Research Article Open Access
Evolutionary dynamics of the vapD gene in Helicobacter pylori and its wide distribution among bacterial phyla
Gabriela Delgado-Sapién1, Rene Cerritos-Flores2, Alejandro Flores-Alanis1, José L Méndez1, Alejandro Cravioto1, Rosario Morales-Espinosa1
*1Laboratorio de Genómica Bacteriana, Departamento de Microbiología y Parasitología, Facultad de Medicina, Universidad Nacional Autónoma de México, Mexico City, México 04510.
2Centro de Investigación en Políticas, Población y Salud, Facultad de Medicina, Universidad Nacional Autónoma de México, Mexico City, México 04510.
*Corresponding author: RosarioMorales-Espinosa, PhD, MD, Laboratorio de Genómica Bacteriana, Departamento de Microbiología y Parasitología. Universidad Nacional Autónoma de México. Avenida Universidad 3000, Colonia Ciudad Universitaria, Delegación Coyoacán, C.P. 04510, México City, México.Tel.: +525 5523 2135; Fax: +525 5623 2114, Email: @
Received: 12thAugust , 2020; Accepted: 15th November 2020; Published: 03rd December, 2020
Citation: G Delgado-Sapien, R Cerritos-Flores, Alejandro Flores-Alanis, José L. Méndez et al., (2020) Evolutionary Dynamics of vapD gene in Helicobacter pylori and its wide Distribution among bacterial phyla. SOJ Microbiol Infect Dis 8(1):1-20.
Abstract Top
The vapD gene is present in microorganisms from different phyla and encodes for the virulence-associated protein D (vapD). In some microorganisms, it has been suggested that vapD participates in either protecting the bacteria from respiratory burst within the macrophage or in facilitating the persistence of the microorganism within the respiratory epithelial cell. The aim of this study was to define the phylogenetic relationship of the Helicobacter pylori vapD gene with other vapD genes of different bacterial species from different phyla and to estimate the genealogy of vapD gene within H. pylori species. Sixteen sequences of Helicobacter pylori vapD gene obtained from Mexican patients and 211 vapD sequences from 72 species of six bacterial phyla were analysed. Our results showed that the vapD region is a hot spot that presents a greater diversity in the Mexican strains of H. pylori to that previously reported. Rearrangements in the vapD region led to the formation of new ORFs in Mexican strains, which were not seen as being fortuitous, suggesting that these chromosomal rearrangements might provide some type of advantage to the bacteria. Phylogenetic analysis, codon usage bias and GC content indicated that vapD was acquired by horizontal gene transfer. Then, in some H. pylori strains it was incorporated and fixed into the bacterial chromosome and maintained in these strains in a similar fashion as an essential gene. Genealogical analysis of the H. pylori vapD gene showed two divisions: one that grouped most of the strains from different parts of the world and the other that grouped only Mexican strains together with the 60190 and 26695 reference strains.

Keywords: vapD gene; Helicobacter pylori; Genealogy; Phylogeny; Horizontal Gene Transfer.
Introduction
As has been widely documented, Helicobacter pylori is a genetically diverse pathogen that colonizes human gastric epithelial cells leading to a variety of gastric diseases in susceptible patients. The clinical outcomes of H. pylori infection are diverse, so it is important to understand the genomic variability that enables the microorganism to adapt to the host, whilst at the same time exhibiting a range of potential virulence factors [1,2,3]. Genetic diversity is seen among H. pylori strains from different origins and ethnic populations, as well as within H. pylori populations within a single stomach. It is well known that H. pylori is a highly recombinant microorganism [4-8] and a natural transformant, which explains its genomic variability and diversity that favour a better adaptive capacity and its permanence on the gastric mucosa for decades. The important events that contribute to its adaptive evolution are horizontal gene transfer (HGT) [4,9] and nucleotide insertion-deletion or substitution [10], which result in the polymorphism of individual genes [11]. Additionally, recombination between homologous chromosomal DNA fragments of different H. pylori strains [10] and inversion or translocation of large DNA fragments within the same genome lead to chromosomal rearrangements [12,13]. Another distinctive hallmark is the presence of strain-specific genes [14,15], which are found within the plasticity zone where the highest diversity among H. pylori strains can be seen [16].

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.
Material and Methods Top
Background of H. pylori vapD isolated from Mexican patients
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
Figure 1: Alignment of Helicobacter pylori vapD gene from Mexican strains. (A) Alignment of nucleotide sequences of vapD ORF from Mexican (MxHp) strains and 60190 strain. Sequence analysis showed a large nucleotide polymorphism between the sequences. However, the amino acid alignment of the deduced vapD product showed an identical amino acid sequence among MxHp strains and 26695 strain, and only two aa changes with respect to the 60190 strain. (B) The red cases indicate that the vapD sequences for the MxHp strains were identical but in the MxHp 262c11 and MxHp 22c13 strains, there was an adenine (A) insertion around nucleotide 55, which caused changes in the reading frame and with the subsequent formation of stop codons (*). All vapD genes were amplified with D1 and D2 primer set.
Table 1: Traces of IS elements present in the vapD region of H. pylori strains from Mexican populations

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

and another, the existence of an intermediate haplotype can be assumed, which is represented by a small black circle or a number that represent the mutational steps.

