The diphtheria toxoid vaccination protects against the action of the toxin but does not protect against colonization and invasion by
There are three types of pili in
Pili are composed of three proteins: the main subunit forming the stem of pili, and two smaller subunits located at the base and at the end of the pili, e.g., SpaA-type pili is structured in such way that the SpaA pili protein creates a stem, SpaC is located at the end of pili, while SpaB is located along the stem and at the base (Ton-That and Schneewind 2003; Mandlik et al. 2008; Rogers et al. 2011). SpaA is important for the formation of the pile structure (Ton-That and Schneewind 2004). It has been proven that in the absence of SpaA protein, SpaB and SpaC are anchored in the cell wall as monomers (Mandlik et al. 2007).
The adhesion process of
Initially, the 67–72p adhesive protein was described as a ligand responsible for the adherence of
In our study, we analyzed the nucleotide and amino acid sequences of the genes encoding pili proteins which contribute to bacterial adherence to host cells, and also the gene encoding 67–72p protein involved in adhesion, colonization, and induction of the cell apoptosis in the early stage of infection, which should be used in a preliminary research for the finding of new vaccine antigens.
Strain | Biotype | Site of isolation | Year of isolation |
---|---|---|---|
27/E | Serous cyst contents | 2010 | |
40/E | Blood | 2014 | |
68/E | Endocarditis | 2015 | |
71/E | Wound | 2015 | |
73/E | Blood | 2016 | |
77/E | Wound | 2016 | |
78/E | Blood | 2016 | |
79/E | Blood and joint fluid | 2016 | |
86/E | Blood | 2017 | |
89/E | Wound | 2017 |
Table II Primers used in this study.
Gene | Primer | Sequence | Length of the amplified fragment |
---|---|---|---|
6772p1L | TGAAAAATAATTTAAGGAGTTCCAA | 695 bp | |
6772p1R | CAACCCACCAGTAACAGCAA | ||
6772p2L | CTGTTTTGCTGGTCGTAGCA | 841 bp | |
6772p2R | ACCTCATCAACCTGGTTTGC | ||
6772p3L | GAATCGTTGCAGCCCAAG | 699 bp | |
6772p3R | CCTTAAGCACTGGGTCGTTT | ||
6772p4L | CACCGACAACGTTGGTTACA | 844 bp | |
6772p4R | TTCTGGCTTGTCCCTGTTCT | ||
6772p5L | TCAAGCCGGAGTCCCAGA | 692 bp | |
6772p5R | TCAGTTGTGTCTGGTGAAAGG | ||
SpaI1L | GCGGAATCAACACCAACAC | 600 bp | |
SpaI1R | AAGCGCTTACGATCCAAGAA | ||
SpaI2L | ACACGGCCTTCCAAACTTC | 482 bp | |
SpaI2R | TGATATTGAGGCGTCGCTAA | ||
SapD1L | TCGCGAAGGTAAGAAATACTCA | 698 bp | |
SapD1R | CGTTTGTATCCGAGCCACTT | ||
SapD2L | GTCCAAAACAAGAGCGGAAA | 814 bp | |
SapD2R | GGTTCAGTGAAAACCCAGTTG | ||
SpaC1L | GCCTACTCTCACTGGCAAGG | 824 bp | |
SpaC1R | ACATGGCGATCTCCTGAAGT | ||
SpaC2L | TCGTGCAGGACGTACCAATA | 838 bp | |
SpaC2R | AACTGCACTGTGACCGAAAA | ||
SpaC3L | GGCATCATAAAGTGCAATCG | 808 bp | |
SpaC3R | TCACGTTGAGTTCTTCGTTCA | ||
SpaC4L | CATTCGTTTTTGTTCCGTGA | 850 bp | |
SpaC4R | GGTGTAGAAACGCCTCGAAA | ||
SpaC5L | CCAAATTCAACAGTTTGATTATCACT | 850 bp | |
SpaC5R | TTCCTGTCACTTACACCTGTCG | ||
SpaC6L | CAAAATACGGATTGGTTTCTGG | 845 bp | |
SpaC6R | AGCTGGCTGGAATTTCGAT | ||
SpaC7L | CAAAGGTGTCTTGGCCATTT | 687 bp | |
SpaC7R | TCACGCCAGTAAGTCTTGCTAA | ||
SpaC8L | CTGGCATCTGGATGTCATTG | 578 bp | |
SpaC8R | ACCGAACGTGCCTAGCGTA |
The PCR reaction was conducted in a total volume of 25 μl and the reaction mixture contained 0.5 μl genomic DNA, 12.5 μl HotStarTaq Master Mix (Qiagen), and 1 μl of 10 μM solution of each primer (Table II). The cycling conditions were as follows: initial denaturation at 95°C/10 min and 29 cycles of denaturation at 94°C/1 min, primer annealing at 52°C/45 s, primer extension at 72°C/1 min and a final elongation at 72°C/10 min.
