2Centro de Investigación en Políticas, Población y Salud, Facultad de Medicina, Universidad Nacional Autónoma de México, Mexico City, México 04510.
Keywords: vapD gene; Helicobacter pylori; Genealogy; Phylogeny; Horizontal Gene Transfer.
Several virulence factors of H. pylori have been well described and in specific populations (Anglo-Saxon) have been shown to have a clear association with gastric pathologies [17-20]. However, in Latin American populations, H. pylori infection is often characterized by mixed genotypes without any clear association between a specific genotype and gastric pathology [11,21,22]. It has been shown that the CagA antigen and specific vacA (s1a/m1) genotype are the accepted virulence markers for gastric and peptic ulcer disease, as well as for gastric cancer, and the chronicity of infection suggests that some H. pylori strains have strategies allowing it to persist inside the cells for decades or even longer [3,23,24]. Various studies have reported that some H. pylori strains can invade the gastric cells and remain inside for an indefinite time [25,26], although the molecular mechanism related to this phenomenon has not been fully understood. Recently, Morales-Espinosa et al. demonstrated that vapD is expressed in the intracellular environment of adenocarcinoma gastric (AGS) cells and high levels of vapD expression were detected in gastric biopsies of patients with severe gastric pathologies. This suggests that vapD is necessary to support the long-term persistence of H. pylori in gastric cells and maintain the chronicity of the infection [3].
The vapD gene encodes for a virulence-associated protein D (vapD) that is found in various microorganisms from different phyla. Reports suggest that in some microorganisms, vapD participates in protecting the bacteria from respiratory burst with in the macrophage or in facilitating the persistence of the microorganism within the respiratory epithelial cell [27-29]. Virulence-associated protein (vap) genes were first identified in pathogenic strains of Dichelobacter nodosus [30,31] as part of a pathogenicity island of a plasmid. Subsequently, vap genes have been recognized in other microorganisms, such as Haemophilus influenza [29], where vapD is present in the bacterial chromosome as part of a Toxin-Antitoxin (TA) module with ribonuclease activity that promotes bacterial persistence inside respiratory cells. The action of Toxin and Antitoxin (TA) module systems favour bacterial persistence within epithelial cells enabling them to adapt to environmental conditions and antibiotic treatment [32-35]. Since persistence is a major factor contributing to the chronic state of infections and tolerance to antibiotic treatment, it was proposed that one of the roles of TA was to contribute to dormancy, i.e., making the cells metabolically inactive [36,37].
In Rhodococcus equi, vapA and vapD genes are present in a virulence plasmid and are highly induced in an acid tolerance response within the macrophage. The ability to withstand a stressful environment is an important factor in the virulence of an intracellular bacterium [27,28]. In Actinobacillus actinomycetemcomitans [38], the product of the orf2 gene is known to have 78.9% amino acid identity with vapD of D. nodosus. The vapD gene is a strain-specific gene that contributes to the higher genetic diversity of H. pylori. It was first described in the 60190 strain by Cao and Cover in 1997, in a variable chromosomal region of 3.8 kb downstream from vacA gene. In the 26695 strain, it is found in the HP0315 locus while in the J99 strain, vapD is located in the JHP0829 locus, but the ORF in this case is truncated encodes for a non-functional protein. Subsequent data suggests that vapD is present in 36% to 61% of H. pylori strains [38,39]. In 2012, Kwon et al [40]determined the structural and biochemical characteristics of vapD and found that this protein displeyed a purine-specific endoribonuclease activity, which was later shown to be structurally related to the Cas2 proteins [41].
Although an endoribonuclease function has been attributed to vapD in H. pylori, it is not clear what the mechanism of action is, or how important the vapD gene is in this specific bacterium, since it is widely distributed among microorganisms of different phyla [28,42,43]. Furthermore, previous studies in some microorganisms of different genera have suggested that the vapD gene, originates either from phages through integration events, or from a plasmid containing bacteriophage-related int genes [44]. Therefore, the aim of this study was to determine the phylogenetic relationship of the Helicobacter pylori vapD gene with the vapD gene present in other bacterial species from different phyla in order to infer its possible horizontal transfer, and to estimate the genealogy from vapD gene within the H. pylori species.
In a previous study, we characterized the vapD gene from a group of Mexican H. pylori strains (MxHp) isolated from adults and children [39]. In that study, a set of primers (D1 and D2), were used to obtain a 485 bp PCR product (expected size), but we also obtained vapD gene amplicons (from 800 bp to 1300 bp) that were larger than had been expected. In terms of the chromosomal vapD region of the 60190 strain, our D1 and D2 primer sequences were located at 100 bp upstream from the vapD ORF 5’ end to 100 bp downstream from the vapD ORF 3’ end. According to previous results, we considered that there could be a greater variability in the vapD gene from Mexican strains, as well as in the vapD region, than had been described previously [38,39].
vapD gene sequencing
From the previously mentioned study [39], we randomly selected 16 amplicons of vapD gene, 7 of which had an expected size of 498 bp and nine had a larger than expected PCR products. All the products were cloned into a pCR 2.1 vector (Invitrogen, Carlsbad, CA) and sequenced on both strands using the M13 sequencing primers and the Sanger method. The SeqMan program of the DNASTAR Lasergene 7 package (DNASTAR, Inc., Madison, WI) was used to edit, trim and assemble each sequence.
The GenBank ID for the vapD gene sequences obtained from our laboratory [39] were as follows: MxHp72a6 strain (AY781665), MxHp72a10 strain (AY781666), MxHp252a2 strain (AY781671), MxHp254a8 strain (AY781670), MxHp254c9 strain (AY781669), MxHp128 strain (EU822947), MxHp563 strain (EU822948), MxHp262c11 strain (AY781668), MxHp262c13 strain (AY781667), MxHp21.23a strain (EU818714), MxHp21.23c strain (EU822949), MxHp248 strain (EU826974), MxHp249 strain (EU826975), MxHp54 strain (EU826976), MxHp84 strain (EU826977) and MxHp118 strain (EU818713). The vapD sequences of the H. pylori 26695, 60190 and J99 strains were used as reference sequences. For Helicobacter species and the other bacterial species included in the study, the sequences were downloaded from the NCBI (Table S1).
