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that the disruption of H.pylori tolB and pal severely affected membrane integrity and tolB mutants displayed increased amounts of Outer membrane vesicles (OMV)
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release. OMVs have been implicated as mediators of various types of responses by the host immune system, including the production of pro-inflammatory cytokines,
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activation of antigen presenting cells (APCs), and the induction of B and T-cell responses (Schroeder and Aebischer, 2009). TolB is an allosteric β-propeller protein
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that acts in the bacterial periplasmic space and may interact with other proteins. In the biofilm- forming bacterium Pseudomonas aeruginosa, tolB is one of the most
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abundant proteins and is essential for bacterial growth, resistance and pathogenicity (Lo Sciuto A et al.,2014), meanwhile, tolB is required for the growth of E. coli at high hydrostatic pressure (Black et al.,2013). Since TolB is a soluble protein residing in the
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periplasmic space, it should be more accessible to drugs than cytosolic targets, and drug binding to TolB could delay later extrusion by efflux pumps. This suggesting that antiTolB therapy could be ultimately beneficial for the treatment of different bacterial infections.
Bioinformatics, a new interdisciplinary subject emerging with the development of the human genome project, has now become a powerful tool for genomics and proteomics research (Romano et al.,2011). Through analysis of amino acid sequence, basic physical and chemical properties, transmembrane structure, signal peptide cleavage site, subcellular localization, protein post-translational modification, 3D
ACCEPTED MANUSCRIPT structure and antigenicity, we are able to screen out potential antigen epitopes, and therefore speed up vaccine research so as to avoid wasteful duplication of effort and ineffective experimental process. Due to its high efficiency and economy, Biophotonics has been widely applied in protein structure analysis, protein function research and antigenic epitope prediction. In this study, bioinformatics software was
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used to analyze the basic physical and chemical properties, transmembrane domain, signal peptide cleavage site, subcellular localization and post-translational
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modification of TolB protein. The basic information acquired will help to determine whether TolB is suitable to be an excellent antigen. Furthermore, special prediction
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software was also used to predict the 2-D and 3-D structures of TolB protein, and the potential B cell epitopes and T cell epitopes on it. The more advanced information
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will play a guiding role for the follow- up experimental design and process, and
animal immunity experiment.
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5. Conclusion
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improve the feasibility of the experiment. At the same time, it lays a foundation for
In this study, we successfully amplified and cloned TolB genes from the standard
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strains and five clinical isolates, verified that it was highly conserved among these strains, and finally designed a good recombinant epitope vaccine. Those results further increased its possibilities to become an excellent antigen. In conclusion, it is a
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long and difficult process to develop a vaccine that could be effective against a pathogenic microbial infection，which requires a large number of screening and comparison experiments, while the application of bioinformatics can promote this process. Funding This work was supported in part by grants from Shandong Provincial Natural Science Foundation (Grant No. ZR2017BH097) and the Research Foundation of Qilu Hospital of Shandong University (Grant No. QDKY2015QN01 and QDKY2016LH02).
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ACCEPTED MANUSCRIPT Fig.1 Prediction of flexibility, antigenicity, accessibility and epitope of TolB protein. Fig.2 Prediction of B-cell epitopes in the TolB protein. Fig.3A-B Modeling the 3-D structure of TolB. (A) The modeling and 3D structure of TolB was based on 1crz.1.A, which encoding Escherichia coli TolB protein; (B)
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Rotate 90 degrees clockwise.
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Fig.4A-B Label the selected epitope on the 3D structure. (A) The distribution of potential epitopes on TolB is marked with yellow lines; (B) The distribution of
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potential epitopes on TolB is marked with yellow balls.
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Fig.5 Amplification of TolB gene. M:marker, 1: ATCC17978, 2: Ab1, 3:Ab2, 4: Ab3, 5: Ab4, 6: Ab5.
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Fig.6 Alignment of TolB gene sequences from A. baumannii ATCC17978 and the other five clinical isolates.
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Fig.7 Bioinformatics analysis of the recombinant epitopes.
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Fig.8A-B Verification of antigenic epitopes. (A) Experimental verification of B cell epitopes; (B) Experimental verification of T cell epitopes. PBS: Phosphate Buffered Saline, ConA: Concanavalin A, P1: epitope1, P2: epitope2, P3: epitope3, P4: epitope4,
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ACCEPTED MANUSCRIPT Table 1 Prediction of Physical and chemical characteristics, protein modification sites
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of TolB.