The PCR products were enzymatically cleaned using an Exo-BAP Mix kit (EURx), according to the manufacturer’s procedure and then sent for sequencing.
The isolates from invasive diseases and wound infections were included in the study because a wound can be a portal of entry for invasive infections. Moreover, future vaccines against non-toxigenic
Comparison of the nucleotide sequences of all strains tested against the reference strain, given in percent (%). The sequences are presented according to the analysed fragments.
Target protein | Fragment | 27E | 40E | 68E | 71E | 73E | 77E | 78E | 79E | 86E | 89E |
---|---|---|---|---|---|---|---|---|---|---|---|
67–72p | 1 | 98.35 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 |
2 | 99.5 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 99.87 | |
3 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | |
4 | 99.01 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 97.90 | 97.90 | |
5 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | |
SapD | 1 | 98.91 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 |
2 | 98.37 | 27.52 | 100 | 100 | 100 | 100 | 100 | 100 | 99.86 | 99.86 | |
SpaI | 1 | − | 100 | 100 | 100 | 100 | 100 | 100 | 62.77 | 100 | 100 |
2 | − | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | |
SpaC | 1 | 72.78 | 99.87 | 99.87 | 99.87 | 99.87 | 99.87 | 99.87 | 99.87 | 99.87 | 99.87 |
2 | 97.11 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | |
3 | − | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | |
4 | − | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 99.75 | IS* | |
5 | 91.47 | 99.21 | 100 | 100 | 99.87 | 100 | 100 | 100 | 100 | 100 | |
6 | − | 100 | 100 | 100 | 100 | 100 | 99.87 | 99.87 | 100 | 100 | |
7 | 50.16 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | |
8 | − | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 |
In the fragment 4 of the gene encoding SpaC protein, the insertion sequence has been transposed
The nucleotide sequences of the genes investigated were translated into the amino acid sequences. We revealed that the identified mutations resulted in a reading frame shift or were synonymous and nonsynonymous substitutions (Table IV).
Comparison of amino acid sequences of all strains tested against the reference strain, given in percent (%).
Target protein | 27E | 40E | 68E | 71E | 73E | 77E | 78E | 79E | 86E | 89E |
---|---|---|---|---|---|---|---|---|---|---|
67–72p | 99 | 100 | 100 | 100 | 100 | 100 | 100 | 98 | 98 | 100 |
SapD | 97 | 99 | 100 | 100 | 100 | 100 | 100 | 100 | 99 | 99 |
SpaI | − | 100 | 100 | 100 | 100 | 100 | 100 | 84 | 100 | 100 |
SpaC | 48 | 99 | 99 | 99 | 69 | 99 | 69 | 69 | 69 | 51 |
In addition, we observed that a fragment of 1380 bp was inserted in the place of the gene encoding the SpaC protein in the 89/E strain (Table III). After sequencing the fragment No. 4 of
Analysis of the 67–72p, SpaC, SpaI and SapD protein affinity to MHC classes I and II, and linear B-cell epitopes in the first stage relied on the determination of the position of the proteins tested in the cell membrane and confirmation that all selected proteins were at least partially membranous or extracellular (Table V). Then, using the IEDB platform, it was observed that all proteins have high-affinity areas for MHC receptors of both classes and the fragments, which can be recognized by antibodies. The output for the prediction of the high- affinity MHC binding peptides is typically given either in the units of a predicted affinity (IC50 nanomolar) or as a percentile score reflecting the relative affinity of a selected peptide compared with a universe of random sequences. According to Paul et al. (2013), there are four categories of percentile ranks: 1) 0–0.30; 2) 0.30–1.25; 3) 1.25–5.0; and 4) 5.0–15.0. Their study proved that four pools of predicted peptides derived from the first two categories (0–0.30; 0.30–1.25) were immunogenic but finally, the transgenic mice in their study recognized only one peptide pool from the first category (0–0.30) (Paul et al. 2013). We can say that the smaller the percentile rank value, the higher the affinity. As for the IC50 value, according to the IEDB Solutions Centre guidelines, IC50 < 50 designs very high affinity, IC50 < 500 – high affinity, and IC50 < 5000 means low affinity (Fleri 2013). Accepting even the threshold of cutting off the percentile rank below 1 or IC50 below 50, we still could have at least 100 to several hundred regions with high affinity for each of the proteins (Table VI, Table VII).
