Analysis of the Amino Acid Sequence Variation of the 67-72p Protein and the Structural Pili Proteins of Corynebacterium diphtheriae for their Suitability as Potential Vaccine Antigens.

The aim of this study was to identify the potential vaccine antigens in Corynebacterium diphtheriae strains by in silico analysis of the amino acid variation in the 67-72p surface protein that is involved in the colonization and induction of epithelial cell apoptosis in the early stages of infection. The analysis of pili structural proteins involved in bacterial adherence to host cells and related to various types of infections was also performed. A polymerase chain reaction (PCR) was carried out to amplify the genes encoding the 67-72p protein and three pili structural proteins (SpaC, SpaI, SapD) and the products obtained were sequenced. The nucleotide sequences of the particular genes were translated into amino acid sequences, which were then matched among all the tested strains using bioinformatics tools. In the last step, the affinity of the tested proteins to major histocompatibility complex (MHC) classes I and II, and linear B-cell epitopes was analyzed. The variations in the nucleotide sequence of the 67-72p protein and pili structural proteins among C. diphtheriae strains isolated from various infections were noted. A transposition of the insertion sequence within the gene encoding the SpaC pili structural proteins was also detected. In addition, the bioinformatics analyses enabled the identification of epitopes for B-cells and T-cells in the conserved regions of the proteins, thus, demonstrating that these proteins could be used as antigens in the potential vaccine development. The results identified the most conserved regions in all tested proteins that are exposed on the surface of C. diphtheriae cells. The aim of this study was to identify the potential vaccine antigens in Corynebacterium diphtheriae strains by in silico analysis of the amino acid variation in the 67–72p surface protein that is involved in the colonization and induction of epithelial cell apoptosis in the early stages of infection. The analysis of pili structural proteins involved in bacterial adherence to host cells and related to various types of infections was also performed. A polymerase chain reaction (PCR) was carried out to amplify the genes encoding the 67–72p protein and three pili structural proteins (SpaC, SpaI, SapD) and the products obtained were sequenced. The nucleotide sequences of the particular genes were translated into amino acid sequences, which were then matched among all the tested strains using bioinformatics tools. In the last step, the affinity of the tested proteins to major histocompatibility complex (MHC) classes I and II, and linear B-cell epitopes was analyzed. The variations in the nucleotide sequence of the 67–72p protein and pili structural proteins among C. diphtheriae strains isolated from various infections were noted. A transposition of the insertion sequence within the gene encoding the SpaC pili structural proteins was also detected. In addition, the bioinformatics analyses enabled the identification of epitopes for B-cells and T-cells in the conserved regions of the proteins, thus, demonstrating that these proteins could be used as antigens in the potential vaccine development. The results identified the most conserved regions in all tested proteins that are exposed on the surface of C. diphtheriae cells.

Introduction

Corynebacterium diphtheriae is the etiological agent of a serious infectious disease – diphtheria. The diphtheria vaccine is highly effective but is directed only against the diphtheria toxin. The non-toxigenic C. diphtheriae strains may cause many severe invasive diseases, e.g., endocarditis, septic arthritis, bacteremia, and noninvasive wound infections. The notified increasing number of non-toxigenic C. diphtheriae infections indicates that infections are a growing problem in Europe (Belko et al. 2000; Zasada et al. 2010; Zasada 2013; Fricchione et al. 2014; Dangel et al. 2018). It is hypothesized that high vaccination coverage has resulted in the emergence of non-toxin-producing (non-toxigenic) C. diphtheriae strains, which acquired new virulence factors.

The diphtheria toxoid vaccination protects against the action of the toxin but does not protect against colonization and invasion by C. diphtheriae. Little is known about C. diphtheriae virulence factors other than the diphtheria toxin. The degree of adhesion of microorganisms to host cells has been shown to be an important pathogenicity factor for both toxigenic and non-toxigenic strains (Colombo et al. 2001). The virulence factors that facilitate bacterial colonization to specific host tissues and are related to pathogenesis include pili and fimbriae (Reardon-Robinson and Ton-That 2014). These structures are covalently attached to the bacterial cell wall and are recognized by the related host receptors (Sauer et al. 2000; Rogers et al. 2011). The surface structures are potential candidates for the development of new vaccines and antimicrobial therapies, due to their significant role in pathogenesis (Maione et al. 2005; Soriani and Telford 2010). Pili are found in both Gram-positive and Gram-negative bacteria, albeit with different folding mechanisms (Thanassi et al. 1998; Ton-That and Schneewind 2004). The function of pili is not only the involvement in adhesion, but they also act as bacteriophage receptors and participate in DNA transfer, biofilm formation, cell aggregation, host cell penetration, and motility (Proft and Baker 2009).