Genealogical analyses of vapD gene among H. pylori strains
In order to assess the genealogical relationship of the vapD gene within H. pylori species, we selected vapD sequences from strains that had been isolated from different parts of the world, including our sequences reported in the GenBank database (Table S1), to create a network of haplotypes. This assessment used the most conserved region of the vapD gene (158 bp) sequence. Genealogy was reconstructed using TCS 1.21 software [45] and based on individual allele (haplotype) sequences. The haplotype network shows the connection between each allele, inferring the number of mutational steps between the connecting axes of each. When there is more than one mutational step between one haplotype
Table 1: Traces of IS elements present in the vapD region of H. pylori strains from Mexican populations |
||||||
Strain |
IS family |
Group |
IS |
Origin |
*E. value |
Sequence |
21.23a |
IS 5 |
IS 5 |
IS Pto 9 |
Psycroflexus torquis |
0.54 |
GTTTAGCCCTATCTTA |
54 |
IS 1595 |
ISPna 2 |
IS Aur 1 |
Actinobacillus ureae |
0.8 |
AACAAAAAAATTAG |
54 |
IS 3 |
IS 3 |
IS Cb3 |
Clostridium beijerinncki |
0.8 |
TAACAAAAAAATTAGAAAT |
54 |
IS 4 |
IS 231 |
IS Cb2 |
Clostridium beijerinncki |
0.8 |
AAAAGATCAATAACAAAAA |
84 |
IS 1595 |
ISPna 2 |
IS Aur1 |
Actinobacillus ureae |
0.8 |
TAACAAAAAAATTAGG |
84 |
IS 3 |
IS 3 |
IS Cb3 |
Clostridium beijerinncki |
0.79 |
TAACAAAAAAATTAGAAAT |
84 |
IS 4 |
IS 231 |
IS Cb2 |
Clostridium beijerinncki |
0.79 |
AAAAGATCAATAACAAAAA |
248 |
IS 607 |
- |
IS 607 |
Helicobacter pylori |
1 |
TAAAAAACTTATTAAA |
249 |
IS 607 |
- |
IS 607 |
Helicobacter pylori |
1 |
TAAAAAACTTATTAAA |
21.23c |
IS 200/IS 605 |
IS 1341 |
IS Hasp2 |
Halobacterium sp. |
0.11 |
CGGTGAATGGTTCGCT |
21.23c |
IS 1595 |
ISPna 2 |
IS Aur1 |
Actinobacillus ureae |
0.8 |
TAACAAAAAAATTAG |
21.23c |
IS 3 |
IS 3 |
IS Cb3 |
Clostridium beijerinncki |
0.8 |
TAACAAAAAAATTAGAAAT |
21.23c |
IS 4 |
IS 231 |
IS Cb2 |
Clostridium beijerinncki |
0.8 |
AAAAGATCAATAACAAAAA |
118 |
IS 1595 |
ISPna 2 |
IS Caje5 |
Campylobacter jejuni |
0.09 |
TTTAAAAAAGGAATAA |
118 |
IS 3 |
IS 51 |
ISS pr1 |
Serratia proteamaculans |
0.09 |
GATATTTTAAAAAAGG |
118 |
IS 1595 |
ISPna 2 |
IS Pto1 |
Psycroflexus torquis |
0.39 |
CAAACCCTTTATAAC |
*E. value: expected value |
Horizontal transfer inference of vapD gene
Currently, the methods for inferring horizontal transfer events are based on the analysis of gene sequences or complete genomes. In this current study, we analysed the vapD gene sequences using a phylogenetic method based on the reconstruction and comparison of phylogenetic trees. Two phylogenetic trees (16S rRNA and vapD) from 6 bacterial phyla and several H. pylori strains isolated from different parts of the world were constructed. This was followed by two parametric methods that use specific characteristics of the gene sequence, such as GC content and codon usage [46,47].
16S rRNA phylogenetic reconstruction of bacterial phyla
The phylogeny of different bacterial phyla was inferred from 16S rRNA sequence comparisons. Sequences of 152 strains from six bacterial phyla and 72 species were analysed (Table S1). These species were selected because they contained the vapD gene. The sequences were retrieved from the GenBank database and were aligned with Muscle software v.3.8.3 [48]. jModelTest [49] was used to select the optimal evolutionary model by evaluating the selected parameters using a corrected version of the Akaike Information Criteria (AIC). This approach suggested the HKY + I + G substitution model. Phylogeny was performed using MrBayes software v 3.0 [50] with five Markov chain Monte Carlo (MCMC) being run for five million generations. After discarding the first 20% iterations, the phylogenetic tree was constructed using Figtree v1.4.3 [51].
Phylogenetic analysis of the vapD gene from different bacterial species
In order to explore the phylogenetic relationships of the vapD gene among different bacterial phyla, as well as among H. pylori strains, we selected 211 vapD sequences from a wide variety of bacterial species found in GenBank. All the sequences were aligned using the Muscle software v.3.8.3 [48]. The substitution model was the same as for 16S rRNA. vapD phylogeny was constructed using MrBayes software v 3.0 [50]. An independent run of MrBayes was performed and consisted of five MCMC for ten million generations, the first 20% iterations being discarded as burn-in. Finally, the phylogenetic tree was drawn using Figtree v1.4.3 [51].
Codon usage bias and GC content
Using the online Codon Usage Database program [52], we obtained the Relative Synonymous Codon Usage (RSCU) of the genome Coding Regions (CDS’s) for the H. pylori 26695 strain. For the vapD gene, the RSCU values were obtained by DnaSP v5.1 software [53]. Methionine (Met) and tryptophan (Trp) that have unique codons, and the stop codons UAG, UAA and UGA, were all excluded from further analyses.
For the H. pylori genomic GC content, we collected information for 125 complete genome assemblies from the NCBI database. For the vapD gene, we used three sequences obtained during the previously mentioned study [39], and 26 sequences from the NCBI database. The GC content for each sequence was calculated using DnaSP v5.1 software [53].
X-squared was used to examine the significance (P<0.05) of codon usage differences and GC content between genomes and the vapD gene. Rstudio v3.2.2 [54] was employed for all statistical analyses.
Determination of the insertion sequences present in the vapD region
Mobile genetic elements, such as Insertion Sequences (IS’s), have played an important role in moving genetic material between organisms, including those during the early stages of evolution. To explore the presence of IS in the vapD region, we used the database from the ISfinder website [55].
Genetic diversity associated with the H. pylori vapD gene from Mexican strains
The strains MxHp72a6, MxHp72a10, MxHp254a8, MxHp254c9, MxHp252a2, MxHp262c11, and MxHp262c13 contained the complete vapD ORF, and among these sequences, three polymorphic sites were found. Strains MxHp72a6, MxHp72a10, MxHp254a8 and MxHp254c9 showed nucleotide substitutions in only two positions, at nucleotides 171 and 216 of the vapD ORF. Meanwhile, the MxHp252a2 strain presented a third substitution at position 10. When the nucleotide sequences of these Mexican strains were compared against the nucleotide sequences of the reference strains, 26695 and 60190, a higher polymorphism was observed throughout all the vapD sequences (Fig. 1A). However, vapD amino acid sequence analysis (Mexican and reference strains) showed high homology among all proteins with only two amino acid changes (Fig. 1A), which reflected more synonymous than non-synonymous substitution events (dN/dS= -1.403), thereby suggesting that these genes were under purifying selection. On the other hand, the nucleotide sequence analysis of the MxHp262c11 and MxHp262c13 strains showed a nucleotide (adenine) insertion around position 55. This nucleotide insertion caused a frame shift with the formation of several internal stop codons yielding a truncated protein (Fig. 1B) [39].
Genetic rearrangements associated to H. pylori vapD gene and vapD region from Mexican strains
Sequence analysis of MxHp21.23a, MxHp54, MxHp84, MxHp248, MxHp249, MxHp21.23c, MxHp563 and MxHp118 strains, all of which yielded a larger than the expected PCR product (from 860 bp to 1259 bp), showed high nucleotide identity with two discontinued chromosomal regions of the J99 strain, from nucleotide 911502 to 910648 and from nucleotide 908908 to 908521. In our strains, complete DNA fragment analysis showed a large deletion of 1738 bp with respect to the J99 strain (from 910647 to 908909), which corresponds to jhp0827 (tnpA) and jhp0826 (tnpB) loci present in the J99 strain, but not in our strains (Fig. 2). The deletion in our strains led to this chromosomal portion being rearranged in their ORFs, generating up to four new ORFs. Particularly ORF2, which was present in all of our strains, encodes for a hypothetical protein comprising of 93 amino acids, and exhibits high homology (93%) with proteins encoded for ORFs of different plasmids, such as ORF1 of pHac1 of the Helicobacter acinonychis strain Sheeba, H. pylori ORF12 of pAL226; ORF5 of pHe15; HPAG1poo6; pHP69; pHe14-07 and R4 pHP666 (Fig. 2). ORF1 and ORF4 were also formed and found in MxHp21.23a, MxHp54, MxHp84 and MxHp21.23c strains. ORF1 has a 134 bp size and encodes for a hypothetical protein of 44 amino acids. ORF1 exhibits no homology with any protein reported in the database, although it overlaps with the amino-terminal region (20 amino acids) of the hypothetical protein JHP0829, as well as with the vapD gene (from 5 to 14 amino acids) of H. pylori. ORF4 (present in the complementary strand) is between 203 bp and 233bp in size and is similar (94%) to the hypothetical protein PMM1631 of Prochlorococcus marinus. The fourth ORF present was ORF3, which was detected in a complete form (98 amino acids) in the MxHp 21.23a strain. ORF3 is similar to several other genes or ORFs, including H. pylori JHP0825, ORF2 of pHAC1 of H.