Extracellular regions of individual proteins.
Region* | 67–72p | SpaC | SpaI | SapD | |||
---|---|---|---|---|---|---|---|
Start | 44 | 138 | 235 | 310 | 36 | 1 | 1 |
Stop | 57 | 169 | 257 | 987 | 1845 | 236 | 631 |
Amino acid positions
MHC class I epitopes predicted from the target proteins.
MHC I | |||||
---|---|---|---|---|---|
Target protein | Alleles | Start | End | Peptide | Percentile rank |
67–72p | HLA-A*02:06 | 6 | 14 | FTNDRFWSV | 0.06 |
67–72p | HLA-B*44:02 | 34 | 42 | SENDSSVEY | 0.06 |
67–72p | HLA-A*30:02 | 68 | 77 | RMASYWLDRY | 0.06 |
67–72p | HLA-B*44:02 | 9 | 17 | AEALSQVGI | 0.07 |
67–72p | HLA-A*02:06 | 27 | 36 | MILGALVPTV | 0.07 |
67–72p | HLA-A*68:01 | 1 | 9 | YAFTLPALR | 0.11 |
67–72p | HLA-A*01:01 | 43 | 51 | DTDSSTYTY | 0.11 |
67–72p | HLA-A*01:01 | 48 | 56 | YTTLTSLPY | 0.11 |
67–72p | HLA-B*44:03 | 34 | 42 | SENDSSVEY | 0.11 |
67–72p | HLA-B*57:01 | 58 | 66 | SSLAIGNAW | 0.12 |
SapD | HLA-B*44:03 | 65 | 74 | AEWQELDTWW | 0.06 |
SapD | HLA-B*07:02 | 4 | 13 | RPIWAGIGAF | 0.11 |
SapD | HLA-B*44:03 | 28 | 36 | KEGAYGLEY | 0.11 |
SapD | HLA-A*68:01 | 47 | 55 | NVFFKNNSR | 0.12 |
SapD | HLA-B*40:01 | 10 | 18 | IEAQISGSL | 0.17 |
SapD | HLA-A*24:02 | 70 | 79 | VWYAPQNIPF | 0.18 |
SapD | HLA-A*68:01 | 24 | 32 | DTVGSESAR | 0.2 |
SapD | HLA-B*51:01 | 50 | 58 | YPLHISYLV | 0.2 |
SapD | HLA-A*68:01 | 41 | 50 | EPAFGVTIPK | 0.22 |
SapD | HLA-A*68:01 | 17 | 26 | EAYVKNGAFK | 0.26 |
SpaC | HLA-A*11:01 | 658 | 666 | STNSVWIPK | 0.06 |
SpaC | HLA-A*01:01 | 220 | 229 | LSDDKPFDLY | 0.07 |
SpaC | HLA-B*53:01 | 233 | 241 | LPSEDDYYW | 0.1 |
SpaC | HLA-A*68:02 | 191 | 199 | EVVELENAV | 0.1 |
SpaC | HLA-A*02:06 | 1859 | 1867 | LVAAALWLV | 0.11 |
SpaC | HLA-A*23:01 | 88 | 96 | PYRFGIYTF | 0.11 |
SpaC | HLA-A*68:01 | 1436 | 1444 | NTTYSITYK | 0.11 |
SpaC | HLA-A*31:01 | 363 | 371 | RFKNARCQR | 0.11 |
SpaC | HLA-B*44:02 | 1054 | 1063 | AENTLSADAI | 0.11 |
SpaC | HLA-A*23:01 | 1578 | 1587 | SYTCTMPHLF | 0.12 |
SpaI | HLA-A*30:01 | 2 | 11 | KKTHLFRIPA | 0.08 |
SpaI | HLA-B*07:02 | 9 | 17 | IPAATTAAV | 0.1 |
SpaI | HLA-B*07:02 | 147 | 155 | RPAEYRRTL | 0.1 |
SpaI | HLA-B*57:01 | 109 | 117 | RSRLSDEVW | 0.12 |
SpaI | HLA-A*30:02 | 129 | 137 | VTGLPMGVY | 0.18 |
SpaI | HLA-A*02:01 | 137 | 145 | YLVSETPPA | 0.2 |
SpaI | HLA-A*02:03 | 20 | 29 | LLASGPIASA | 0.2 |
SpaI | HLA-A*02:06 | 153 | 161 | RTLDFLITV | 0.21 |
SpaI | HLA-B*51:01 | 196 | 205 | FPPVESSVTL | 0.24 |
SpaI | HLA-A*68:01 | 252 | 261 | LAIAGFLVQR | 0.32 |
MHC class II epitopes predicted from the target proteins.
