J-Western forms of Helicobacter pylori caga constitute a distinct phylogenetic group with a. widespread geographic distribution

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1 JB Accepts, published online ahead of print on 13 January 2012 J. Bacteriol. doi: /jb Copyright 2012, American Society for Microbiology. All Rights Reserved. 1 2 J-Western forms of Helicobacter pylori caga constitute a distinct phylogenetic group with a widespread geographic distribution Stacy S. Duncan 1, Pieter L. Valk 1, Carrie L. Shaffer 2, Seth R. Bordenstein 2,3 *, Timothy L. Cover 1, 2,4 * Department of Medicine 1 and Department of Pathology, Microbiology and Immunology 2, Vanderbilt University School of Medicine, Nashville, TN; Department of Biological Sciences 3, Vanderbilt University, Nashville, TN; and Veterans Affairs Tennessee Valley Healthcare System, Nashville, TN 4 Running title: Molecular evolution of H. pylori caga *Mailing address for TLC: Division of Infectious Diseases, A2200 Medical Center North, Vanderbilt University School of Medicine, Nashville, TN timothy.l.cover@vanderbilt.edu Mailing address for SRB: Department of Biological Sciences, Vanderbilt University, U7215 BSB / MRB III, st Avenue South, Nashville, TN s.bordenstein@vanderbilt.edu 1

2 Abstract Chronic infection with Helicobacter pylori strains expressing the bacterial oncoprotein CagA confers an increased risk of gastric cancer. While much is known about the ancestry and molecular evolution of Western, East Asian, and Amerindian caga sequences, relatively little is understood about a fourth group, known as J-Western, which has been detected mainly in strains from Okinawa, Japan. We show here that J-Western caga sequences have a more widespread global distribution than previously recognized, occur in strains with multiple different ancestral origins (based on MLST analysis), and did not arise recently. When comparing Western and J-Western forms of CagA, there are 45 fixed or nearly fixed amino acid differences, and J-Western forms contain a unique 4-amino-acid insertion. The mean nucleotide diversity of synonymous sites (π s ) is slightly lower in the J-Western group than in Western or East Asian groups (0.066, 0.086, and respectively), which suggests that the three groups have comparable, but not equivalent, effective population sizes. The reduced π s of the J-Western group is attributable to ancestral recombination events within the 5' region of caga. Population genetic analyses suggest that within the caga region encoding EPIYA motifs, the East Asian group underwent a marked reduction in effective population size compared to the Western and J- Western groups, in association with positive selection. Finally, we show that J-Western caga sequences are found mainly in strains producing m2 forms of the secreted VacA toxin, and propose that these functionally interacting proteins co-evolved to optimize the gastric colonization capacity of H. pylori. 2

3 Introduction Helicobacter pylori is a Gram-negative, microaerophilic bacterium that colonizes more than half of the world s human population (4, 16). H. pylori infection is associated with an increased risk of gastric cancer and peptic ulcer disease (51). H. pylori strains isolated from unrelated humans display a high level of genetic diversity (9, 20, 50) and the intraspecies recombination rate in H. pylori is higher than in most other bacteria (52). Multilocus sequence typing (MLST) has revealed multiple populations and subpopulations of strains with distinct geographic distributions (20, 35, 39). It is proposed that H. pylori had an African ancestral origin, and subsequently spread throughout the world concurrently with human migrations (4, 25, 35, 50). One of the important virulence factors of H. pylori is a highly antigenic protein known as CagA (15, 27, 56, 61). CagA is translocated into gastric epithelial cells through a type IV secretion system, and upon entry into cells, it causes a complex set of alterations in signal transduction (2, 8, 11, 27, 33, 42, 49, 54, 56). Many of the alterations caused by CagA are associated with malignant transformation of cells (2, 8, 11, 27-29, 33, 49, 54, 56). CagA expression in a transgenic mouse model leads to the development of tumors, and CagA is required for H. pylori-induced gastric carcinoma in a Mongolian gerbil model (23, 38, 43). Therefore, CagA is considered to be a bacterial oncoprotein (26). CagA as well as components of the type IV secretion system required for CagA translocation into host cells are encoded by the cag pathogenicity island (cag PAI), a ~40 kb chromosomal region that is present in some H. pylori strains but not others (12, 13, 22, 56). The cag PAI has a lower G+C content than the rest of the H. pylori chromosome and is flanked by 63 direct repeats, which suggest that it was acquired through a horizontal transfer event (13). H. 3