There are three types of pili in C. diphtheriae strains (SpaA, SapD, and SpaH), each containing the LPXTG motif (Ton-That and Schneewind 2003; Gaspar and Ton-That 2006; Swierczynski and Ton-That 2006). The genes involved in the production of pili encode nine pili proteins, defined in the successive letters from SpaA to SpaI, and five sortases defined from SrtA to SrtE, which are organized in three separate clusters. The sixth sortase SrtF (class-D homolog) is now referred to as the housekeeping sortase, located in a different region of the chromosome (Ton-That and Schneewind 2003). C. diphtheriae, like other Gram-positive bacteria (e.g. Streptococcus pneumoniae, group A and B streptococci or Actinomycetes), have the gene encoding cysteine transpeptidase (sortase) conserved in the genome, which is necessary for the assembly of pili (Ton-That and Schneewind 2004; Marraffini et al. 2006).

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 C. diphtheriae strains to the surface of human cells is multifactorial. Functions and mechanisms of action of fimbriae (Mandlik et al. 2007), non-fimbrial 67–72p adhesin (Colombo et al. 2001), trans-sialidase (Mattos-Guaraldi et al. 1998), hydrophobins, and sugar residues (Mattos-Guaraldi et al. 1999a; 2000; Moreira et al. 2003) are poorly understood, especially how they jointly participate in the adherence to the host cells and in the colonization of these cells during bacterial infection.

Initially, the 67–72p adhesive protein was described as a ligand responsible for the adherence of C. diphtheriae to human erythrocytes (Colombo et al. 2001). Later, the participation of this protein in the adherence of bacteria to HEp-2 cells was described (Hirata et al. 2004). The presence of the 67–72p protein has been confirmed in C. diphtheriae strains isolated from various sources, e.g., on the surface of the cells of invasive HC01 strain isolated from the blood of a patient with endocarditis (Sabbadini et al. 2012).

C. diphtheriae strains exhibit cell surface hydrophobicity and autoaggregation. Thanks to these features, microorganisms avoid immune defenses and are able to survive on the surface of the skin and mucosal membranes (Mattos-Guaraldi et al. 1999b). In addition, it has been proven that the 67–72p protein has the ability to induce host cell death, giving a signal for apoptosis in the early stages of infection. The occurrence of 67–72p hemagglutinin is one of the characteristics of the potentially invasive strains because it can contribute to the cytotoxicity and apoptosis of the infected cells (Sabbadini et al. 2012).

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.

Experimental

Materials and Methods

Bacterial strains. In total, 10 C. diphtheriae non-toxigenic isolates were used in this study (Table I). Strains were isolated in Poland in 2010–2017 from patients with bacteremia, wound infection, septic arthritis, endocarditis, and serous cyst contents. The strain NCTC 13129 was used as the control strain.

Table I

C. diphtheriae strains used in this study.

Strain	Biotype	Site of isolation	Year of isolation
27/E	mitis	Serous cyst contents	2010
40/E	gravis	Blood	2014
68/E	gravis	Endocarditis	2015
71/E	gravis	Wound	2015
73/E	gravis	Blood	2016
77/E	gravis	Wound	2016
78/E	gravis	Blood	2016
79/E	gravis	Blood and joint fluid	2016
86/E	gravis	Blood	2017
89/E	gravis	Wound	2017

DNA isolation. C. diphtheriae strains were grown on Columbia agar with 5% sheep blood (BioMerieux) for 24 h at 37°C under aerobic conditions. Genomic DNA was isolated using a Wizard Genomic DNA Purification Kit (Promega) according to the Gram-positive bacteria procedure provided by the manufacturer.