MHC II | |||||
---|---|---|---|---|---|
Target protein | Alleles | Start | End | Peptide | Percentile rank |
67–72p | HLA-DRB3*01:01 | 660 | 674 | DGSVDLYEFDENDPV | 0.01 |
67–72p | HLA-DRB3*01:01 | 713 | 727 | MLARYHVDDARDFFT | 0.01 |
67–72p | HLA-DPA1*03:01/DPB1*04:02 | 15 | 29 | PQRRLTWLIPLLMIL | 0.01 |
67–72p | HLA-DPA1*03:01/DPB1*04:02 | 173 | 187 | STFSVLLVVAFLIAL | 0.01 |
67–72p | HLA-DRB1*07:01 | 49 | 63 | VDFRGVFNKVIATRI | 0.01 |
67–72p | HLA-DPA1*01/DPB1*04:01 | 165 | 179 | LPALRLVVSTFSVLL | 0.01 |
67–72p | HLA-DPA1*01/DPB1*04:01 | 267 | 281 | VISAVVAISFFSVIV | 0.01 |
67–72p | HLA-DRB1*09:01 | 100 | 114 | PVVQYRAAVEKGVHR | 0.02 |
67–72p | HLA-DRB3*01:01 | 712 | 726 | KMLARYHVDDARDFF | 0.02 |
67–72p | HLA-DPA1*01:03/DPB1*02:01 | 172 | 186 | VSTFSVLLVVAFLIA | 0.02 |
SapD | HLA-DRB3*01:01 | 181 | 195 | GKDSIPEHLDKNMYF | 0.01 |
SapD | HLA-DRB1*03:01 | 531 | 545 | PLHISYLVGDATIAR | 0.03 |
SapD | HLA-DQA1*04:01/DQB1*04:02 | 535 | 549 | SYLVGDATIARAKEI | 0.09 |
SapD | HLA-DRB1*03:01 | 444 | 458 | PSDALLPDSKMTVSL | 0.12 |
SapD | HLA-DQA1*03:01/DQB1*03:02 | 616 | 630 | VQDEAVTTAAEWQEL | 0.13 |
SapD | HLA-DRB1*03:01 | 442 | 456 | DLPSDALLPDSKMTV | 0.13 |
SapD | HLA-DQA1*03:01/DQB1*03:02 | 615 | 629 | DVQDEAVTTAAEWQE | 0.16 |
SapD | HLA-DRB5*01:01 | 640 | 654 | LLGILGIVGAFVLFR | 0.24 |
SapD | HLA-DQA1*04:01/DQB1*04:02 | 537 | 551 | LVGDATIARAKEILA | 0.27 |
SapD | HLA-DRB1*03:01 | 346 | 360 | TGTPKTIINDGHMDL | 0.29 |
SpaC | HLA-DRB3*01:01 | 157 | 171 | NDIDRGIKYDAVYFI | 0.01 |
SpaC | HLA-DRB3*01:01 | 505 | 519 | DNGTYRFKADTDAFK | 0.01 |
SpaC | HLA-DRB3*01:01 | 1427 | 1441 | EHSVDPWLLNTTYSI | 0.01 |
SpaC | HLA-DRB3*01:01 | 1699 | 1713 | VVINNVYTTDAEINI | 0.01 |
SpaC | HLA-DRB3*01:01 | 1804 | 1818 | EVTLDNYDADSGLIT | 0.01 |
SpaC | HLA-DRB3*01:01 | 1450 | 1464 | IKDRSYSNDVDIQAD | 0.02 |
SpaC | HLA-DRB1*03:01 | 769 | 783 | AGDIVKVVVDNTAKR | 0.03 |
SpaC | HLA-DRB1*09:01 | 1845 | 1859 | NGYLRWLLAGAAGLL | 0.04 |
SpaC | HLA-DPA1*03:01/DPB1*04:02 | 21 | 35 | LAMVMSIVLVPLIAA | 0.05 |
SpaC | HLA-DRB3*01:01 | 1432 | 1446 | PWLLNTTYSITYKCD | 0.07 |