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Tools
n Number 381
ProtPar
of amino
am
acids
( Gastei
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ger et
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Molecula 41828.94
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r weight Theoretic 8.81
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al pI Aliphatic 74.54
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index Grand -0.463
5) ProtPar am ProtPar am ProtPar am ProtPar am
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average
al.,200
of
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hydropat hicity
Instabilit 36.33
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y index
Estimated 30 hours (mammalian reticulocytes) half-life
ProtPar am ProtPar am
>20 hours (yeast) >10 hours (Escherichia coli)
signal No peptide
SignalP 4.1 ( Peters
en
et
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NetNGl
lation
yc 1.0
sites
( Joshi
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Phosphor Tyrosine:10,51,68,103,167,246
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Gupta , 2015) NetOGlyc 4.0
( Steent
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al.,201 3) NetPhos 3.1 ( Blom
et al.,199 9)
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Threonine:24,107,126,132,138,159,172,191,212,228,235,242 ,247,279,289,324,326,358 Serine:20,25,26,89,114,148,155,169,192,203,208,214,227,23 8,262,274,283,291,304,327,331,335,354
N-termin No al acetylatio n
NetAcet 1.0 (Kieme
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ACCEPTED MANUSCRIPT Table 2 Prediction of epitope onTolB protein. Type
Start position
End position
Sequence
epitope1
B cell
261
265
GSPQI
epitope2 epitope3
B cell B cell
138 328
144 335
TDGEQPK LDESPSFS
epitope4
B cell
241
248
DTEARYTP
epitope5
+
CD 8 T cell
209
216
MLFTASMN
epitope6
CD+4 T cell
116
130
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ACCEPTED MANUSCRIPT Highlights Bioinformatics is a powerful tool for predicting antigenic epitopes on proteins TolB has excellent B-cell epitopes and potential Th-cell epitopes
A good recombinant epitope vaccine was designed and verified by experiments
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Xiaojie Song, Hua Zhang, Dongsheng Zhang, Weifeng Xie, Guanghui Zhao PII: DOI: Reference:
S1567-1348(18)30197-7 doi:10.1016/j.meegid.2018.04.019 MEEGID 3485
To appear in:
Infection, Genetics and Evolution
Received date: Revised date: Accepted date:
21 September 2017 9 April 2018 13 April 2018
Please cite this article as: Xiaojie Song, Hua Zhang, Dongsheng Zhang, Weifeng Xie, Guanghui Zhao , Bioinformatics analysis and epitope screening of a potential vaccine antigen TolB from Acinetobacter baumannii outer membrane protein. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Meegid(2018), doi:10.1016/j.meegid.2018.04.019
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Bioinformatics analysis and epitope screening of a potential vaccine antigen TolB from Acinetobacter baumannii outer membrane protein #
#
Xiaojie Song 1 , Hua Zhang 2 , Dongsheng Zhang 3, Weifeng Xie4, Guanghui Zhao5
Department of Respiratory, Qilu Hospital of Shandong University, Qingdao,
Shandong Province 255036, People’s Republic of China
Department of Geriatric, Qingdao Municipal Hospital, Qingdao, Shandong Province
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2
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266011, People’s Republic of China
Department of Hepatobiliary Surgery, Qingdao Municipal Hospital, Qingdao,
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3
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1
*
Shandong Province 266011, People’s Republic of China Department of ICU, Qingdao Municipal Hospital, Qingdao, Shandong Province
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4
266011, People’s Republic of China
Clinical Laboratory Medicine Center, Qilu Hospital of Shandong University, Jinan,
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5
*
Equal contribution
Corresponding author
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#
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Shandong Province 250012, People’s Republic of China
Correspondence Address: Guanghui Zhao, Ph.D Clinical Laboratory Medicine Center, Qilu Hospital of Shandong University, Jinan, Shandong Province 250012, PR China. Fax: +86 532 66850876. Email:[email protected]
ACCEPTED MANUSCRIPT Abstract The clinical isolation rate of multidrug-resistant or pan-resistant Acinetobacter baumannii (A. baumannii) is increasing, resulting that optional antibiotics are very limited in clinical practice. To deal with such a dilemma in treatment, the development of effective vaccines serves as a good strategy. Outer membrane proteins
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(Omp) often contain potential excellent vaccine antigens, and NCBI has published more than 300 Omp sequences of A. baumannii (including the duplicates). To
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accurately screen out the potential excellent antigen molecules from a large number of
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sequences, and avoid repetitive experimental processes is of great significance. In this study, we used the bioinformatics software to give extensive predictions of TolB
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protein. Results suggest it is a potential vaccine antigen. We then cloned the TolB gene fragments and confirmed it was highly conserved among the strains. Finally, we
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designed a good recombinant epitopes and conducted experimental verification. These findings provided grounds for animal immunology experiments in the future, and
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showed an orientation for the efficient development of A.baumannii vaccine.
