Zonula occludens toxins and their prophages in Campylobacter species

Background We previously showed that zonula occludens toxin (Zot) encoded by Campylobacter concisus zot808T gene has the potential to initiate inflammatory bowel disease. This Zot protein caused prolonged intestinal epithelial barrier damage, induced intestinal epithelial and macrophage production of tumor necrosis factor-α and enhanced the responses of macrophages to other microbes. In order to understand the potential virulence of Zot proteins in other Campylobacter species, in this study we examined their presence, similarities, motifs and prophages. Methods The presence of Zot proteins in Campylobacter species was examined by searching for the Zot family domain in multiple protein databases. Walker A and Walker B motifs in Zot proteins were identified using protein sequence alignment. A phylogenetic tree based on Campylobacter zot genes was constructed using maximum-likelihood method. Campylobacter Zot proteins were compared using protein sequence alignment. The zot-containing prophages in Campylobacter species were identified and compared with known prophage proteins and other viral proteins using protein sequence alignment and protein BLAST. Results Twelve Zot proteins were found in nine Campylobacter species/subspecies. Among these Campylobacter species, three species had two Zot proteins and the remaining six species/subspecies had one Zot protein. Walker A and Walker B motifs and a transmembrane domain were found in all identified Campylobacter Zot proteins. The twelve Campylobacter zot genes from the nine Campylobacter species/subspecies formed two clusters. The ZotCampyType_1 proteins encoded by Cluster 1 Campylobacter zot genes showed high similarities to each other. However, ZotCampyType_2 proteins encoded by Cluster 2 Campylobacter zot genes were more diverse. Furthermore, the zot-containing Campylobacter prophages were identified. Conclusion This study reports the identification of two types of Campylobacter Zot proteins. The high similarities of ZotCampyType_1 proteins suggest that they are likely to have similar virulence. ZotCampyType_2 proteins are less similar to each other and their virulent properties, if any, remain to be examined individually. Electronic supplementary material The online version of this article (doi:10.1186/s13099-016-0125-1) contains supplementary material, which is available to authorized users.


Background
Campylobacter concisus is a Gram-negative spiral shaped motile bacterium [1]. Their growth under both anaerobic and microaerobic conditions is largely determined by the presence of H 2 [2]. This bacterium usually colonizes the human oral cavity [3,4]. However, it may also colonize the intestinal tract of some individuals and its prevalence in intestinal biopsies has been associated with human inflammatory bowel disease (IBD) [5][6][7][8][9]. Translocation of C. concisus from the oral cavity to intestinal tract has been suggested to be a cause of a subgroup of IBD [10]. Campylobacter concisus has also been suggested to be involved in diarrheal disease due to the frequent isolation of this bacterium from diarrheal stool samples [8,[11][12][13].
Campylobacter concisus zonula occludens toxin (Zot) is encoded by the zot gene located in CON_phi2 prophage [14,15]. The zot genes in different C. concisus strains have polymorphisms [14]. In a recent study, we examined the effects of C. concisus Zot encoded by zot 808T gene on human intestinal epithelial cells and macrophages using cell line

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Gut Pathogens *Correspondence: L.Zhang@unsw.edu.au School of Biotechnology and Biomolecular Sciences, University of New South Wales, Kensington, Sydney 2052, Australia models. In that study, we found that C. concisus Zot caused prolonged intestinal epithelial barrier damage, induced intestinal epithelial and macrophage production of proinflammatory cytokines such as tumor necrosis factor-α, and enhanced the responses of macrophages to Escherichia coli [16]. These data suggest that C. concisus Zot may play a role in initiating chronic intestinal inflammatory conditions such as IBD through damaging the intestinal barrier and enhancing the immune responses to luminal microbes.
In addition to C. concisus, four additional Campylobacter species including Campylobacter ureolyticus, Campylobacter corcagiensis, Campylobacter gracilis and Campylobacter iguaniorum were recently reported to possess the zot gene [17][18][19][20][21]. The similarities of Zot proteins in different Campylobacter species have not been examined. In this study, we examined Zot proteins and their genes in Campylobacter species, which revealed two clusters of Campylobacter zot genes and two types of Campylobacter Zot proteins. Furthermore, we identified the prophages that contain zot genes in Campylobacter species.