Genealogical analyses of Helicobacter pylori vapD gene
The genealogical network estimation with statistical parsimony showed a haplotype network with the formation of two main clades, namely clade A and B (Fig. 3). Clade A formed a homogenous and compact cluster that diverged from clade B at 31 mutational steps, and comprised most of the analysed sequences, which were isolated from different geographical regions, and from different genetic populations (hpAfrica2 and hpAsia2) and subpopulations (hspAmerind, hspAfrica1NAmerica, hspEuropeColombia, hspEuropeN, hspEuropeS, hspSAfrica and hspWAfrica) [7,56,57]. Nevertheless, several samples of this group (50%) could not be assigned to any genetic population or subpopulation. The presence of loops into clades A and B suggests signals of recombination among members of each clade, but not between clades. A diversification process was observed in clade A. The Mexican strains with the complete vapD ORF and the vapD gene haplotypes from 60190 and 26695 (hspEuropeN) strains were grouped into clade B. Further investigation into clade B showed that two divergent clades, namely C and D were present. Clade C was positioned at approximately 44 mutational steps from clade B and was formed by 3 strains from Europe, which belonged to the hspEuropeN, hspEuropeS and hspEasia subpopulations (Fig. 3). Meanwhile, clade D diverged at more than 60 mutational steps from clade B and was formed by 6 Mexican strains that presented structural reorganization in the vapD region. Strain J99 (hspWAfrica), which has a truncated vapD, was also found in this clade D group (Fig. 3).
Analysis of the 16S rRNA phylogenetic tree showed that the cluster corresponding to the Proteobacteria phylum was grouped into four well defined and separate Proteobacteria Classes: alpha, beta, gamma and epsilon (α, β, γ and ε) (Fig. 4A). In this same cluster, there were also two other phyla: Firmicutes and Actinomycetales. In the second and third clusters, we found the Cyanobacteria and Fusobacterium phyla. The last cluster (bottom of the tree) was formed by the Bacteroidetes phylum that included 3 families (Bacteroidetes, Sphingobacteria and Flavobacteria) [58-60].
The phylogeny of the 16S rRNA gene reflects the relationships (ancestor-offspring) of the species at the level of large taxonomic groups. When this phylogeny was compared against the phylogeny of the vapD gene, it showed a great phylogenetical incongruence among bacteria of both the same phylum and different phyla, suggesting that the vapD gene could have participated in horizontal gene transfer. This idea is supported by the fact that this gene is present in different species that belong to very divergent phyla.
Relative Synonymous Codon Usage (RSCU) values and GC content were calculated for the complete genome of H. pylori and for the vapD gene (Table S2). RSCU values greater than 1, reflected a preference for the use of a specific codon to determine amino acids. RSCU values for the H. pylori complete genome were as follows: 26 codons had a value greater than 1 and 29 had a value less than 1. When the codon usage of the vapD gene was obtained, 16 had a value greater than 1, while none were detected with a value less than 1. The following codons were shared between the H. pylori chromosome and vapD gene: UUU (F), AAU (N), GUG (V), AGC (S), ACC (T), UAU (Y), AAA (K), GAU (D) and GAA (E); only leucine (L) showed a significant difference (p=0.0487). Histidine (His) and cysteine (Cys) were removed from the table because there were no RSCU values for the vapD gene (Table S2).
The average GC content in the whole H. pylori genome was 38.84 ± 0.19. This was lower than for the vapD gene, which had a content of 41.23 ± 0.92 (Fig. S1). These results suggest that vapD could be foreign DNA acquired from an external source other than H. pylori.
Presence of IS in H. pylori vapD region
Insertion sequence (IS) analysis in the vapD region of our strains showed traces of different IS sequences from different families. In the vapD region of the MxHp21.23a strain, we found remnants of a 16 bp of IS Pto9 (transposase) from the IS 5 family (Table 1).
MxHp54 and MxHp84 strains presented traces of different ISs: 15 bp of IS Aurf from the IS 1594 family, 19 bp of IS Cb3 from the IS 3 family, and 19 bp of IS Cb2 from the IS4 family (Table 1). With respect to MxHp248 and MxHp249 strains, we found scars of IS 607 (16 bp), which is specific to H. pylori. The vapD region of the MxHp21.23c strain was the region with most traces of ISs, where we identified IS 3 and IS 4, which are families of Clostridium beijerinncki. This strain also carried 15 bp of IS Aur1 belonging to the IS 1595 family from Actinobacillus ureae and 16 bp belonging to the IS 200/IS 605 family. Finally, we identified traces of the IS 1595 (16 bp) family from Campylobacter jejuni and Psycroflexus torquis and 16 bp from the IS 3 family of Serratia proteomaculans in strain MxHp118 (Table 1).
Table S1: GenBank accession number for the sequences used in this study. |
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GenBank accession number |
Isolate |
Locus |
NP_207113 |
H. pylori 26695 |
vapD |
ASYV01000064 |
H. pylori PZ5080 |
vapD |
AKHP02000000 |
H. pylori FD535 |
vapD |
EQL49894 |
H. pylori FD430 |
vapD |
OOP87307 |
H. pylori CA22327 |
vapD |
PDW75985 |
H. pylori 22311 |
vapD |
EKE81371 |
H. pylori R030b |
vapD |
PUD52554 |
H. pylori GC65HL |
vapD |
AEN17075 |
H. pylori SNT49 |
vapD |
KNX43647 |
H. pylori UM300 |
vapD |
EJC13024 |
H. pylori HP23 |
vapD |
ANH42653 |
H. pylori L7 |
vapD |
PUD05235 |
H pylori 38:2 |
vapD |
EKE84337 |
H. pylori R32b |
vapD |
PUB97335 |
H. pylori 55:2 |
vapD |
OOP75995 |
H. pylori CA2231 |
vapD |
ANH45646 |
H pylori CC33C |
vapD |
PUD18190 |