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Keywords: Acinetobacter baumannii; TolB; Bioinformatics; T cell Epitope; B cell Epitope
ACCEPTED MANUSCRIPT 1. Introduction Acinetobacter baumannii is conditioned pathogens that can cause pneumonia, bloodstream infections, urinary tract infections, secondary meningitis, wound infections (Munoz-Price and Weinstein, 2008). The clinical isolation rate of multi-drug and pan-drug resistance strains is increasing, resulting that optional
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antibiotics are very limited in clinical practice. To deal with such a dilemma in treatment, the development of effective vaccines serves as a good strategy. The
multiple antigenic
vaccine, single subunit protein
vaccine,
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cell vaccines,
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development process of Acinetobacter baumannii Vaccine includes inactivated whole
polysaccharide vaccine and DNA vaccine. McConnell and Pachón (2010) and Harris
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et al. (2013) prepared inactivated whole cell vaccines using ATCC19606 strain and LAC-4 strain respectively. Through intramuscular injection, it can induce specific
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antibody production, alleviate tissue bacterial load and improve mouse survival. The results showed that inactivated whole cells had partial immunoprotection. However, it
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also has shortcomings, such as low immunogenicity, excessive invalid ingredients, potential virulence recovery. In the case of multiple antigenic component vaccines,
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McConnell et al. (2011) used the outer membrane complex to immunize mice and induce humoral and cellular immune responses. Huang et al. (2014) used a clinical multidrug-resistant strain to prepare outer membrane vesicles, which significantly
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increased the IgG and IgA in mice through intramuscular injection and intranasal perfusion. In the case of single protein component vaccines, Fattahian et al. (2011) immunized mice three times with recombinant biofilm-associated proteins, resulting with a humoral immune response, a significant increase in specific IgG antibody titers, and a significant decrease in the mortality of infected mice. Goel and Kapil (2001) immunized mice with iron regulators to obtain monoclonal antibodies. The results showed that antibody has the effect of antibiotics and regulators, which can effectively prevent iron absorption process. Luo et al. (2012) used recombinant outer membrane protein A to immunize the diabetic mouse model and induced humoral immune response. The 28-day survival rate of the infected mice was increased to 50%,
ACCEPTED MANUSCRIPT and the bacterial load of multiple tissues was significantly decreased. rOmpA antibody has an immune conditioning effect, which can enhanced phagocytosis of macrophages. Bentancor et al. (2012) immunized mice with trimeric autotransporters as candidate vaccines. The results showed that Ata antibody could reduce the adhesion of A.baumannii, enhance the phagocytosis of multinucleated neutrophils, mediate
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complement-dependent bactericidal effect and reduce tissue bacterial load. Russo et al. (2013) used the purified K1 capsular polysaccharide to immunize mice. The results
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showed that K1 antiserum could enhance phagocytosis mediated by PMNs and improve the clearance rate of bacteria. Although single protein component vaccine
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can induce a humoral or cellular immune response, it does not have sufficient protective power so it is difficult to apply to multiple serotypes of infection. DNA
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vaccine is a new type of vaccine. Compared with the traditional inactivated whole cell vaccine or single protein component vaccine, it showed superiority: firstly, DNA
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vaccine introduced into the host can continue to express specific protein antigen components, and then stimulate the host to produce a stro ng humoral immune and
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cellular immune response (Lever et al.,2017); Secondlly, the DNA vaccine is easy to construct recombinant multi- gene fragments, construction of recombinant; DNA
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vaccines with excellent epitopes can significantly improve the effectiveness against polyclonal infection (Wu et al.,2016). So far, NCBI has published more than 300 strains of acinetobacter baumannii
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outer membrane protein gene sequences (including repetitive sequences), involving how to accurately screen out potential excellent antigen molecules from a large number of sequences so as to further build its recombinant epitope vaccine to verify its immune protection effect, which is a very meaningful job. Based on that, bioinformatics software was applied to screen out potential antigen with good hydrophilicity, strong plasticity, high accessibility, high antigenicity index and numerous T cells and B cell epitopes. We finally screened the TolB protein, one of the components of the Tol-Pal system for follow-up studies.