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].
ResultsTop
Based on the PCR product size, 16 vapD sequences from H. pylori Mexican strains were chosen to be cloned, sequenced, and analysed. Among the 16 analysed sequences, we found genetic variations and rearrangements that affected the structure of vapD gene. In 9 strains, the vapD gene was complete, while in 6 strains only a fragment of the gene was found, and in one strain the entire gene was deleted.

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.
Figure 2: Chromosomal arrangements of the vapD region present in Mexican strains. Using D1 /D2 primers set, the different chromosomal arrangements of the vapD region in Mexican (MxHp) strains, presented PCR products that were larger than expected. Comparison and analysis were made against the J99 type strain. The position of the “scars” present in each sequence for different IS´s is also shown.
acinonychis strain Sheeba, H. pylori ORF13, addiction module toxin, RelE/StbE family and the plasmid stabilization system of Xylella fastidiosa. In MxHp54, MxHp84, MxHp249 and MxHp248 strains, ORF3 is truncated and overlaps with the N-terminal region of the hypothetical protein ORF13, JHP0825 of H. pylori, and pHAC1_2 of H. acinonychis. ORF1 and ORF4 were not present in strains MxHp248 and MxHp249. Arrangements that have commonality with the strains described above are those of ORF2, that are also found in a complete form, the truncated ORF3, and 30 nucleotides that correspond to 10 conserved amino acids (AFDLKIEILK) of the vapD protein. Sequence analysis of the MxHp563 strain showed both a complete vapD ORF and a different ORF that was not previously formed in our strains, although it was described in NCBI databases in other H. pylori strains isolated from Latin American countries. This ORF encodes for a hypothetical protein of 76 amino acids, which to date has not been assigned a function. The MxHp128 strain contained the complete vapD ORF, which was identical to the vapD gene of the 60190 strain. Nonetheless, seven polymorphic sites were found with respect to the H. pylori 26695 strain. Finally, sequence analysis of the MxHp118 strain showed a greater chromosomal variability in the vapD region with the formation of 3 ORFs: the first corresponded to a hypothetical protein of 88 amino acids, which identifies with HP0894, HPAG1_0873, type II toxin-antitoxin system mRNA interferase toxin, RelE/StbE family of H. pylori and type II toxin-antitoxin system YafQ family toxin; the second ORF encodes a hypothetical protein of 96 amino acids; and the third ORF encodes a protein of 90 amino acids, which identifies with the type II toxin-antitoxin system mRNA interferase toxin, RelE/StbE family [Helicobacter pylori] or the type II toxin-antitoxin system YafQ family toxin [Helicobacter pylori].

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).
Figure 3: Genealogical relationship of the vapD gene of Helicobacter pylori strains isolated from different parts of world. This figure shows the network of haplotypes that form two well-defined subpopulations, A and B, and two smaller groups, C and D. The numbers between bars show the mutational steps that diverge between one subpopulation and another; the small black circles indicate a hypothetical intermediate haplotype and the circle size refers to the number of sequences that have that haplotype. Subpopulation A clusters most of the sequences arising from strains from different parts of the world; the cluster of subpopulation B corresponds to strains isolated from Mexico and the collection strains, 26695 and 60190. Subpopulations C and D are very divergent groups. More specifically, cluster D enclosed all those vapD sequences that are truncated, such as J99, and those that presumably suffered different genetic events that resulted in the fragmentation of the gene.
vapD gene phylogenetic relationship among different bacterial phyla, codon usage and GC content
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.
Figure 4: Markov Chain Monte Carlo (MCMC) phylogenetic trees of 16S rRNA and vapD genes. (A) 16S rRNA MCMC tree. This phylogeny reflects the ancestor-offspring relationships of the species at the large, taxonomic group level. The branch colours depict the taxonomy of the 16S rRNA based lineages: Proteobacteria (green), Firmicutes (dark green), Actinomycetales (red), Cyanobacteria (blue), Fusobacterium (brown) and Bacteroidetes (lilac). (B) vap DMCMC tree. The vapD phylogenetic tree shows the relationship of this gene present in species of divergent phyla. Significant conflict between the 16S rRNA and vapD trees is shown by the alternative diagonal lines. These phylogenetic incongruities could be explained by subsequent ancient horizontal transfer events.
On the other hand, the phylogeny of the vapD gene showed a large dispersion without a genetic relationship between species nor grouping within a defined phylum, as was seen in the case of the 16S rRNA gene. In general, vapD phylogeny showed a great intragenic diversity within the same species that can be extrapolated to describe the diversity of the gene within the same phylum. Even though most of the sequences of the Helicobacter vapD gene were in the same branch, they did not form a compact cluster. Some Helicobacter species were observed closer to bacteria from the Fusobacterium phylum, Firmicutes phylum or to Actinomycetales. This issue was not unique to the Helicobacter genus but also observed in members of other phyla. In the 16S rRNA phylogeny of the Bacteroidetes phylum, the species were grouped into a single cluster, while in the vapD phylogeny they were divided into five clusters. The first three vapD clusters were located near to the α and γ-proteobacteria, while the other two clusters were in a separate and independent branch close to both the ε-proteobacteria group and members of the Synergistetes phylum. Meanwhile, strains of the Xylella fastidiosa and Aggregatibacter sp. form different and distant clusters in the 16S rRNA phylogeny but in the vapD phylogeny, they were grouped into the same cluster (Fig. 4).