H. pylori 56599 |
vapD |
AAC45241 |
H. pylori 60190 |
vapD |
PUD52921 |
H. pylori GC23HL |
vapD |
EJB78246 |
H. pylori HpA27 |
vapD |
PUD75866 |
H. pylori B31 |
vapD |
PUD39986 |
H. pylori 3755 |
vapD |
PUB99539 |
H. pylori 38:5 |
vapD |
PUD86084 |
H. pylori B25 |
vapD |
PDX33282 |
H. pylori 2061 |
vapD |
PDW33764 |
H. pylori 3053 |
vapD |
KNX48507 |
H. pylori UM408 |
vapD |
EMG86822 |
H. pylori GAM114Ai |
vapD |
AUZ23548 |
H. pylori dRdM2addM2 |
vapD |
BAW56543 |
H. pylori F55 |
vapD |
CP002953 |
H. pylori ELS37 |
vapD |
CP003419 |
H. pylori XZ274 |
vapD |
CP000241 |
H. pylori HPAG1 |
vapD |
EQD88536 |
H. pylori SouthAfrica50 |
vapD |
CP000012 |
H. pylori 51 |
vapD |
PDX44683 |
H. pylori 2006 |
vapD |
OOQ10045 |
H. pylori CC26100 |
vapD |
PUD90214 |
H. pylori B23+S27R2:R48 |
vapD |
CP002073 |
H. pylori SJM180 |
vapD |
AFX90414 |
H. pylori Aklavik86 |
vapD |
OPG59870 |
H. pylori 2036 |
vapD |
EMH03913 |
H. pylori GAM245Ai |
vapD |
PDW32774 |
H. pylori 3056 |
vapD |
EQD94110 |
H. pylori PZ5026 |
vapD |
AP014523 |
H. pylori NY40 |
vapD |
PDW41337 |
H. pylori 22316 |
vapD |
PDW34671 |
H. pylori 3046 |
vapD |
CP003486 |
H. pylori HUPB14 |
vapD |
AGT74177 |
H. pylori SouthAfrica20 |
vapD |
WP_100949690 |
H. pylori |
vapD |
WP_033596798 |
H. pylori |
vapD |
WP_050545892 |
H. pylori |
vapD |
WP_001988454 |
H. pylori |
vapD |
WP_080025382 |
H. pylori |
vapD |
AFI03858 |
Helicobacter cetorum MIT 00-7128 |
vapD |
WP_104748587 |
Helicobacter cetorum |
vapD |
EEO24159 |
Helicobacter bilis ATCC 43879 |
vapD |
KGL25492 |
Helicobacter bilis ATCC 49314 |
vapD |
WP_104747237 |
Helicobacter bilis |
vapD |
WP_104696282 |
Helicobacter salomonis |
vapD |
WP_027327662 |
Helicobacter pametensis |
vapD |
WP_018450011 |
Leptotrichia shahii |
vapD |
WP_071124930 |
Leptotrichia massiliensis |
vapD |
WP_021744953 |
Leptotrichia sp. oral taxon 879 |
vapD |
WP_018497980 |
Leptotrichia wadei |
vapD |
WP_026747354 |
Leptotrichia trevisanii |
vapD |
WP_064615231 |
Streptobacillus |
vapD |
CP001779 |
Streptobacillus moniliformis DSM 12112 |
vapD |
CP027400 |
Streptobacillus moniliformis strain FDAARGOS_310 |
vapD |
WP_064608975 |
Streptobacillus moniliformis |
vapD |
WP_012858896 |
Streptobacillus moniliformis |
vapD |
SDF71462 |
Sporolituus thermophilus DSM 23256 |
vapD |
WP_093691362 |
Sporolituus thermophilus |
vapD |
KGQ40318 |
Gallibacterium anatis |
vapD |
KGQ53121 |
Gallibacterium anatis |
vapD |
OKZ70694 |
Clostridiales bacterium 41_12_two_minus |
vapD |
CDE43797 |
Clostridium sp. CAG:411 |
vapD |
DQ517426 |
Actinobacillus pleuropneumoniae 12494 plasmid p12494 |
vapD |
AEJN02000103 |
Aggregatibacter actinomycetemcomitans |
vapD |
CP007502 |
Aggregatibacter actinomycetemcomitans HK1651 |
vapD |
CP001733 |
Aggregatibacter actinomycetemcomitans D11S-1 |
vapD |
CP012958 |
Aggregatibacter actinomycetemcomitans strain VT1169 |
vapD |
CP003099 |
Aggregatibacter actinomycetemcomitans ANH9381 |
vapD |
CP016553 |
Aggregatibacter actinomycetemcomitans strain IDH781 |
vapD |
CP003496 |
Aggregatibacter actinomycetemcomitans D7S-1 |
vapD |
CP012959 |
Aggregatibacter actinomycetemcomitans strain 624 |
vapD |
AP014520 |
Aggregatibacter actinomycetemcomitans NUM4039 |
vapD |
PCGW01000011 |
Aggregatibacter actinomycetemcomitans 310b |
vapD |
CP001607 |
Aggregatibacter aphrophilus |
vapD |
AKS65690 |
Aggregatibacter aphrophilus NJ8700 |
vapD |
AAC37126 |
Aggregatibacter actinomycetemcomitans Plasmid pVT7361 |
vapD |
WP_025141941 |
Pedobacter jeongneungensis |
vapD |
KIA92090 |
Pedobacter kyungheensis |
vapD |
CP018790 |
Campylobacter sp. plasmid pSUIS6137 |
vapD |
ARR01447 |
Campylobacter sp. RM6137 |
vapD |
CP004067 |
Campylobacter coli CVM N29710 |
vapD |
KGI22836 |
Prevotella timonensis |
vapD |
CCY64135 |
Prevotella sp. CAG:1124 |
vapD |
CDA67310 |
Prevotella copri CAG:164 |
vapD |
CP002006 |
Prevotella ruminicola 23 |
vapD |
AP018051 |
Prevotella melaninogenica GAI 07411 plasmid pPME0001 |
vapD |
WP_005845454 |
Prevotella dentalis |
vapD |
CP003368 |
Prevotella dentalis DSM 3688 |
vapD |
CDC29181 |
Prevotella sp. CAG:386 |
vapD |
KGI22838 |
Prevotella timonensis |
vapD |
PVX43535 |
Prevotella colorans |
vapD |
OAV75707 |
Bacteroidales bacterium Barb7 |
vapD |
OAV68226 |
Bacteroidales bacterium Barb6XT |
vapD |
OAV70465 |
Bacteroidales bacterium Barb4 |
vapD |
WP_013619538 |
Bacteroides salanitronis |
vapD |
CP002531 |
Bacteroides salanitronis DSM 18170 plasmid pBACSA01 |
vapD |
CP018939 |
Bacteroides fragilis |
vapD |
L22307 |
Dichelobacter nodosus |
vapD |
M74565 |
Dichelobacter nodosus |
vapD |
L22308 |
Dichelobacter nodosus |
vapD |
Q46565 |
Dichelobacter nodosus virulence |
vapD |
L31763 |
Dichelobacter nodosus tRNASer |
vapD |
CP000513 |
Dichelobacter nodosus |
vapD |
CP031475 |
Dichelobacter nodosus VCS1703A |
vapD |
CP022124 |
Fusobacterium nucleatum subsp. animalis strain ChDC F332 |
vapD |
CP007062 |
Fusobacterium nucleatum subsp. animalis 7_1 |
vapD |
RRD31709 |
Fusobacterium nucleatum |
vapD |
EGQ80846 |
Fusobacterium nucleatum subsp. animalis ATCC 51191 |
vapD |
FP929056 |
Fretibacterium fastidiosum draft genome |
vapD |
CBL28245 |
Fretibacterium fastidiosum |
vapD |
AF364087 |
Riemerella anatipestifer plasmid pRA34/901 |
vapD |
AF048718 |
Riemerella anatipestifer plasmid pCFC1 |
vapD |
AF082180 |
Riemerella anatipestifer plasmid pCFC2 |
vapD |
CP018939 |
Bacteroides fragilis strain Q1F2 plasmid Q1F2p2 |
vapD |
CP012938 |
Bacteroides ovatus ATCC 8483 |
vapD |
LT622246 |
Bacteroides ovatus V975 |
vapD |
CP022384 |
Capnocytophaga leadbetteri H6253 |
vapD |
CP022384 |
Capnocytophaga leadbetteri H6253 |
vapD |
CP022022 |
Capnocytophaga sp. ChDC OS43 |
vapD |
CP012589 |
Capnocytophaga sp. oral strain F0383 |
vapD |
CP027232 |
Capnocytophaga oral F0512 |
vapD |
CP001632 |
Capnocytophaga ochracea DSM 7271 |