ACCEPTED MANUSCRIPT 2. Materials and Methods 2.1.Basic physical and chemical properties prediction The gene sequences (Gene ID: 4917606) and amino acids (WP_011859984.1) sequences of TolB were obtained from the GenBank (https://www.ncbi.nlm.nih.gov).
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GENSCAN program was used to search the open reading frame (ORF) of TolB gene (http://genes.mit.edu/GENSCAN). The online server ProtParam (http://web.expasy.
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org/protparam/) was used to analyze basic physical and chemical properties of TolB
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protein, including molecular weight, theoretical isoelectric point, estimated half life in mammals, yeast, Escherichia coli, instability index, aliphatic index and grand average
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of hydropathicity. TMHMM server (http://www.cbs.dtu.dk/services/ TMHMM2.0/) and SignalP server (http://www.cbs.dtu.dk/services/SignalP/) were used to analyze its
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transmembrane structure and signal peptide, respectively. While NetNGlyc 1.0 server(http://www. cbs. dtu.dk/services/NetNGlyc) and NetPhos 3.1 server (http://ww w.cbs.dtu. dk/services/ NetPhos/) were used to analyze the post-translational
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2.2.Structural prediction
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modification sites of it, including N-glycosylation sites and phosphorylation sites.
Two online services, SWISS-MODEL (http: // swissmodel. Expasy. Org /) and
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SOPMA (http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/ npsa_ sopma. html), were employed to predict secondary structure and 3D structure of TolB, the protein structure acquired was marked with Vector NTI software. 2.3.Bacterial strains and mice In order to investigate the conservation of TolB protein between different strains, we randomly selected five clinical isolates, named Ab1, Ab2, Ab3, Ab4 and Ab5 respectively, and one standard strain ATCC 17978. Six-week-old female BALB/c mice were purchased from the Shandong University Laboratory Animal Center. All the mice were maintained under specific-pathogen- free conditions. All the animal
ACCEPTED MANUSCRIPT experiments were approved by the Animal Ethics Committee of Shandong University Qilu Hospital. 2.4.DNA extraction and PCR Bacterial DNA was extracted from the six A. baumannii strains through DNA
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Extraction Kit. A pair of primers were designed according to the TolB gene of A. baumannii ATCC 17978. Restriction enzyme recognition sequence and protective
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base were inserted into the 5ˊ-end of both the forward and the reverse primers
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(Forward primer: 5ˊ- CGGGGTACCGTGCCTTTTAATAACGACAATGGA -3ˊ , Reverse primer 5ˊ - CCCAAGCTTTTATTTAGGTGCCCAGGCTGG -3ˊ , Kpn I
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and HindIII recognition sites were introduced and underlined). The TolB gene of the other five clinical isolates were also amplified by this primer. Specifically, the primer
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concentration was diluted to 10 μM with sterile ddH2 O, a 50μl PCR reaction system was prepared in a 0.5 ml tube: Forward Primer 1μl, Reverse Primer 1μl, PCR Mix (TransGen, China) 25μl, DNA template 2μl, ddH2 O 21μl. Then run the PCR program
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of initial denaturation at 94℃for 5 min, then undergo a proceeding of 35 reaction cycles as denaturation at 94℃for 30 s, anneal at 57℃ for 30 s, polymerization at 72℃
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for1 min, followed by a final polymerization at 72℃ for 10 min. After amplification, the PCR products were identified by 1% agarose gel electrophoresis.