Examination of the presence of Zot proteins in Campylobacter species
The presence of Zot proteins in Campylobacter species was examined by searching for proteins in Campylobacter species that have the Zot family domain in multiple protein databases including NCBI protein database, InterPro and Pfam [22][23][24]. The Zot proteins in these databases were annotated based on the presence of Zot family domain.

Examination of the presence of Walker A and Walker B in Campylobacter Zot proteins
It is shown in the InterPro database that the Zot family proteins (InterPro entry identity: IPR008900) belong to p-loop containing nucleoside triphosphate hydrolase (p-loop NTPase) superfamily. The proteins of p-loop NTPase superfamily have Walker A and Walker B motifs [25]. In this study, we examined the presence of Walker A and Walker B motifs in Campylobacter Zot proteins by protein alignment using Clustal Omega software [26]. The Walker A motif has a sequence of GxxxxGK[S/T], where x is any residue and the Walker B motif has a sequence of hhhh[D/E], where h is a hydrophobic residue [25].

Generation of a phylogenetic tree based on zot genes in Campylobacter species
To examine the genetic relationship between the zot genes in different Campylobacter species, the nucleotide sequences of the zot genes in Campylobacter species identified above were obtained from NCBI genome database. These sequences were used to generate a phylogenetic tree using the maximum likelihood method implemented in molecular evolutionary genetics analysis software version 6.0 [27]. In order to differentiate the zot genes in different Campylobacter species, the zot genes found in different Campylobacter species were indicated by the last four digits of their locus tag numbers.

Comparison of zot-containing prophages in Campylobacter species
To examine whether the zot genes in different Campylobacter species are carried by prophages similar to that in C. concisus, the zot-containing prophages in these Campylobacter species were identified by examination of the genes adjacent to their zot genes. The prophages were defined based on the presence of integrase, hypothetical proteins and attachment sites [28]. The attachment sites were identified by the presence of repetitive sequences located at both ends of the prophages [28].
The proteins in the identified Campylobacter prophages in this study were compared with the corresponding proteins in C. concisus prophages CON_phi2 and CON_phi3. The comparison was performed using Clustal Omega [26]. Proteins sharing more than 40 % sequence identity were considered to have high similarities and were recorded [29].

Comparison of the proteins of zot-containing prophages in Campylobacter species with other viral proteins
To examine whether the zot containing prophages identified in Campylobacter species are similar to previously reported prophages, proteins of Campylobacter prophages were compared with viral proteins in the NCBI non-redundant protein sequence database (taxonomy identity for viruses: 10,239) using protein BLAST with default settings [22]. The identified viral proteins with E-values lower than 10 were noted. For prophage proteins which shared sequence similarities with multiple viral proteins, the viral proteins with the lowest E-values were noted. The full length sequences of the identified viral proteins were then aligned with the Campylobacter prophage proteins using Clustal Omega [26]. The protein identities were calculated by dividing the number of identical amino acids by the total number of amino acids in proteins from Campylobacter prophages. The Campylobacter prophage proteins were also compared with viral proteins in the virus pathogen database and analysis resource (ViPR) using BLAST with a cut-off E-value of 10. In addition to C. concisus, the zot gene was also found in other bacterial species such as Vibrio cholerae and Neisseria meningitis [30,31]. The identities between Campylobacter Zot, V. cholerae Zot and N. meningitidis Zot proteins were also compared in this study.

Prediction of secreted proteins and transmembrane proteins in Campylobacter prophages
Secreted proteins in zot-containing prophages in different Campylobacter species were predicted using Sig-nalP 4.1 and SecretomeP 2.0. The software SignalP 4.1 predicts secreted proteins based on the presence of the signal peptide at the N-terminus [32]. The software SecretomeP 2.0 predicts non-classical (not signal peptide triggered) protein secretion based on analysis of posttranslational and localizational aspects of the proteins [33]. Transmembrane proteins were predicted using the software Phobius [34].