Polymerase Chain Reaction (PCR). The oligonucleotide primers for amplification of the genes coding 67–72p protein and structural pili proteins (SpaC, SpaI, SapD) were designed based on the nucleotide sequence of the C. diphtheriae NCTC 13129 whole genome, available from GenBank under the number BX248353 (Table II).

Table II

Table II Primers used in this study.

Gene	Primer	Sequence	Length of the amplified fragment
67–72p	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
spaI	SpaI1L	GCGGAATCAACACCAACAC	600 bp
	SpaI1R	AAGCGCTTACGATCCAAGAA
	SpaI2L	ACACGGCCTTCCAAACTTC	482 bp
	SpaI2R	TGATATTGAGGCGTCGCTAA
sapD	SapD1L	TCGCGAAGGTAAGAAATACTCA	698 bp
	SapD1R	CGTTTGTATCCGAGCCACTT
	SapD2L	GTCCAAAACAAGAGCGGAAA	814 bp
	SapD2R	GGTTCAGTGAAAACCCAGTTG
spaC	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.

Sequencing of fragment 4 of the gene encoding SpaC protein in strain 89/E. Based on the result of the first round of sequencing, new primers were designed to completely sequence a fragment of approximately 2000 bp, as follows: SpaC4L 5’-CATTCGTTTTTGTT CCGTGA-3’ and SC1pR 5’-GAGCTGCTTGAAGTT GCAGA-3’ (391 bp), SC2pL 5’-CCCGAACACGTTTG GTAAGT-3’ and SC2pR 5’-GGGTAGTGGGTCAGGG TTTT-3’ (687 bp), SC3pL 5’-CGCACACAATCAGTGA CTAAAA-3’ and SC3pR 5’-ACAACGTATTCGCAGCA GTG-3’ (850 bp), SC4pL 5’-ATGGTTATCGCCGTATC TGG-3’ and SpaC4R 5’-GGTGTAGAAACGCCTCGA AA-3’ (764 bp). The PCR reaction was carried out according to the above conditions, the PCR products were then enzymatically purified and sent for sequencing.

Comparing the sequenced fragments and the translated nucleotide sequences for proteins. The sequences of the particular gene fragments were obtained as fluorograms. The BioEdit program was used for the gene assembly, alignment of sequences and comparative calculations. The translation of DNA into protein sequences was performed using the BLASTx program.

Analysis of 67–72p, SpaC, SpaI and SapD protein affinity to MHC classes I and II and linear epitopes. Amino acid sequences of C. diphtheriae NCTC 13129 reference strain available from the National Center for Biotechnology Information (NCBI) were used for analysis. To predict the cellular localization of the proteins, the software packages CELLO v.2.5 (Yu et al. 2006) and PSORTb version 3.0.2 (Yu et al. 2010) were used. The analysis for the transmembrane domains was performed using TMHMM Server v. 2.0. The MHC classes I and II binding prediction was performed using the immune epitope database (IEDB) (Kim et al. 2012) for the recommended human leukocyte antigen (HLA) allele set (Bui et al. 2005; Nielsen et al. 2003). Analysis has also been carried out using Propred-I (for MHC class I) and Propred (for MHC class II) regarding the number of alleles for which epitopes were found in the proteins analyzed (Singh and Raghava 2001; 2003). The prediction of linear B-cell epitopes has been carried out using the Bepipred Linear Epitope Prediction 2.0 (Jespersen et al. 2017). VaxiJen 2.0 was used for prediction of the protective antigens. The results page on VaxiJen server creates lists of the selected target, the protein sequence, its prediction probability, and a statement of protective antigen or non-antigen, according to a predefined cut-off. Since more of the models had their highest accuracy at a threshold of 0.5, this threshold value was chosen for all bacterial models types (Doytchinova and Flower 2007).