vapD |
YP007366462 |
Citrobacter freundii CFSTE plasmid pMobC |
vapD |
LK985408 |
Escherichia coli FHI100 |
vapD |
LM996818 |
Escherichia coli FHI71 |
vapD |
LM995883 |
Escherichia coli FHI30 |
vapD |
CP006992 |
Methylobacterium sp. AMS5 |
vapD |
AP014809 |
Methylobacterium populi |
vapD |
CP001298 |
Methylobacterium extorquens CM4 |
vapD |
FP103042 |
Methylobacterium extorquens DM4 |
vapD |
CP021054 |
Methylorubrum zatmanii PSBB041 |
vapD |
LT962688 |
Methylobacterium extorquens TK 0001 |
vapD |
FN995097 |
Neisseria lactamica 02006 |
vapD |
CP019894 |
Neisseria lactamica Y921009 |
vapD |
CP015886 |
Neisseria meningitidis strain 38277 |
vapD |
SBO77403 |
Neisseria gonorrhoeae strain WHO Y |
vapD |
SBO57104 |
Neisseria gonorrhoeae strain WHO K |
vapD |
SBQ20831 |
Neisseria gonorrhoeae strain WHO F |
vapD |
SBO57445 |
Neisseria gonorrhoeae strain WHO M |
vapD |
AGU85211 |
Neisseria gonorrhoeae MS11 plasmid |
vapD |
NC_011034 |
Neisseria gonorrhoeae NCCP11945 plasmid Pngk |
vapD |
SBO74338 |
Neisseria gonorrhoeae strain WHO P |
vapD |
SBO58433 |
Neisseria gonorrhoeae strain WHO O |
vapD |
SBO57901 |
Neisseria gonorrhoeae strain WHO N |
vapD |
SBO57823 |
Neisseria gonorrhoeae strain WHO L |
vapD |
SBO57377 |
Neisseria gonorrhoeae strain WHO G |
vapD |
NP040415 |
Neisseria gonorrhoeae strain UM01 plasmid pJD1 |
vapD |
CP034034 |
Neisseria gonorrhoeae FQ01 plasmid |
vapD |
CP022278 |
Neisseria sp. 10023 |
vapD |
OFN85042 |
Neisseria sp. HMSC064E01 |
vapD |
AWP54479 |
H. influenzae strain 10P129H1 |
vapD |
CP005967 |
H. influenzae KR494 |
vapD |
CP007472 |
H. influenzae 723 |
vapD |
ARB90438 |
H. influenzae FDAARGOS_199 |
vapD |
AIT67183 |
H. influenzae Hi375 |
vapD |
CKG85575 |
H. influenzae NCTC8143 |
vapD |
PRK52554 |
H. influenzae 84P36H1 |
vapD |
NP438611 |
H. influenzae Rd KW20 |
vapD |
BR00016 |
H. influenzae PittGG |
vapD |
CBW28768 |
H. influenzae 10810 |
vapD |
AKA46443 |
H. influenzae 2019 |
vapD |
AAX87502 |
H. influenzae 86-028NP |
vapD |
AJO88533 |
H. influenzae 477 |
vapD |
CP005384 |
H. parasuis ZJ0906 |
vapD |
CP001321 |
H. parasuis SH0165 |
vapD |
CP009158 |
H. parasuis SH03 |
vapD |
CP015099 |
H. parasuis SC1401 |
vapD |
CP009237 |
H. parasuis KL0318 |
vapD |
EU714231 |
Xylella fastidiosa isolate AZ04 |
vapD |
EU714230 |
Xylella fastidiosa isolate NM02 |
vapD |
AP018005 |
Candidatus Rickettsiella viridis ApRA04 |
vapD |
ABB28825 |
Chlorobium chlorochromatii CaD3 |
vapD |
PIE55217 |
Dethiosulfovibrio peptidovorans |
vapD |
OOO00658 |
Epulopiscium sp. Nele67Bin004 |
vapD |
CP006942 |
Mannheimia sp. 1261 |
vapD |
OUP06775 |
Mediterranea sp. An20 |
vapD |
CP024450 |
Moraxella osloensis NP7 |
vapD |
LADV01000113 |
Peptococcaceae bacterium BRH_c23 |
vapD |
WP_108831492 |
Peptoniphilus sp. MarseilleP3761 |
vapD |
CP012903 |
Providencia rettgeri strain N1501091 pNDM15109 |
vapD |
EJW09502 |
Rhodovulum sp. PH10 |
vapD |
LK931667 |
Thiomonas sp. CB2 |
vapD |
CP000542 |
Verminephrobacter eiseniae EF012 |
vapD |
AP018248 |
Tolypothrix tenuis PCC 7101 |
vapD |
AP018307 |
Aulosira laxa NIES-50 |
vapD |
AGC23530 |
plasmid Citrobacter freundii |
vapD |
WP_015353868 |
Citrobacter freundii |
vapD |
STM47029 |
Escherichia coli NCTC10757 |
vapD |
KSW14140 |
Proteus mirabilis PM655 |
vapD |
KSW14422 |
Proteus mirabilis PM593 |
vapD |
EII22447 |
Escherichia coli 9.0111 |
vapD |
NC_004854 |
Rhodococcus equi ATCC33701 |
vapD |
JN990998 |
Rhodococcus equi BBG163 |
vapD |
JN990997 |
Rhodococcus equi SNP89 |
vapD |
P018172 |
Calothrix sp. NIES-2098 |
vapD |
EU127530 |
Campylobacter coli 8693/04 |
16S rRNA |
DQ174144 |
Campylobacter jejuni LMG 8843 |
16S rRNA |
AF372091 |
Campylobacter jejuni NCTC 11351 |
16S rRNA |
AF372092 |
Campylobacter coli LMG 6440 |
16S rRNA |
L04312 |
Campylobacter coli |
16S rRNA |
AY554142 |
Campylobacter sp. BTP1Tcr |
16S rRNA |
AY554143 |
Campylobacter sp. EQ1 |
16S rRNA |
AY554144 |
Campylobacter sp. WB1 |
16S rRNA |
EU781617 |
Campylobacter ureolyticus 4 |
16S rRNA |
GQ167665 |
Campylobacter ureolyticus UNSWR |
16S rRNA |
L04321 |
Campylobacter ureolyticus ATCC 33387 |
16S rRNA |
GQ167666 |
Campylobacter ureolyticus UNSWCD |
16S rRNA |
DQ174173 |
Campylobacter mucosalis ATCC 43264 |
16S rRNA |
M35016 |
Dichelobacter nodosus 198A ATCC 27521 |
16S rRNA |
DQ016290 |
Dichelobacter nodosus AN363/05 |
16S rRNA |
DQ016291 |
Dichelobacter nodosus AN484/05 |
16S rRNA |
CP000513 |
Dichelobacter nodosus VCS1703A4 |
16S rRNA |
AB078974 |
Haemophilus parasuis 322 |
16S rRNA |
AB078973 |
Haemophilus parasuis 319 |
16S rRNA |
CP000672 |
Haemophilus influenzae PittGG |
16S rRNA |
L42023 |
Haemophilus influenzae Rd KW20 |
16S rRNA |
M88148 |
Helicobacter acinonychis 901193 |
16S rRNA |
U18766 |
Helicobacter bilis Hb1 MIT 931909 |
16S rRNA |
AF292378 |
Helicobacter cetorum 9956 |
16S rRNA |
M88147 |
Helicobacter pametensis B9A |
16S rRNA |
U89351 |
Helicobacter salomonis |
16S rRNA |
U01330 |
Helicobacter pylori ATCC 43504 |
16S rRNA |
Z25744 |
Helicobacter pylori NCTC 11916 |
16S rRNA |
KF297892 |
Helicobacter pylori Hp1 |
16S rRNA |
AF277832 |
Helicobacter sp. LU1 |
16S rRNA |
JX001468 |
Citrobacter freundii 7/A10 |
16S rRNA |
LN589732 |
Citrobacter freundii MF3631 |
16S rRNA |
FJ971857 |
Citrobacter freundii ATCC 8090 |
16S rRNA |
AF025363 |
Citrobacter rodentium CDC 184373 |
16S rRNA |
AF025364 |
Citrobacter sedlakii CDC 469686 |
16S rRNA |
AF025367 |
Citrobacter gillenii CDC 469386 |
16S rRNA |
EU888872 |
Citrobacter gillenii A8P19 |
16S rRNA |
KR088351 |
Citrobacter gillenii BK16 |
16S rRNA |
AF025368 |
Citrobacter braakii CDC 8058 |
16S rRNA |
JN118501 |
Citrobacter braakii C274 |
16S rRNA |
JX518488 |
Citrobacter braakii UAAD6 |
16S rRNA |
AF025369 |
Citrobacter murliniae CDC 297059 |
16S rRNA |
KC017346 |
Citrobacter murliniae amBHI1 |
16S rRNA |
AF025371 |