SpaI | HLA-DRB3*01:01 | 147 | 161 | RPAEYRRTLDFLITV | 0.07 |
SpaI | HLA-DRB1*08:02 | 3 | 17 | KTHLFRIPAATTAAV | 0.18 |
SpaI | HLA-DRB3*01:01 | 151 | 165 | YRRTLDFLITVPAGM | 0.19 |
SpaI | HLA-DPA1*01/DPB1*04:01 | 248 | 262 | LGIALAIAGFLVQRR | 0.28 |
SpaI | HLA-DRB3*01:01 | 43 | 57 | ISDIRCDTGSLTLIK | 0.29 |
SpaI | HLA-DRB5*01:01 | 155 | 169 | LDFLITVPAGMRTAD | 0.42 |
SpaI | HLA-DRB1*09:01 | 95 | 109 | AGWDAAKALTIQEAR | 0.44 |
SpaI | HLA-DRB1*11:01 | 50 | 64 | TGSLTLIKRPPAAFE | 0.44 |
SpaI | HLA-DQA1*01:02/DQB1*06:02 | 242 | 256 | VLGIAALGIALAIAG | 0.48 |
SpaI | HLA-DPA1*02:01/DPB1*05:01 | 150 | 164 | EYRRTLDFLITVPAG | 0.6 |
Table VIII contains data on the number of alleles for which epitope that was found in the proteins analyzed. Overall, 40 HLA alleles of human origin encoded by HLA-A and HLA-B were selected for ProPred1. The HLA 7 from mouse s-derived (MHC-Db, MHC-Db revised, MHC-Dd, MHC-Kb, MHC-Kd, MHC-Kk, and MHC-Ld) was omitted. The Proteasome Filter and Immunoproteasome filters were included in the analysis and for both, the threshold score of 4% was used. The ProPred1 cut-off threshold was also set at 4%. At the ProPred, 51 alleles related to MHC class II were considered. These were HLA-DR alleles. These molecules were encoded by DRB1 and DRB5 genes containing HLA DR1 (2 alleles), DR3 (7 alleles), DR4 (9 alleles), DR7 (2 alleles), DR8 (6 alleles), DR11 (9 alleles), DR13 (11 alleles), DR15 (3 alleles), and DR51 (2 alleles). The cut-off threshold was set at 3%.
Number of alleles for which epitopes were found in the proteins tested.
Target protein | Number of MHC alleles of class I (per 40) | % Bound alleles MHC class I | Number of MHC alleles of class II (per 51) | % Bound alleles MHC class II |
---|---|---|---|---|
67–72p | 40 | 100 | 51 | 100 |
SpaC | 37 | 92.5 | 51 | 100 |
SpaI | 34 | 85 | 50 | 98 |
SapD | 33 | 82.5 | 51 | 100 |
The target protein sequences were scanned for B-cell epitopes using the Bepipred Linear Epitope Prediction 2.0. The selected B-cell linear epitopes of the proteins analyzed are shown in Table IX.
B-cell epitopes predicted from the target proteins.