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2.5.TolB gene cloning and identification PCR products were cloned into the pEASY-T1 simple vector (TransGen Biotech, China) to generate a recombinant cloning plasmid. After preparation of DH5α competent cells, 50μl recombinant plasmid was incubated with it on ice for 20 min, heat shock at 42℃ for 30s, then immediately was put on ice for 2min. The reaction system was added to 250 μl of LB liquid medium and incubated at 200 rpm, 37℃for 1h, and then transferred to the LA plate. A single white colony was picked and verified by PCR, positive colonies were inoculated into 3 ml LK medium and shaken overnight at 180 rpm, 37℃. The recombinant cloned plasmid pEASY-TolB was
ACCEPTED MANUSCRIPT extracted from the cultures, and then identified by double digestion using Kpn I and HindIII. The digested product was sequenced to obtain the gene sequence. 2.6.Sequencing and alignment of gene sequence The PCR products of A. baumannii ATCC17978 and the other five clinical
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isolates were sequenced and compared for homology. Briefly, nucleotide BLAST of NCBI was used to analyze the TolB gene conserve among different strains. A highly
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conserved gene sequence will help the potential vaccine antigen to maintain
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suitability for polyclonal strains.
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2.7.Epitope screening
The protein sequence of A. baumannii ATCC17978 was selected as analysis object.
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IEDB server（http://tools. immuneepitope.org/main/ html/tcell _tools.html）was used to analyze potential B cell epitopes on TolB protein and predicted items are mainly related to flexibility, antigenicity, accessibility and epitope, meanwhile, two online
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servers ABCpred and BepiPred were used to predict peptide segment of B cell epitopes. T cell epitopes were also predicted by IEDB server, including potential
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CD4+ T cell epitopes binding to MHC class Ⅱ molecules and CD8+ T cell epitopes binding to MHC classⅠwere screened out.
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2.8.Epitope recombination
In order to achieve the best immunogenicity, predicted epitopes are reorganized by series connection, the conjugate was a flexible peptide GGGG. All the arrangement schemes were analyzed and compared by DNAstar software, The optimal combination of recombinant epitopes selected by DNAstar software. The ideal combination is that all epitopes in the recombinants remain relatively independent, no new epitope is formed, no epitope is drifted, and antigenicity index of each epitope are high.
ACCEPTED MANUSCRIPT 2.9. Experimental verification of B cell epitopes B cell epitopes of TolB were detected by ELISA. Briefly, Blood was collected from the eyelids of BALB/c mice that had been infected with A.baumannii ATCC 17978 and then the serum was separated by centrifugation. The B-cell epitopes (epitopes 1 to 4) were coated in 96-well plates (10μg/ml) and PBS was used as a
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control well, then add 50μl mouse serum to each well. After washing, continue to add goat anti- mouse IgG horseradish peroxidase labeled secondary antibody (100μl/well).
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450 nm using a microplate reader.
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2.10. Experimental verification of T cell epitopes
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The reaction was terminated after 30 minutes at 37℃. The OD value was detected at
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Lymphocyte proliferation assays were used to identify TolB protein CD8+ T cell epitopes and CD4+ T cell epitopes. The BALB/c mice infected with A.baumannii ATCC 17978 selected and the spleen was removed in a sterile environment. The
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spleen was grinded into cell suspension with a 200- mesh copper mesh, and the red blood cells in the spleen were removed using red blood cell lysate. The lymphocyte
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separation solution was added to separate the lymphocytes and resuspended in RMPI-1640 cell culture medium containing 10% fetal bovine serum. The cell suspension was added into a 96-well plate (100 μl/well) and incubated at 37℃, 5%
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CO2 for 24 h. The predicted CD8+T cell epitope (epitope 5) and CD4+ T cell epitope (epitope 6) were added into the plate and stimulated with 1 μg/well ConA as a positive control. After 24 hours, CCK-8 reagent (10 μl/well) was added and the culture was continued for 4h. The OD value at 450 nm was detected by a microplate reader. 3. Results 3.1.Physical and chemical characteristics of TolB
ACCEPTED MANUSCRIPT The TolB protein has 381 amino acids with a molecular weight of 41.8289 kDa, the theoretical isoelectric point was 6.34, the percentage of basic amino acid residue (Arg + Lys) was 64%, and the percentage of acidic amino acid residue (Asp+Glu) was 61%. The half- lives (in vivo) in mammals, yeasts and Escherichia coli were 30h, 20h and 10h respectively, the instability index was 42.98%, the fat solubility index was
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84.29%, the amphiphilic index was -0.072. Protein post-translational modification (PTM) plays a critical role in cellular control mechanism, mainly including
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phosphorylation, glycosylation and acylation. We found 25 hits on the TgIMP1
and 2 sumoylation site (Table 1).