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.

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

DiscussionTop
One of the most intriguing aspects of H. pylori is its genetic diversity at both the genomic level, as well as in terms of homologous genes from different strains, but its biological significance is still not well understood. H. pylori is a highly variable microorganism in its genetic content and its influence on gene genesis, horizontal gene transfer and gene loss. Gene variability is observed more frequently in specific regions called plasticity zones, which are distributed along the chromosome [18].

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
Figure S1: Average GC content of the whole genome of Helicobacter pylori strains compared with the average GC content of H. pylori vapD gene. There is a marked variation in GC content between the whole genome and vapD gene despite the amelioration process to which the gene has been subjected.
Table S2: Codon usage bias comparisons in whole genome and vapD gene of Helicobacter pylori

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

that participated in the transfer of genetic material, yielding an important impact on genome architecture and function. The ISs play an important role in prokaryote chromosomes, promoting gene inactivation or modulation of neighbouring gene expression, as well as promoting foreign DNA insertion, thereby increasing genome diversity and plasticity [55]. The presence of IS remnants suggests ancestral horizontal gene transfer in our strains. In the J99 strain, we observed the presence of the complete IS606 (jhp0827 and jhp0826 loci) with its tnpA and tnpB genes, and the formation of different ORFs (JHP0829, JHP0828, JHP0827, JHP0826, JHP0825) when compared with those found in the vapD region of our strains. However, there were strains, including the 60190 strain, that presented a complete vapD ORF and did not contain any ISs or traces of IS in the vapD region (flanked by D1 and D2 primers set). Although the MxHp563 strain presented a complete vapD ORF, this strain presented traces of ISs (IS 200/IS 605 and IS 4) in its vapD region and a DNA fragment towards its 5´ end with high homology to a DNA fragment of the J99 strain. It is likely that the MxHp563 strain is an intermediate strain between those strains with the complete vapD gene and those strains that are losing it. Its presence in these strains could aid in the survival of H. pylori inside gastric cells, as seen previously [3], which indicates that vapD is transcribed only when there is H. pylori-epithelial cell interaction. Its transcription is elevated in severe gastric pathologies, reaching the highest expression levels in patients above 57 years of age, demonstrating that vapD is overexpressed during chronic infection. This may suggest that vapD gene detection in Helicobacter pylori strains could be associated with chronic infection.

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.
ConclusionTop
This study documents the evolutionary dynamics of the vapD gene of H. pylori which in other microorganisms has been shown to participate in the virulence of infectious diseases with a high rate of horizontal transfer through mobile elements. In H. pylori, the vapD gene not only fixes on the host chromosome, but it is also functional, allowing the bacterium to acquire adaptive characteristics from other microorganisms, independently of its phylogenetic distance. It is not surprising to find alleles of a gene or specific genes from strains associated with virulence, such as the s1-m1 genotype of vacA and the cag-PAI pathogenicity island, where a clear association has been found between the vacA genotype and the presence of cag-PAI with the development of gastric pathology. The presence of vapD in different strains of H. pylori and its expression in gastric biopsies [3] suggest that vapD is a strain-specific gene, that could confer particular characteristics on survival in a stressful environment, as has been described in R. equi and H. influenzae.
Funding and AcknowledgmentsTop
This work was supported by DGAPA-PAPIIT grant IN213816 and CONACYT grant CB-255574. We thank Dr Carlos Alberto Santiago Olivares and Dr Luisa Sandner-Miranda for helpful discussions.
DeclarationsTop
Conflict of interest statement: The authors declare that they have no competing interests Ethical approval: NA
ReferencesTop
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