Target protein | No. | Start | End | Peptide | Length |
---|---|---|---|---|---|
6772p | 1 | 4 | 20 | GFTRPAAAPKRPQRRLT | 17 |
6772p | 2 | 48 | 53 | EVDFRG | 6 |
6772p | 3 | 87 | 111 | GRPDELEFFDPDSPVVQYRAAVEKG | 25 |
6772p | 4 | 143 | 156 | NRQDFGVSDQQFGM | 14 |
6772p | 5 | 194 | 211 | GGIRAGNQAAGVKGSITN | 18 |
6772p | 6 | 281 | 287 | VTKDLRI | 7 |
6772p | 7 | 316 | 329 | SPNRAEKESEYISR | 14 |
6772p | 8 | 337 | 369 | AYGITDDAVTYKDNWGAKGASSEKVASDSATVS | 33 |
6772p | 9 | 381 | 410 | PTFTQQQQLRNFYGFPKSLAMDRYVIDGEL | 30 |
6772p | 10 | 421 | 434 | DPNALKENQRDWIN | 14 |
6772p | 11 | 452 | 467 | QVDEVARDVGSARGGY | 16 |
6772p | 12 | 474 | 490 | DLQTTDKEAQELGIVVK | 17 |
6772p | 13 | 498 | 507 | PVIASATDGA | 10 |
6772p | 14 | 514 | 541 | SENDSSVEYDTDSSTYTYQGKGGVNIGN | 28 |
6772p | 15 | 562 | 566 | RVNGN | 5 |
6772p | 16 | 573 | 581 | RDPRERVHN | 9 |
6772p | 17 | 612 | 645 | TSLPYAERTSLSEATNDTTAQVGNSAQRLVTDNV | 34 |
6772p | 18 | 680 | 705 | GVFPGTVKAKSEISEELMNHLRYPED | 26 |
6772p | 19 | 714 | 749 | LARYHVDDARDFFTNDRFWSVPSDPSATEGQKDVAQ | 36 |
6772p | 20 | 760 | 763 | DTGK | 4 |
6772p | 21 | 773 | 777 | RGLQR | 5 |
6772p | 22 | 803 | 837 | TDTLTQGPKQAQDTMMSSDQIASDRTLWKDTNDLF | 35 |
6772p | 23 | 861 | 868 | RKNQASAF | 8 |
6772p | 24 | 896 | 944 | GIDPKEAQDLGEAKGLKPESQNRDKPEDKEGKAPSTPSAPASGSGTTGE | 49 |
6772p | 25 | 956 | 976 | LQSAKNGSNEEYGRALDELDK | 21 |
SpaC | 1 | 38 | 49 | ANAEPLPKKEFE | 12 |
SpaC | 2 | 64 | 69 | SLSASD | 6 |
SpaC | 3 | 100 | 113 | SPAAGNKNFTPVSL | 14 |
SpaC | 4 | 131 | 146 | MPAIRENKKGSPNGGT | 16 |
SpaC | 5 | 176 | 184 | PTWDNNGRN | 9 |
SpaC | 6 | 225 | 228 | PFDL | 4 |
SpaC | 7 | 231 | 234 | PILP | 4 |
SpaC | 8 | 246 | 254 | WKIDRSLTG | 9 |
SpaC | 9 | 324 | 332 | PSIETDKNG | 9 |
SpaC | 10 | 355 | 358 | TGDQ | 4 |
SpaC | 11 | 371 | 387 | RYSYGQAPTDIPIKTSD | 17 |
SpaC | 12 | 417 | 432 | KVNVNTPQLLEELNNQ | 16 |
SpaC | 13 | 455 | 468 | GVHNGESKEIGKVA | 14 |
SpaC | 14 | 478 | 507 | VTPKVDDSRMKLSTTWSSENTTADANQDNG | 30 |
SpaC | 15 | 512 | 522 | KADTDAFKNKK | 11 |
SpaC | 16 | 531 | 537 | NYEAQTA | 7 |
SpaC | 17 | 545 | 561 | IINRDKIPATKLPEKFP | 17 |
SpaC | 18 | 569 | 591 | VPHPNARPEHGGLPETNPYFVDS | 23 |
SpaC | 19 | 601 | 610 | SIEIGPFPVG | 10 |
SpaC | 20 | 619 | 659 | ARLDPNVQADAKIPGFSLKTEWNSNICFGNTIDNNSQDCST | 41 |
SpaC | 21 | 664 | 672 | IPKPGQYSL | 9 |
SpaC | 22 | 676 | 684 | NTYTRELAS | 9 |
SpaC | 23 | 690 | 702 | TVSGDASDLTNSH | 13 |
SpaC | 24 | 712 | 731 | DSGVEVYSQDNIVVKKDGRQ | 20 |
SpaC | 25 | 746 | 754 | EKQPEQKGV | 9 |
SpaC | 26 | 761 | 769 | PFHLRASTA | 9 |
SpaC | 27 | 779 | 786 | NTAKRQVA | 8 |
SpaC | 28 | 792 | 812 | KKVHKKDTFSPEISASIDALT | 21 |
SpaC | 29 | 819 | 846 | CTVPGVETPRKVLKTVSDNQTVEFGNFP | 28 |
SpaC | 30 | 857 | 861 | TEAPA | 5 |
SpaC | 31 | 881 | 885 | TPINK | 5 |