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3.2.TolB protein secondary structure prediction
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sequence: 3 N-glycosylation sites, 4 O-glycosylation sites, 47 Phosphorylation sites
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We used three online services PRABI, CFSSP and Jpred to predict the secondary structure of TolB. The results showed that Alpha helix accounted for 5.25% to 25.98%, with the average 14.74%, Extended strand accounted for 23.26% to 54.59%,
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with the average 31.89%, Random coil accounted for 16.27% to 65.62%, with the average 49.02%, Beta turn accounted for 0 to 14.44%, with the average 2.38%.
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Extended strand and random coil took up a large proportion. These secondary structures are conducive to the formation of antigenic epitopes (Table S1).
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3.3.Modeling of three-dimensional structure We used an on- line service SWISS-MODEL to predict the 3D structures of TolB. The modeling and 3D structure of TolB was based on 1crz.1.A, which encoding Escherichia coli TolB protein. This sequence has a 39.05% identity with TolB (Fig.1A-B). There are many β-turns and random coils on TolB protein. Furthermore, we labeled the previously selected epitopes on the 3D model using Vector NTI software (Fig.2A-B). The results showed that TolB is a barrel- like structure and the surface exposed a lot of flexible areas, which is suitable for the formation of antigenic epitopes.
ACCEPTED MANUSCRIPT 3.4.Amplification of TolB gene The TolB gene was amplified from the genome of A.baumannii ATCC 17978 and five clinical isolates by PCR. The gene fragment of about 1145bp was obtained by 1% agarose gel electrophoresis, which was consistent with the expectation (Fig.3).
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3.5.Alignment of TolB gene sequence
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The 4 gene fragments were sequenced and aligned with the software, including ATCC17978, Ab1, Ab2 and Ab3, the TolB proteins from these strains share 96.2%
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similarity (Fig.4). The results showed that TolB gene was highly conserved among
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different strains. 3.6.Prediction of Linear-B cell epitopes on TolB
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Online service IEDB was used to analyze the flexibility, antigenicity, accessibility and epitope of TolB protein (Fig.5). In order to improve the reliability of the
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prediction results, we intercept the positive value higher than the threshold set by the software itself. Based on the prediction results of epitope, and considering the good
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flexibility, strong accessibility and high antigenicity index, we finally selected 7 peptides as the most promising epitopes (Fig.6).
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3.7.Prediction of T cell epitopes on TolB We used online service IEDB to predict the T cell epitopes on TolB. The results are given in units of IC50nM for combinatorial library and SMM_align. Therefore a lower number indicates higher affinity. This high affinity is necessary for potential T cell epitopes. The predicted output showed that there were many potential T cell epitopes on TolB protein, including CD4+ T cell epitopes binding to MHC class Ⅱ molecules and CD8+ T cell epitopes binding to MHC classⅠ(Table S2). 3.8.Screening of antigen epitopes
ACCEPTED MANUSCRIPT Six epitopes, including four B cell epitopes, one CD4+ T cell epitope and one CD8+ T cell cell epitope, were screened by IEDB server, ABCpred and BepiPred softwares (Table 2). Further, these epitopes are labeled on the predicted 3D structure and found that they all exist on the protein surface.
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3.9.Recombination of antigen epitopes These six antigenic epitopes can form a total of Seven hundred and twenty
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combinations schemes, through the DNAstar Protean analysis, and finally found this
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arrangement: epitope2-epitope5-epitope4-epitope6-epitope1-epitope3, was the best combination scheme (Fig.7). The polypeptide sequence of the designed recombinant
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epitope vaccine was: TDGEQPKGGGGMLFTASMNGGGGDTEARYTPGGGGRIA YVLR NPATPAERGGGGGSPQIGGGGLDESPSFS. Corresponding
nucleotide
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sequence was: CAGATGGTGAACAACCAAAAGGTGGCGGTGGCATGCTATTT ACAGCATCGATGAATGGTGGCGGTGGC GATACTGAAGCGCGTTACACACCAGG TGGCGGTGGCCGTATCGCTTATGTATTACGTAATCCTGCCACTCCTGCTGA
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ACGTGGTGGCGGTGGC GGCTCGCCTCAAATTGGTGGCGGTGGCTTAGATG
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AATCACCAAGTTTCTCA.