The Zot proteins in different Campylobacter species and their Walker A and Walker B motifs
Twelve Zot proteins were found in nine Campylobacter species/subspecies. Three Campylobacter species including C. concisus, C. ureolyticus and C. corcagiensis had two Zot proteins and the remaining six Campylobacter species/subspecies including C. gracilis, Campylobacter jejuni subsp. doylei, Campylobacter jejuni subsp. jejuni, Campylobacter hyointestinalis subsp. hyointestinalis, C. hyointestinalis subsp. lawsonii and C. iguaniorum had one Zot protein ( Table 1).
The Zot family domains were localized at the N-terminal side of the identified Campylobacter Zot proteins, prior to the transmembrane domains. The Zot family proteins had p-loop NTPase domains and the entry identity for p-loop NTPase domain was IPR027417 in InterPro database. We identified the Walker A and Walker B motifs in Campylobacter Zot proteins, which were at the N-terminal side of the Zot proteins (Fig. 1).

The phylogenetic tree formed based on zot genes in Campylobacter species
The Campylobacter zot genes formed two clusters. Cluster 1 contained three zot genes, including C. concisus zot2276, C. ureolyticus zot3935 and C. corcagiensis zot6485. Cluster 2 contained nine Campylobacter zot genes (Fig. 2).

Comparison of Zot CampyType_1 and Zot CampyType_2 proteins
The Zot proteins encoded by Cluster 1 and Cluster 2 Campylobacter zot genes were referred to as Zot CampyType_1 and Zot CampyType_2 proteins respectively. The three Zot CampyType_1 proteins had high similarities; they shared 171 identical amino acids and 77 conservative mutations (Fig. 3). The Zot CampyType_2 proteins were less similar to each other as compared to Zot CampyType_1 proteins; they had 71 identical amino acids and 65 conservative mutations (Fig. 4).

The zot-containing prophages in different Campylobacter species and their similarities to C. concisus prophages CON_phi2 or CON_phi3
We identified the zot-containing prophages in different Campylobacter species. Each of these prophages began with an integrase and had a number of hypothetical proteins ( Fig. 5; Additional file 1). The attachment sites were found ( Table 2). These prophages were inserted within either tRNA-Met or tRNA-Ser genes, except for URE_ phiZB, which was inserted into tRNA-Leu gene ( Table 2). For the prophage in C. iguaniorum, two tRNA genes were found after the integrase, suggesting that multiple insertions have occurred. Prophages containing Zot CampyType_1 proteins had high similarities to CON_phi2. Eight proteins in URE_phiZA and nine proteins in COR_phiZA were found to have more than 40 % identities (41-73 %) with proteins in CON_phi2 ( Fig. 5a; Additional file 1). Proteins in prophages containing Zot CampyType_2 proteins were more diverse. However,   three to five proteins in these prophages had more than 40 % identities with that in CON_phi3 (40-72 %) ( Fig. 5b; Additional file 1).

The similarities between proteins in Campylobacter prophages and viral proteins
The zot-containing Campylobacter were compared with known viral proteins in NCBI non-redundant protein sequence database. The proteins within each individual prophage showed low similarities to multiple phage proteins, except for CCC13826_0188 in CON_phi3 that shared 43 % identity with a phage transferase from an uncultured phage (Additional file 2). The Zot proteins in Campylobacter prophages had low similarities with the Zot proteins in V. cholerae and N. meningitidis (15-21 %) (Additional files 3, 4). None of the Campylobacter prophage proteins shared significant similarities with viral proteins in ViPR database. These data suggest that the zot-containing prophages in Campylobacter species are new prophages that have not been previously characterized.

Secreted and transmembrane proteins in Campylobacter prophages
Proteins secreted via both classical secretory pathway (with signal peptides) and non-classical secretory pathway (without signal peptides), as well as transmembrane proteins were found in all Campylobacter prophages (Fig. 5).