SpaC | 32 | 891 | 895 | FENAR | 5 |
SpaC | 33 | 904 | 948 | VLDGDMPQALVDQIPSSFTVNVACSITGNHSITLQKDEQKAVPGV | 45 |
SpaC | 34 | 957 | 968 | SEEVTPITGATH | 12 |
SpaC | 35 | 971 | 991 | HWIKGELLEVADSTDITINPN | 21 |
SpaC | 36 | 1001 | 1007 | HYETDAV | 7 |
SpaC | 37 | 1012 | 1037 | TKRVRVIDQVGNDVNSELKNAVVRPE | 26 |
SpaC | 38 | 1043 | 1052 | RYRCEINGQV | 10 |
SpaC | 39 | 1059 | 1073 | SADAINTGATKVPRG | 15 |
SpaC | 40 | 1079 | 1131 | EEDSSSVELSNATLSHVEFFVHGTKTNDKASVAINSDHNRLDATNTFTLKTGS | 53 |
SpaC | 41 | 1135 | 1146 | KKKVDGEGVSTI | 12 |
SpaC | 42 | 1157 | 1164 | RCTLGDWK | 8 |
SpaC | 43 | 1174 | 1188 | FDSAESHSVKDIPVG | 15 |
SpaC | 44 | 1195 | 1204 | EDSEKAQEPN | 10 |
SpaC | 45 | 1210 | 1240 | RWTHTDSTNGWGDTEAACENHAACEVDPKNE | 31 |
SpaC | 46 | 1250 | 1255 | NEKENF | 6 |
SpaC | 47 | 1276 | 1288 | KVLTNDGPELAGK | 13 |
SpaC | 48 | 1298 | 1346 | TDPRFAGSDLADKHSIPDPTITVALNAKGQSRASYQVADERHDSVEVPV | 49 |
SpaC | 49 | 1357 | 1360 | IALY | 4 |
SpaC | 50 | 1378 | 1401 | AVQRTSSNSASARFVTEKQENNGT | 24 |
SpaC | 51 | 1409 | 1413 | DYIRP | 5 |
SpaC | 52 | 1424 | 1437 | AKPEHSVDPWLLNT | 14 |
SpaC | 53 | 1443 | 1483 | YKCDDPYIKDRSYSNDVDIQADAEKPTPIFADPTAHVKIPA | 41 |
SpaC | 54 | 1492 | 1498 | NTEGHLP | 7 |
SpaC | 55 | 1506 | 1555 | DETNKVAEFAGEHEKRSYFTPEIKDVVLSESEPTRIEFTNSYVMPQRILS | 50 |
SpaC | 56 | 1560 | 1569 | VEGDPGHAVI | 10 |
SpaC | 57 | 1582 | 1605 | TMPHLFPNQPNPMSQEVGNKVARG | 24 |
SpaC | 58 | 1614 | 1622 | TWRSPEVPI | 9 |
SpaC | 59 | 1630 | 1643 | EEDDPALRTKLENN | 14 |
SpaC | 60 | 1645 | 1687 | LRMVPTYLFPTERAGAASAPVIPPLTDRPIYNGTEPRLQMPES | 43 |
SpaC | 61 | 1718 | 1723 | ADNSPL | 6 |
SpaC | 62 | 1734 | 1755 | GENGQRKELPEVADAPAKSAKS | 22 |
SpaC | 63 | 1808 | 1825 | DNYDADSGLITVEHPQGK | 18 |
SpaC | 64 | 1837 | 1842 | STLPLT | 6 |
SapD | 1 | 23 | 72 | PVSASEDAALDATGHKKGEPAFGVTIPKGTTYRDSDGKEVPHPCVDRKIG | 50 |
SapD | 2 | 86 | 96 | YSVKEPATDLP | 11 |
SapD | 3 | 104 | 113 | DGQQVVPQES | 10 |
SapD | 4 | 122 | 145 | AGEDGEELSRIRIPDDEEFSFLGK | 24 |
SapD | 5 | 157 | 162 | IPFANG | 6 |
SapD | 6 | 174 | 190 | DPHHEPKGKDSIPEHLD | 17 |
SapD | 7 | 224 | 234 | SNDEELKTIEY | 11 |
SapD | 8 | 264 | 269 | AFKVKT | 6 |
SapD | 9 | 281 | 350 | DEEVGLPEGTTTNLNKITKPLDKDATNEPPTDPSEKKKPPRPEKGHSETSSPSA LDDSIERAWKLTGTPK | 70 |
SapD | 10 | 371 | 380 | TVINREGKKY | 10 |
SapD | 11 | 392 | 418 | SGGDQGGPLVKTDSWKDRIEAQISGSL | 27 |
SapD | 12 | 441 | 451 | EDLPSDALLPD | 11 |
SapD | 13 | 525 | 529 | GKQES | 5 |
SapD | 14 | 542 | 606 | TIARAKEILAGEKLGGSLKKKPQEKETKKPASVQNKSGKHNKDTVGSESARK RQQLAATSGSDTN | 65 |
SapD | 15 | 624 | 632 | AAEWQELDT | 9 |
SpaI | 1 | 22 | 50 | ASGPIASADSRTITGATDGLNISDIRCDT | 29 |
SpaI | 2 | 55 | 75 | LIKRPPAAFEGVDKADLPAGT | 21 |
SpaI | 3 | 86 | 124 | IEGIDLTKQAGWDAAKALTIQEARSRLSDEVWKAVSGRD | 39 |