3.10. Verification of antigenic epitopes
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Antibody recognition experiments showed that the serum of mice infected with A.baumannii ATCC 17978 could specifically recognize the four B cell epitopes, and the specificity of epitope 1 is the strongest, as shown in Figure 8A. Lymphocyte proliferation assay results showed that both epitopes 5 and 6 were able to stimulate lymphocyte proliferation, and epitope 6 stimulated a higher level of lymphocyte proliferation, as shown in Figure 8B. More importantly, the recombinant epitopes performed better than the single epitopes in both experiments. Therefore, the antigen epitopes screened by bioinformatics software was proved to be effective. 4. Discussion
ACCEPTED MANUSCRIPT The Tol-Pal complex is composed of five core proteins: TolQ, TolR, TolA, TolB and Pal. The most classic function of the Tol-Pal system is in maintaining the integrity of the cell envelope of E. coli (Godlewska et al.,2009). In addition, the Tol-Pal system is potentially involved in cell division. It was reported that the disruption of any of the genes within the tol-pal cluster resulted in a variety of
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phenotypic changes in E. coli (Bernadac et al.,1998), Caulobacter crescentus (Yeh et al.,2010), Erwinia chrysanthemi (Dubuisson et al.,2005), and Pseudomonas putida
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(Llamas et al.,2000). These changes included as follows: a reduction in the kinetics of bacterial growth, changes in motility, decreased virulence. Turner et al. (2015) found
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that the disruption of H.pylori tolB and pal severely affected membrane integrity and tolB mutants displayed increased amounts of Outer membrane vesicles (OMV)
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release. OMVs have been implicated as mediators of various types of responses by the host immune system, including the production of pro-inflammatory cytokines,
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activation of antigen presenting cells (APCs), and the induction of B and T-cell responses (Schroeder and Aebischer, 2009). TolB is an allosteric β-propeller protein
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that acts in the bacterial periplasmic space and may interact with other proteins. In the biofilm- forming bacterium Pseudomonas aeruginosa, tolB is one of the most
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abundant proteins and is essential for bacterial growth, resistance and pathogenicity (Lo Sciuto A et al.,2014), meanwhile, tolB is required for the growth of E. coli at high hydrostatic pressure (Black et al.,2013). Since TolB is a soluble protein residing in the
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periplasmic space, it should be more accessible to drugs than cytosolic targets, and drug binding to TolB could delay later extrusion by efflux pumps. This suggesting that antiTolB therapy could be ultimately beneficial for the treatment of different bacterial infections.
Bioinformatics, a new interdisciplinary subject emerging with the development of the human genome project, has now become a powerful tool for genomics and proteomics research (Romano et al.,2011). Through analysis of amino acid sequence, basic physical and chemical properties, transmembrane structure, signal peptide cleavage site, subcellular localization, protein post-translational modification, 3D
ACCEPTED MANUSCRIPT structure and antigenicity, we are able to screen out potential antigen epitopes, and therefore speed up vaccine research so as to avoid wasteful duplication of effort and ineffective experimental process. Due to its high efficiency and economy, Biophotonics has been widely applied in protein structure analysis, protein function research and antigenic epitope prediction. In this study, bioinformatics software was
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used to analyze the basic physical and chemical properties, transmembrane domain, signal peptide cleavage site, subcellular localization and post-translational
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modification of TolB protein. The basic information acquired will help to determine whether TolB is suitable to be an excellent antigen. Furthermore, special prediction
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software was also used to predict the 2-D and 3-D structures of TolB protein, and the potential B cell epitopes and T cell epitopes on it. The more advanced information
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will play a guiding role for the follow- up experimental design and process, and
animal immunity experiment.
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5. Conclusion
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improve the feasibility of the experiment. At the same time, it lays a foundation for
In this study, we successfully amplified and cloned TolB genes from the standard
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strains and five clinical isolates, verified that it was highly conserved among these strains, and finally designed a good recombinant epitope vaccine. Those results further increased its possibilities to become an excellent antigen. In conclusion, it is a
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long and difficult process to develop a vaccine that could be effective against a pathogenic microbial infection，which requires a large number of screening and comparison experiments, while the application of bioinformatics can promote this process. Funding This work was supported in part by grants from Shandong Provincial Natural Science Foundation (Grant No. ZR2017BH097) and the Research Foundation of Qilu Hospital of Shandong University (Grant No. QDKY2015QN01 and QDKY2016LH02).