Results

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 C. diphtheriae should protect against all kinds of infections. The analyses conducted in this study showed the variability of C. diphtheriae strains in terms of the nucleotide sequence of the genes encoding the 67–72p protein (99.37–100% average similarity) and structural proteins of pili SpaC (38.94–99.97% average similarity), SpaI (81.39–100% average similarity), and SapD (63.78–100% average similarity). The 27/E strain, the only representative of the mitis biotype, was the most different from the other strains tested. This strain did not have the gene coding for the SpaI protein, while the average similarities of the nucleotide sequence of genes encoding the 67–72p, SapD, and SpaC proteins were 99.37%, 98.64%, and 38.94%, respectively when compared to the reference strain. Despite the significant variation, we managed to locate two fragments of the 67–72p protein (fragments No. 3 and 5), where the nucleotide sequences were 100% identical for all C. diphtheriae strains tested (the most conserved in the genome). In contrast, for C. diphtheriae biotype gravis we found as many as nine fragments located in the sequence of the gene encoding 67–72p protein (fragment No. 1, 3, 5) and the genes coding for the pili SapD (fragment No. 1), SpaI (fragment No. 2), and SpaC (fragment No. 2, 3, 7, 8), which were 100% identical in all nine strains tested (Table III).

Table III

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).

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 spaC, we obtained the exact nucleotide sequence, which we compared to the available sequences in the GeneBank using the Nucleotide Basic Local Alignment Search Tool (BLASTn). In this way, we proved that the 1380 bp insertion sequence (IS) was transposed, which interrupted the continuity of the tested gene between 3072 bp and 3073 bp of the reference sequence of the NCTC 13129 strain. The result from the BLAST search revealed that the insertion fragment we detected was similar to the sequence of IS3 family transposase also identified in other C. diphtheriae strains but in different locations.

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).

Table V

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

Table VI

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

Table VII

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%.

Table VIII

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.

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).

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

Discussion

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 C. diphtheriae infections has been observed. For example, Poland is a country where the last case of diphtheria was recorded in the year 2000 and where the vaccination level of over 95% is achieved (Zasada et al. 2010). The first case of non-toxigenic infection with C. diphtheriae biotype gravis was recorded in Poland in 2004, where this bacterium induced sepsis and endocarditis in a patient (Zasada et al. 2005) and since then, practically every year, several cases of invasive C. diphtheriae infections have been diagnosed. In northern Germany, the number of non-toxigenic C. diphtheriae infections increased from five in 2013 to 23 in 2016, and 24 in only the first half of 2017 (Dangel et al. 2018). In England and Wales, a dramatic increase in infections was recorded since 1986, peaking at almost 300 cases in the year 2000 (Edwards et al. 2011). These examples indicate that the development of a new vaccine against non-toxigenic C. diphtheriae infection is of very important and necessary demand.

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 S. pneumoniae have proved that those strains, which have a pili island, adhere better to lung epithelial cells than do the strains that lack this island. In the invasive disease model, the piliated strain is more virulent and has a competitive advantage over the pili-negative strain after the mixed intranasal infection. Infection with the piliated strain induces a stronger inflammatory response and a higher level of the tumor necrosis factor in the bloodstream of mice, which may be due to the higher adhesion of the piliated bacteria to the cells involved in the innate immune response and their detection by host cell pattern-recognition receptors (Barocchi et al. 2006). The pili proteins are used as antigens in vaccines, for example in some acellular pertussis vaccines (Mosley et al. 2016).

The adhesion of Corynebacterium to host cells was observed for the first time for C. renale pili, which caused agglutination of trypsinized sheep red blood cells (Honda and Yanagawa 1974). It was not until more than thirty years later that Mandlik and colleagues identified adhesins, which were involved in adherence to pharyngeal host cells – the minor pilins SpaB and SpaC of C. diphtheriae (Mandlik et al. 2007). Subsequent studies showed that wild type C. diphtheriae cells bind to human lung epithelial, laryngeal, and pharyngeal cells, whereas mutants that lacked SrtA (i.e. they did not polymerize the SpaA-type pili) showed more than a 90% lower ability to adhere to human pharyngeal cells. Moreover, mutants that lacked only the major pili subunit, SpaA, showed a 10% reduction in adherence to these cells. In contrast, mutants that lacked either of the minor pilin subunits, SpaB or SpaC, showed a 70–75% reduction in adhesion. In addition, the latex beads coated only with SpaB or SpaC were sufficient to adhere to the host pharyngeal cells, while the SpaA-coated beads did not bind. SpaB and SpaC are anchored in the cell wall as monomers independent of the pilus structures. It is likely that the long pili mediate the initial attachment, while the monomeric pilins on the surface of the bacteria participate in the formation of an adhesion zone allowing the delivery of toxins and other virulence factors and may even play a significant role in host cell signaling (Rogers et al. 2011).

