A Messenger Ribonucleic Acid: A Platform for
Protein Synthesis

Gabriela Delgado-Sapién; Rene Cerritos-Flores; Alejandro Flores-Alanis; AlejandroCravioto; Rosario Morales-Espinosa

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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: 12^thAugust , 2020; Accepted: 15^th November 2020; Published: 03^rd 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
Phe	UUC	0.42	0	1
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
Tyr	UAC	0.6	0	1
Gln	CAA	1.7	0	1
Gln	CAG	0.3	2	1
Asn	AAU	1.14	0	1
Asn	AAC	0.86	2	1
Lys	AAA	1.54	2	1
Lys	AAG	0.46	0	1
Asp	GAU	1.46	2	1
Asp	GAC	0.54	0	1
Glu	GAA	1.46	2	1
Glu	GAG	0.54	0	1
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