ACCEPTED MANUSCRIPT References Black,S.L., Dawson,A., Ward,F.B. and Allen,R.J. (2013) PLoS One ,8, e73995. Blom, N., Gammeltoft, S. and Brunak, S. (1994) Journal of Molecular Biology,294,1351-1362. Bernadac, A., Gavioli, M., Lazzaroni, J.C., Raina, S. and Lloubès, R. (1998) J Bacteriol, 180,4872-4878. Bentancor, L.V., Routray, A., Bozkurt-Guzel, C., Camacho-Peiro, A., Pier,G.B. and Maira-Litrán, T. (2012) Infect Immun,80,3381-3388.
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Dubuisson,J.F., Vianney, A., Hugouvieux-Cotte-Pattat,N. and Lazzaroni,J.C. (2005) Microbiology, 151,3337-3347.
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Fattahian, Y., Rasooli, I., Mousavi Gargari, S.L., Rahbar, M.R., Darvish Alipour Astaneh,S.and Amani, J. (2011) Microb Pathog, 51,402-406.
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Yeh, Y.C., Comolli,L.R., Downing,K.H., Shapiro,L. and Mcadams,H.H. (2010) J Bacteriol, 192, 4847-4858. Zhao, Q., Xie, Y., Zheng, Y., Jiang, S., Liu, W., Mu, W., Zhao, Y., Xue,Y. and Ren, J. (2014) Nucleic Acids Research, 42,W325-W330.
ACCEPTED MANUSCRIPT Fig.1 Prediction of flexibility, antigenicity, accessibility and epitope of TolB protein. Fig.2 Prediction of B-cell epitopes in the TolB protein. Fig.3A-B Modeling the 3-D structure of TolB. (A) The modeling and 3D structure of TolB was based on 1crz.1.A, which encoding Escherichia coli TolB protein; (B)
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Fig.4A-B Label the selected epitope on the 3D structure. (A) The distribution of potential epitopes on TolB is marked with yellow lines; (B) The distribution of
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potential epitopes on TolB is marked with yellow balls.
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Fig.5 Amplification of TolB gene. M:marker, 1: ATCC17978, 2: Ab1, 3:Ab2, 4: Ab3, 5: Ab4, 6: Ab5.
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Fig.6 Alignment of TolB gene sequences from A. baumannii ATCC17978 and the other five clinical isolates.
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Fig.7 Bioinformatics analysis of the recombinant epitopes.
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Fig.8A-B Verification of antigenic epitopes. (A) Experimental verification of B cell epitopes; (B) Experimental verification of T cell epitopes. PBS: Phosphate Buffered Saline, ConA: Concanavalin A, P1: epitope1, P2: epitope2, P3: epitope3, P4: epitope4,
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ACCEPTED MANUSCRIPT Table 1 Prediction of Physical and chemical characteristics, protein modification sites
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of TolB.
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Tools
n Number 381
ProtPar
of amino
am
acids
( Gastei
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Molecula 41828.94
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r weight Theoretic 8.81
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al pI Aliphatic 74.54
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5) ProtPar am ProtPar am ProtPar am ProtPar am
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average
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Instabilit 36.33
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Estimated 30 hours (mammalian reticulocytes) half-life
ProtPar am ProtPar am
>20 hours (yeast) >10 hours (Escherichia coli)
signal No peptide
SignalP 4.1 ( Peters
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NetNGl
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Phosphor Tyrosine:10,51,68,103,167,246
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Threonine:24,107,126,132,138,159,172,191,212,228,235,242 ,247,279,289,324,326,358 Serine:20,25,26,89,114,148,155,169,192,203,208,214,227,23 8,262,274,283,291,304,327,331,335,354
N-termin No al acetylatio n
NetAcet 1.0 (Kieme
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ACCEPTED MANUSCRIPT Table 2 Prediction of epitope onTolB protein. Type
Start position
End position
Sequence
epitope1
B cell
261
265
GSPQI
epitope2 epitope3
B cell B cell
138 328
144 335
TDGEQPK LDESPSFS
epitope4
B cell
241
248
DTEARYTP
epitope5
+
CD 8 T cell
209
216
MLFTASMN
epitope6
CD+4 T cell
116
130
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ACCEPTED MANUSCRIPT Highlights Bioinformatics is a powerful tool for predicting antigenic epitopes on proteins TolB has excellent B-cell epitopes and potential Th-cell epitopes
A good recombinant epitope vaccine was designed and verified by experiments
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