Citrobacter farmeri CDC 299181 |
16S rRNA |
KC429579 |
Citrobacter farmeri TERIYE |
16S rRNA |
KP036922 |
Citrobacter farmeri TCS20 |
16S rRNA |
AF025373 |
Citrobacter werkmanii CDC 087658 |
16S rRNA |
KM268970 |
Citrobacter werkmanii C18 |
16S rRNA |
HQ238425 |
Citrobacter youngae S521B50 |
16S rRNA |
JF939011 |
Citrobacter youngae |
16S rRNA |
AB273741 |
Citrobacter youngae GTC 1314 |
16S rRNA |
HF558364 |
Citrobacter koseri CDC813286 |
16S rRNA |
JF935072 |
Citrobacter koseri CtST1 |
16S rRNA |
HQ992945 |
Citrobacter koseri LMG 5519 |
16S rRNA |
JX297468 |
Citrobacter amalonaticus TTK014 |
16S rRNA |
KC689291 |
Citrobacter amalonaticus DD2 |
16S rRNA |
KT027832 |
Citrobacter amalonaticus E91 |
16S rRNA |
AM947041 |
Citrobacter hormaechei 615 |
16S rRNA |
JQ390124 |
Citrobacter sp. F11 |
16S rRNA |
AY513502 |
Escherichia coli O157 |
16S rRNA |
AE014075 |
Escherichia coli CFT073 |
16S rRNA |
U00006 |
Escherichia coli K12 MG1655 |
16S rRNA |
JN654455 |
Escherichia coli NCTC 50365 |
16S rRNA |
JF508184 |
Escherichia coli ATCC 11303 |
16S rRNA |
KP005067 |
Escherichia coli EC01 |
16S rRNA |
AJ810279 |
Fusobacterium nucleatum animalis OMZ 990 |
16S rRNA |
GQ301042 |
Fusobacterium nucleatum animalis ATCC 51191 |
16S rRNA |
AB573068 |
Fusobacterium nucleatum JCM 8532 |
16S rRNA |
AJ133496 |
Fusobacterium nucleatum ATCC 25586 |
16S rRNA |
AJ006964 |
Fusobacterium nucleatum vincentii ATCC 49256 |
16S rRNA |
X55404 |
Fusobacterium nucleatum NCTC 12276T |
16S rRNA |
X55403 |
Fusobacterium nucleatum NCTC 11326T |
16S rRNA |
FJ717336 |
Leptotrichia wadei F0279 |
16S rRNA |
AY029802 |
Leptotrichia wadei LB16 |
16S rRNA |
KP192296 |
Leptotrichia wadei KA00185 |
16S rRNA |
GU561360 |
Leptotrichia trevisanii TG9 |
16S rRNA |
AY029805 |
Leptotrichia trevisanii LB06 |
16S rRNA |
AF206305 |
Leptotrichia trevisanii |
16S rRNA |
CP012410 |
Leptotrichia oral W10393 |
16S rRNA |
AF287813 |
Leptotrichia oral FAC5 |
16S rRNA |
GU086183 |
Leptotrichia shahii PW1036 |
16S rRNA |
AY029806 |
Leptotrichia shahii LB37 |
16S rRNA |
AJ247245 |
Neisseria meningitidis ATCC 35559 |
16S rRNA |
AY187940 |
Neisseria meningitidis |
16S rRNA |
X74900 |
Neisseria meningitidis NCTC 10025 |
16S rRNA |
X74901 |
Neisseria lactamica NCTC 10617 |
16S rRNA |
FN995097 |
Neisseria lactamica 02006 |
16S rRNA |
AJ247241 |
Neisseria lactamica DSM 4691 |
16S rRNA |
AJ247239 |
Neisseria gonorrhoeae DSM 9189 |
16S rRNA |
EU233796 |
Neisseria gonorrhoeae NG19 |
16S rRNA |
KF410894 |
Neisseria gonorrhoeae NF131677 |
16S rRNA |
AY612187 |
Riemerella anatipestifer TW96015 |
16S rRNA |
KT449829 |
Riemerella anatipestifer EF3 |
16S rRNA |
AY612184 |
Riemerella anatipestifer TRa9 |
16S rRNA |
JQ810973 |
Riemerella columbina M351 |
16S rRNA |
AJ400913 |
Methylobacterium extorquens IAM 1081 |
16S rRNA |
AJ400917 |
Methylobacterium extorquens IAM 12639 |
16S rRNA |
AJ400914 |
Methylobacterium extorquens IAM 12630 |
16S rRNA |
DQ346736 |
Methylobacterium populi 'PapViBa7' |
16S rRNA |
AJ549956 |
Methylobacterium populi BJ001 |
16S rRNA |
AY248705 |
Methylobacterium sp. Mb 49 |
16S rRNA |
AJ400938 |
Methylobacterium sp. NI2 |
16S rRNA |
AF192343 |
Xylella fastidiosa ATCC35879 |
16S rRNA |
CP006696 |
Xylella fastidiosa sandyi Ann1 |
16S rRNA |
CP002165 |
Xylella fastidiosa GB514 |
16S rRNA |
FJ755928 |
Xylella fastidiosa isolate AZ04 |
16S rRNA |
AE009442 |
Xylella fastidiosa Temecula1 |
16S rRNA |
FJ755926 |
Xylella fastidiosa isolate NM02 |
16S rRNA |
AF228001 |
Gallibacterium anatis F 149 |
16S rRNA |
AF228013 |
Gallibacterium anatis BJ3453 |
16S rRNA |
AF302255 |
Actinobacillus pleuropneumoniae N273 |
16S rRNA |
AY017472 |
Actinobacillus pleuropneumoniae HS143 |
16S rRNA |
Y09654 |
Actinobacillus sp. M1933/96/1 |
16S rRNA |
CP001733 |
Aggregatibacter actinomycetemcomitans D11S-1 |
16S rRNA |
AB512012 |
Aggregatibacter actinomycetemcomitans IDH781 |
16S rRNA |
AY362906 |
Aggregatibacter aphrophilus CCUG 3715 |
16S rRNA |
CP001607 |
Aggregatibacter aphrophilus NJ8700 |
16S rRNA |
KC866151 |
Aggregatibacter sp. Melo83 |
16S rRNA |
AY425295 |
Mannheimia sp. BJ3956 |
16S rRNA |
AF053898 |
Mannheimia sp. HPA121 CCUG 38468 |
16S rRNA |
X81876 |
Prevotella dentalis DSM 3688 |
16S rRNA |
AF218618 |
Prevotella ruminicola 223/M2/7 |
16S rRNA |
AY331415 |
Prevotella genomo sp. P6 P4PB_24 |
16S rRNA |
CP002530 |
Bacteroides salanitronis DSM 18170 |
16S rRNA |
AB618792 |
Bacteroides fragilis JCM 17586 |
16S rRNA |
AB599947 |
Bacteroides intestinalis SLC8-5 |
16S rRNA |
X67610 |
Capnocytophaga ochracea ATCC 33596 |
16S rRNA |
DQ012356 |
Capnocytophaga leadbetteri AHN8708 |
16S rRNA |
AY005077 |
Capnocytophaga sp. A47ROY |
16S rRNA |
EF660750 |
Pedobacter daejeonensis PB46 |
16S rRNA |
JN196132 |
Pedobacter kyungheensis THG-T17 |
16S rRNA |
AB666454 |
Pedobacter sp. MaI11-5 |
16S rRNA |
LC062896 |
Streptobacillus moniliformis NMS |
16S rRNA |
CP001779 |
Streptobacillus moniliformis DSM 12112 |
16S rRNA |
Z35305 |
Streptobacillus moniliformis ATCC 14647 |
16S rRNA |
AY337519 |
Clostridium sp. L15 |
16S rRNA |
Y15984 |
Clostridium sp. RXyl1 |
16S rRNA |
AJ229244 |
Clostridium sp. VeCb10 |
16S rRNA |
GQ287651 |
Tolypothrix distorta SAG 93.79 |
16S rRNA |
AB093486 |
Tolypothrix sp. IAM M-259 |
16S rRNA |
HG970653 |
Tolypothrix fasciculata ACOI 3104 |
16S rRNA |
AB325535 |
Tolypothrix tenuis PCC 7101 |
16S rRNA |
KJ920353 |
Aulosira laxa NIES-50 |
16S rRNA |
AP018172 |
Calothrix sp. NIES-2098 |
16S rRNA |
D37876 |
Rhodococcus equi ATCC33701 |
16S rRNA |
AF273613 |
Rhodococcus equi isolate DY1 |
16S rRNA |
MH299445 |
Rhodococcus equi SN26 |
16S rRNA |
KF059848 |
Rhodococcus sp. 209/s |
16S rRNA |
AB720119 |
Proteus mirabilis BSN1 |
16S rRNA |
KJ578727 |
Proteus mirabilis str. D |
16S rRNA |
LR134205 |
Proteus mirabilis NCTC4199 |
16S rRNA |
FJ169187 |