In addition to pili, C. diphtheriae produces the 67–72p protein located on its surface, which is involved in colonization, induction of apoptosis, and epithelial cell necrosis that were once attributed exclusively to the action of the diphtheria toxin (Sabbadini et al. 2012). This finding was also confirmed by Cerdeño-Tárraga et al. (2003) who sequenced the genome of the British clinical isolate (strain NCTC13129 biotype gravis – used in our study as the reference strain) and proved that the recent acquisition of pathogenicity factors went beyond the toxin itself and included the fimbrial proteins and adhesins. The 67–72p can act as an invasive and apoptotic protein for C. diphtheriae strains. The ability to penetrate, survive and induce apoptosis in epithelial cells may explain the endurance and dissemination of C. diphtheriae (Sabbadini et al. 2012). Proteins, which act as adhesins were also detected among other bacteria, e.g., the occurrence of the extracellular Eap protein was confirmed to be involved in colonization of eukaryotic cells by S. aureus strains (Haggar et al. 2003).

Our research is based on reverse vaccination. This method relies on the sequencing of pathogen genomes and determination in silico the most likely protective antigens prior to conducting experiments to prove this. Originally, this method was used to identify antigens as probable candidate vaccines against serogroup B meningococci (Christensen et al. 2013). In another study, Droppa-Almeida et al. (2018) used several available bioinformatics tools to design the efficient immunodominant epitopes for the development of the peptide vaccine against C. pseudotuberculosis for sheep and goats. Thanks to this research, it was possible to highlight the importance of bioinformatics software in the design of vaccines, especially in the identification of appropriate vaccine candidates, immune-informatics analysis and design of peptide vaccine from multi-epitopes (Droppa-Almeida et al. 2018). Bioinformatics tools present a lot of advantages, such as speed and low cost, so we used them at particular stages of the research.

First, we selected the gene encoding the 67–72p protein and the three pili genes spaC, sapD and spaI as the genes most frequently detected in various C. diphtheriae isolates as it was reported in our previous study (Zasada et al. 2012). In the present study, we found two fragments of 594 bp and 215 bp in the nucleotide sequence of the gene coding for the 67–72p surface protein, which were identical in all analyzed strains of C. diphtheriae, and in total, nine identical in C. diphtheriae biotype gravis strains sequences of the genes encoding 67–72p protein and structural proteins pili SpaI, SpaC and SapD (Table III). The sequence stability of these fragments represents a first step toward being the potential vaccine candidates. The analysis of amino acid sequences of these fragments confirmed that the proteins tested are located in the membrane or cell wall and have a large extracellular part (Table V).

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 C. diphtheriae adhesive activity as the result of mutations at the base pili protein SpaB and at the tip pili protein SpaC of the SpaA-type pili. Premature stop codons in the continuity of the genes encoding the proteins responsible for the adhesion of bacteria to host cells inactivate them and limit the colonization process. We did not investigate the adhesive activity of the strain 89/E and, therefore, we can only posit the influence of the insertion on the adhesive properties of the strain based on the data published by other researchers.

Due to the fact that in many European countries the number of infections with non-toxigenic C. diphtheriae strains has recently increased, a key aspect of our research was the understanding of virulence factors other than the diphtheria toxin and identification of new vaccine targets. An important problem is also the understanding of the colonization process and in particular the mechanism of adhesion and structure of the proteins, which participate in this process. Blocking the 67–72p surface protein or pili structural proteins could effectively prevent the adhesion of C. diphtheriae bacteria to host tissues, colonization, and infection development. Due to the comparison of the nucleotide sequences of the C. diphtheriae strains identification of the most conserved sequences in the genome and determination of the variability between strains was achieved. The conserved sequences identified in 67–72p, SpaC, SapD and SpaI in our study are identical for all C. diphtheriae strains tested and contain the epitopes for B-cells and T-cells and will be used in further research on the construction of a new vaccine. The main limitation of this study is the small number of isolates investigated. However, the results obtained here support further studies with a larger number of isolates from different countries. Moreover, the results of in silico analysis should be confirmed by in vivo studies on an animal model. The new vaccine will act to inactivate the antigens responsible for the host cell colonization by C. diphtheriae strains and inhibit the development of infection.