Discussion
In addition to C. concisus, a number of other Campylobacter species were recently reported to have the zot genes [17][18][19][20][21]. In this study, we found that Campylobacter zot genes formed two clusters (Fig. 2). Most of the Campylobacter zot genes were in Cluster 2, and Cluster 1 contained only three zot genes. The three Campylobacter species that had Cluster 1 zot genes also contained Cluster 2 zot genes. The remaining six Campylobacter species/ subspecies contained Cluster 2 zot genes only. These data show that Cluster 2 zot gene is more prevalent in Campylobacter species as compared to Cluster 1 zot genes.
Zot CampyType_1 proteins, which were encoded by Cluster 1 zot genes, were highly similar to each other. However they were less similar to Zot CampyType_2 proteins that were encoded by Cluster 2 Campylobacter zot genes. The zot gene in CON_phi2 prophage in C. concisus belongs to Zot CampyType_1 . Using cell culture models, we previously showed that Zot CampyType_1 in C. concisus encoded by zot 808T polymorphism damaged intestinal epithelial barrier by induction of epithelial apoptosis and induced production of proinflammatory cytokines such as TNF-α in HT-29 cells and THP-1 macrophage-like cells, supporting its role as a potential virulence factor [16]. The high similarities between Zot CampyType_1 proteins in the three different Campylobacter species suggest that they may have similar effects on human cells. Great variations in protein sequences between Zot CampyType_1 and Zot CampyType_2 proteins as well as within Zot CampyType_2 proteins were observed in this study. Given this, the effects of Zot CampyType_2 proteins on human cells, if any, require to be examined individually.
A transmembrane domain was found in all Zot proteins, showing that Zot proteins are transmembrane proteins. Furthermore, all Zot proteins contained Walker A and Walker B motifs, which are conserved motifs of p-loop NTPase superfamily [25]. P-loop NTPase bind to NTP typically ATP or GTP through the Walker A and B motifs, which are involved in diverse cellular functions [25]. Future studies should be conducted to examine whether Campylobacter Zot proteins have NTPase activities.
In this study, we identified a number of zot-containing prophages in other Campylobacter species in additional to previous reported prophages in C. concisus (Fig. 5). These prophages have an integrase, a number of hypothetical proteins and attachment sites ( Fig. 5; Table 2; Additional file 1), which satisfy the previously defined criteria for prophages [28]. The proteins in individual Campylobacter prophages identified in this study have low similarities with multiple viral proteins, suggesting that they are new prophages that have not being characterized previously (Additional file 2).
Campylobacter Zot proteins had very low similarities to V. cholerae Zot and N. meningitis Zot proteins (Additional files 3,4). These data showed that despite having a common name, the amino acid sequences of Zot proteins in different bacterial species vary greatly. Thus, they may not necessarily exhibit the same effects on human cells. Proteins with more than 40 % identical amino acids with proteins in CON_phi2 or CON_phi3 were labeled with the same color. The numbers above the proteins are locus tags of the genes in the NCBI database. Int indicates integrase. Asterisk and Hashtag indicate proteins predicted to be secreted via classic secretory pathway or non-classic secretory pathway respectively. Caret indicates transmembrane proteins. Dollar indicates multiple insertion sites for CON_phi prophages in C. concisus 13826 in which only the first attachment site (for CON_phi1) overlapped with tRNA [15]. Filled triangle indicates attachment sites

Conclusions
This study reports the identification of two types of Campylobacter Zot proteins. The high similarities of Zot CampyType_1 proteins suggest that they are likely to have similar virulence. Zot CampyType_2 proteins were less similar to each other and their virulent properties, if any, remain to be examined individually. This study provides useful information for further examination of Campylobacter Zot proteins as potential virulence factors. Table 2 The attachment sites of zot-containing Campylobacter prophages a The start and end positions for the attachment sites refer to the nucleotide position within the contig containing the prophage genomes, except for C. concisus strains 13826, C. jejuni subsp. doylei 269.97 and C. hyointestinalis subsp. lawsonii CCUG 27631 which refer to the nucleotide position in the full genome b Attachment sites overlapped with 3′ end of tRNA, the overlapped sequences were italic