Sporolituus thermophilus AeG |
16S rRNA |
vapD chromosomal region of H. pylori is considered a variable zone. This region was first described in 1997 by Cao and Cover [38], who reported that the vapD gene was located five ORFs downstream from the vacA gene in strain 60190 and was detected in approximately 60% of their strains. Additionally, they reported a high-level of genetic diversity in the corresponding region of vapD-negative strains. In the 26695 strain, the chromosomal locus of vapD (HP0315) is different from that of 60190, while in the J99 strain, the vapD gene is truncated, suggesting that the vapD region is prone to genetic changes. It is probable that the vapD gene was moved through different mobile genetic elements and inserted at different loci into the H. pylori chromosome after being acquired by horizontal gene transfer.
Previous characterization of the vapD gene in our Mexican H. pylori strains showed a lesser frequency (38%) [39], when compared to other studies [38]. The results of the present study showed a greater variability in the vapD region of some of our strains, observing new chromosomal arrangements and new ORFs in this region.
The chromosomal arrangements observed in the vapD region in some of our H. pylori strains suggested horizontal gene transfer with insertions or deletions of genetic material from plasmids, as well as from other mobile element present in different H. pylori strains or other Helicobacter species, such as H. acinonychis strain Sheeba, as shown in the MxHp21.23a, MxHp84, MxHp54, MxHp249, MxHp248, MxHp2123c strains. Additionally, recombination events were observed between loci from different chromosomes resulting in the inversion of DNA segments, as shown in the strain MxHp118, the nucleotide composition of which showed a high degree of similarity to chromosomal regions of the J99 and 26695 strains. Analysis of the complete, continuous sequence of the vapD region in our strains (MxHp21.23a, MxHp84, MxHp54, MxHp249, MxHp248, MxHp21.23c) presented a high degree of nucleotide similarity with two different chromosomal regions of the J99 strain, in which these two regions are joined or separated by the transposase (IS606 and IS605) genes. The role of transposons in the mobility of genetic material is well known; in the J99 strain, the vapD ORF is truncated (JHP0829) and encodes for a hypothetical protein of unknown function. In the Mexican strains (Fig. 2), there are only vestiges of vapD (30 nucleotides), corresponding to a highly conserved region of the vapD gene present in all strains. The extent of variability seen in the vapD region suggests that this region is a hotspot in the H. pylori chromosome, allowing it to become more dynamic and plastic by incorporating or losing genetic material, depending on what is more conducive to the survival of the bacterium.
The vapD gene encodes for the virulence-associated protein D (vapD), which presents homology with other vapD proteins described in other microorganisms, such as Dichelobacter nodosus [31,61], Rhodococcus equi [28], Haemophilus influenza [29], as well as in many other microorganisms of different genera and phyla [42,43,62,63]. In A. actinomycetencomitans, R. anatipestifer, R. equi, N. gonorrhoeae and X. fastidiosa, vapD has been found in plasmids, while in D. nodosus it has been found in both chromosomal and plasmid locations. Strains of pathogenic Rhodococcus equi contain a virulence plasmid that encodes for vapD and for other virulence-associated proteins. Although its function is unknown, it has been suggested that vapD participates in the survival of R. equi within the macrophage, playing a role in acidic tolerance, while R. equi plasmid-cured mutants for vapD fail to induce pneumonia in foals [27,28].
Three copies of the vapD gene have been identified in Dichelobacter nodosus, the essential causative agent of ovine foot rot. Two copies are part of two larger vap regions designated vapABCD, while the third vap region contains only vapD. One of these genes is similar in both length and amino acid sequence to ORF5 found in the N. gonorrhoeae cryptic plasmid, and in the A. actinomycetencomitans plasmid pVT736-1. The similarity between vap encoded-proteins and plasmid-encoded proteins suggests that the vap sequences may have evolved from the sitespecific insertion of an integrative plasmid [44,64].
In the avian pathogen Riemerella anatipestifer, the vapD gene was found in two plasmids, pCFC1 and pCFC2. In pCFC2, the vapD gene was associated with the insertion sequence ISRa1 [42]. In the case of the phytopathogenic bacteria Xylella fastidiosa, the vapD gene (XFa0052) is a strain-specific gene that is located in the plasmid pXF51, which does not have IS elements or transposons [65,66]. It has a great similarity to the vapD protein of R. anatipestifer and A. actinomycetemcomitans. Furthermore, the expression of vapD is induced when the bacteria is under heat stress [43,62]. However, in both microorganisms its function is still unknown.
In H. influenzae, vapD forms part of a toxin-antitoxin(TA) module, where vapD is known as a toxin that on the one hand, helps to promote non-typeable H. influenzae survival within human respiratory cells, while on the other, it enhances virulence during infection using a mechanism of mRNA cleavage [29]. Although its function is still not clear, H. pylori vapD has been reported to have endoribonuclease activity [40], and it may be an evolutionary intermediate of the Cas2 protein in the evolution of the CRISPR-Cas system [67]. Despite several studies indicating the importance of vapD in pathogenesis of different bacterial species, there is a lack of molecular, biochemical and functional data describing the biological role of vapD in the literature.