SpaI | 4 | 144 | 153 | PAKRPAEYRR | 10 |
SpaI | 5 | 166 | 174 | RTADGNVAS | 9 |
SpaI | 6 | 186 | 242 | TDDLPPTVPVFPPVESSVTLTPSSPVPGTPKTPGKPDLPEKFRKEVTDRLGNT GANV | 57 |
SpaI | 7 | 263 | 266 | KKNE | 4 |
The results obtained with the VaxiJen server also confirmed the possibility of using the proteins as antigens in vaccines (Table X).
Prediction of the protective antigens from the VaxiJen server.
Protein | Overall Prediction for the Antigen |
---|---|
6772p | 0.5123 |
SpaC | 0.6757 |
SpaI | 0.5504 |
SapD | 0.5544 |
The huge success of vaccination against diphtheria almost enabled the elimination of the disease in Europe and other developed countries. However, in many countries with high vaccination coverage, i.e. France, Italy, Switzerland, Germany, and Canada an increase in non-toxigenic
In vaccine development, the potential virulence factors exposed on the surface of a pathogen are considered as suitable antigens for an effective acellular vaccine. It has been shown that pili of Gram-positive bacteria play a direct role in the pathogenesis. For example, studies on
The adhesion of
In addition to pili,
Our research is based on reverse vaccination. This method relies on the sequencing of pathogen genomes and determination
First, we selected the gene encoding the 67–72p protein and the three pili genes
An effective vaccine should induce a protective and long-lasting immune response. Therefore, we carried out analyses of the affinity of the tested proteins to MHC classes I and II and linear B-cell epitopes. MHC class I presents antigens to CD8+ T-cells and MHC class II presents antigens to CD4+ T-cells. The antigens, which are recognized by CD4+ and/or CD8+ T-cell receptors, have the potential to stimulate a long-lasting and cytotoxic immune response. B-cell epitopes can induce both primary and secondary immunity. We showed that, in each of the proteins, areas with high affinity to MHC receptors can be distinguished (Table VI, Table VII, Table VIII) and we localized B-cell epitopes from target proteins (Table IX). In addition, the VaxiJen server was used that is a reliable and consistent tool for predicting protective antigens of bacterial, viral and cancer origin. The results obtained also confirmed that the proteins tested by us could be interesting to use as antigens in vaccines (Table X).
Our studies have shown that in the genome of the 89/E strain, the insertion element of 1380 bp was transposed and attached to fragment 4 of the gene encoding the SpaC pili protein. The process of transposition of ISs can inactivate genes (Trost et al. 2012). Mandlik et al. (2007) confirmed the reduced
Due to the fact that in many European countries the number of infections with non-toxigenic