Analysis of nucleotide sequences of the vapD region of the Mexican strains also showed remnants of different Insertion Sequence (IS) families (IS4, IS3, IS1595, IS607, IS200, IS605). This suggests that these IS types are reminiscent of mobile elements
Table S2: Codon usage bias comparisons in whole genome and vapD gene of Helicobacter pylori |
||||
RSCU* |
X2 |
|||
Aminoacid |
Codon |
Genome |
VapD |
P value |
Phe |
UUU |
1.58 |
2 |
1 |
UUC |
0.42 |
0 |
||
Leu |
UUA |
2.34 |
0 |
0.0487 |
UUG |
1.62 |
0 |
||
CCU |
0.9 |
0 |
||
CUC |
0.54 |
0 |
||
CUA |
0.42 |
0 |
||
CUG |
0.24 |
6 |
||
Ile |
AUU |
1.47 |
3 |
0.3581 |
AUC |
1.14 |
0 |
||
AUA |
0.39 |
0 |
||
Val |
GUU |
1.08 |
0 |
0.4338 |
GUC |
0.56 |
0 |
||
GUA |
0.4 |
0 |
||
GUG |
1.96 |
4 |
||
Ser |
UCU |
1.38 |
0 |
0.3987 |
UCC |
0.48 |
0 |
||
UCA |
0.54 |
0 |
||
UCG |
0.36 |
0 |
||
AGU |
0.84 |
0 |
||
AGC |
2.4 |
6 |
||
Pro |
CCU |
1.96 |
0 |
0.0879 |
CCC |
1.04 |
0 |
||
CCA |
0.6 |
0 |
||
CCG |
0.4 |
4 |
||
Thr |
ACU |
1.24 |
0 |
0.2446 |
ACC |
1.28 |
4 |
||
ACA |
0.64 |
0 |
||
ACG |
0.88 |
0 |
||
Ala |
GCU |
1.56 |
0 |
0.2301 |
GCC |
0.8 |
0 |
||
GCA |
0.44 |
0 |
||
GCG |
1.2 |
4 |
||
Tyr |
UAU |
1.4 |
2 |
1 |
UAC |
0.6 |
0 |
||
Gln |
CAA |
1.7 |
0 |
1 |
CAG |
0.3 |
2 |
||
Asn |
AAU |
1.14 |
0 |
1 |
AAC |
0.86 |
2 |
||
Lys |
AAA |
1.54 |
2 |
1 |
AAG |
0.46 |
0 |
||
Asp |
GAU |
1.46 |
2 |
1 |
GAC |
0.54 |
0 |
||
Glu |
GAA |
1.46 |
2 |
1 |
GAG |
0.54 |
0 |
||
Arg |
CGU |
0.84 |
6 |
0.107 |
CGC |
1.44 |
0 |
||
CGA |
0.42 |
0 |
||
CGG |
0.18 |
0 |
||
AGA |
1.62 |
0 |
||
AGG |
1.5 |
0 |
||
Gly |
GGU |
0.68 |
0 |
0.2779 |
GGC |
1.4 |
4 |
||
GGA |
0.44 |
0 |
||
GGG |
1.48 |
0 |
The genealogical relationship of the vapD sequences from 72 H. pylori strains isolated in Mexico and other parts of the world showed 2 main clades (A and B), where the majority of the strains presented little mutational changes, some signals of recombination between them, and slight alteration in the composition of amino acids of the vapD protein (data not shown), as such changes are seen as synonymous substitutions. Synonymous substitutions do not alter the encoded amino acid sequences, since they are almost neutral with respect to fitness and they are not affected by natural selection [68], suggesting that this gene is subject to purifying selection. This makes vapD an interesting gene, since it behaves in a similar fashion to an essential gene, probably because it is necessary for intracellular strains. The designation of the term “essential” is related to functional significance [68]. It is striking that the vapD gene of the Mexican strains, which apparently encode for a functional protein, were grouped together into a single group (B) with 60190 and 26695 (hspEuropeN) strains, whereas most strains isolated from other parts of the world and belonging to different genetic subpopulations clustered in group A. We identified two further clusters, C (includes isolates from hspEuropeN, hspEuropeS and hspEasia subpopulations) and D (integrated by MxHp strains and the J99 strain [hspWAfrica]), which apparently diverged from cluster B over 44 and 73 mutational steps, respectively (Fig. 3). Analysis of the vapD protein from the strains that were grouped into clade C showed a protein without function due to the insertion of various stop codons throughout the open reading frame. Moreover, the haplotype network showed that clade B is more stable, recombination is low between the strains, and there are few diversification traces. Unfortunately, we cannot verify if the strains belonging to clade A had diverged from the strains of clade B or vice versa. However, clade B strains position themselves in the network of haplotypes suggesting that clade A had diverged from these strains (clade B). Contrary to clade B, the strains of clade A presented greater diversification and signals of high recombination rates of the vapD gene leading to many haplotypes. The low diversity and purifying selection detected in the vapD gene among the MxHp strains, and their divergence from the rest of the isolates, could be attributed to two genetic events, namely recombination and genetic drift. Several studies have suggested that H. pylori was introduced to the Americas from Europe and Africa [9,69]. Since then, H. pylori may have evolved alongside its host, generating new independent evolutionary lineages in Latin America [8,69,70]. Finally, there was greater diversification through deletions, insertions, and recombination, giving rise to clades C and D. Clade D strains had important genetic events in the vapD region, as seen in its chromosomal reorganization and formation of new ORFs, and through the elimination of the vapD gene or of truncated ORF, likely resulting in a different phenotype.
The relationship between the phylogenies of 16S rRNA and vapD genes showed substantially different topologies. While the 16S rRNA phylogenetic tree is coherent for species, genus and phylum, the vapD tree reveals a conflicting topology or phylogenetic inconsistency revealing different evolutionary histories. The 16S rRNA gene has enormous repercussions for inferences about phylogenetic relationships among bacteria and in their taxonomy. This gene has relevant characteristics that make it a widely used reference tool in this kind of studies. Some of these unique characteristics include the fact that bacterial genomes contain several copies and more than 99.5% identity between them; it is a very old molecule, present in all current bacteria; and the changes in the sequence occur very slowly, thereby providing information about all prokaryotes along the evolutionary scale. While 16S rRNA genes have high identity between them within the same species, they have enough variability to differentiate between different species and genera [58,71-73], so they have always been used for reference.
However, this harmonious phylogeny is not always reproduced, as has been seen in phylogenies based on many other genes, resulting in conflicting topologies or phylogenetic inconsistencies [74,75]. There are biological factors that may cause these phylogenetic incongruities of a specific gene, especially among different bacteria domains, which may be the result of different homologous recombination or horizontal gene transfer events (HGT), or incomplete lineage sorting through species, genera or phyla. Regarding 16S rRNA and vapD phylogeny of H. pylori, our results showed substantially different topologies between both trees, which revealed different evolutionary histories for both genes. Focusing more closely on the vapD tree, we found that the vapD sequences from Helicobacter species are more closely related to the vapD gene of different phyla, such as Fusobacterium, Actinomycetales or Firmicutes.
GC content analysis of vapD showed a relatively small difference in the content of GC between the whole genome and the vapD sequences. This could be due to the amelioration of the vapD gene over time, making the ancient HGT event even more difficult to detect [47,76]. Our results showed that this gene was inserted and fixed in the host genome over time. Analysis of codon-usage provides insights into the history of genes in a genome. These genes can differ drastically from native genes. Our results showed that while codon usage is compatible between the genome and the vapD gene, only the codon for leucine had a different preferential codon usage, indicating that vapD translates efficiently with the machinery of the host genome. This event occurs throughout successful generations with the vapD gene and when the host genome is placed under the same selection and mutational pressures, resulting in the homogenization of nucleotide composition and codon usage due to amelioration.
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