4 pylori strains containing the cag PAI are associated with a higher risk of gastric cancer and peptic ulcer disease than are strains that lack the cag PAI (10, 21). Within host cells, CagA undergoes tyrosine-phosphorylation by host cell kinases (7, 42, 49). Both tyrosine-phosphorylated CagA and non-phosphorylated CagA can bind to several intracellular target proteins (8, 26, 27, 31, 33, 56). Multiple sites of tyrosine phosphorylation, known as EPIYA motifs, are located in the C-terminal region of the CagA protein (31). These motifs have been classified as EPIYA-A, EPIYA-B, EPIYA-C, and EPIYA-D, based on characteristics of the flanking amino acids (Fig. 1) (27). EPIYA-A and EPIYA-B motifs (in variable numbers) are found in nearly all CagA sequences. Most strains contain either an EPIYA-C motif (in single or multiple copy) or an EPIYA-D motif, but not both. Previous studies have shown that there is a high level of genetic diversity among caga genes from different H. pylori strains, and that caga diversity has been driven by positive selection (19, 24, 25, 44, 59). Diversity among caga genes or CagA proteins has been analyzed using a variety of approaches. One approach has been to examine sequence diversity in EPIYA motifs. EPIYA-D motifs have been identified almost exclusively in H. pylori strains from East Asia, whereas EPIYA-C motifs have been found predominantly in non-asian strains (Fig. 1) (27, 28, 30). Several studies have shown that H. pylori strains with an East Asian form of CagA (EPIYA-D) are associated with an increased risk of gastric cancer, compared to strains with a Western form of CagA (EPIYA-C) (3, 6, 30, 32, 38, 40). In addition to the studies that have analyzed diversity in small regions of CagA such as the EPIYA motifs, several studies have undertaken phylogenetic analysis using larger portions of the caga gene or protein. These studies have detected striking geographic differences in caga sequences (26, 36, 53, 60, 63). For example, caga sequences in H. pylori strains from East Asia 4

5 or H. pylori strains from Amerindian persons within the Amazon are phylogenetically distinct from the caga sequences that are found commonly in H. pylori strains from Western countries (26, 34, 36, 53). Interestingly, two different subtypes of caga have been detected in Japan. One subtype (which encodes an EPIYA-D motif) closely resembles the caga alleles found in other parts of East Asia, whereas a second subtype encodes an EPIYA-C motif, which is typically found in Western forms of CagA (26, 60, 63). The latter subtype has been termed a Japanese subtype of Western CagA, and has been designated J-Western (26, 60). Nearly all of the J-Western caga alleles described in the literature have been detected in H. pylori strains from Okinawa, Japan, and there has been very little study of this caga group (14, 24, 26, 60). One hypothesis is that J-Western forms of caga arose recently, possibly related to the presence of American troops in Okinawa at the end of World War II (26). Another hypothesis is that some of the earliest inhabitants of Japan known as the Jomon people (ancestors of persons currently living in Okinawa) were infected with strains harboring a unique J-Western form of caga (26). The goals of the current study were to conduct an in-depth analysis of the relationships between J-Western caga sequences and other major groups of caga sequences, and to elucidate the origin and evolution of the J-Western caga group. By analyzing a large sequence data set, comprising 129 full-length caga sequences, we show that the J-Western group comprises one of four main groups of caga sequences. We present evidence that the J-Western caga group is not simply a Japanese subtype of Western caga, but in fact has a much broader geographic distribution than previously recognized. By analyzing the mean nucleotide diversity of synonymous sites within caga groups, we show that the J-Western group has an effective population size similar to that of other caga groups; this suggests that the J-Western group did not arise recently. Finally, we show that in an analysis of 5

6 H. pylori strains from the United States, J-Western caga sequences are found mainly in strains producing m2 forms of the secreted VacA toxin; this association is biologically relevant because CagA and VacA are known to be functionally interacting proteins Materials and Methods Selection of caga sequences for phylogenetic analysis. Full-length caga nucleotide sequences and deduced CagA amino acid sequences were identified by performing a BLAST search of GenBank, using the caga nucleotide or CagA protein sequence from a prototype reference strain (26695) as the query. Sequences were aligned using Muscle in Geneious, version (Drummond AJ, Ashton B, Buxton S, Cheung M, Cooper A, Heled J, Kearse M, Moir R, Stones- Havas S, Sturrock S, Thierer T, Wilson A (2010) Geneious v5.1, available from Subsequently, insertions and deletions (indels) and hypervariable regions were removed manually from the sequence alignments, resulting in a final alignment length of 2940 nucleotides (or 980 amino acids). In comparison, the caga gene of a prototype reference sequence (strain 26695) is 3561 nucleotides in length (or 1187 amino acids) prior to indel and hypervariable region removal. Ten caga sequences [from strains v225d (accession numbers FR and FR666857), HUI1769 (accession no. FR and FR666835), CC42C, CUZ20, HUI1692, VietnamHPNo31, VietnamHPNo32 and Shi470 (accession no. ACD48278)] were excluded from the final alignments and were not used for analyses due to absence of large segments (>1 kb) of caga. Phylogenetic analysis of the excluded sequences from Amerindian strains (two truncated copies of caga in strain v225d and the second copy of caga in Shi470) revealed that they were similar to the full-length Amerindian caga sequences 6

7 that were included in the analysis. The final alignment, based on all full-length caga sequences that were available in September, 2010, is comprised of 129 sequences from 129 different strains. Phylogenetic analyses of caga. A model of evolution (model: GTR (I+G)) was selected for analysis of full length caga nucleotide sequences using jmodeltest, version 0.1 ( (46) and the Akaike information criterion (AIC). A model of evolution (model: JTT (I+G)) was selected for analysis of full length CagA amino acid sequences using ProtTest, version 2.4 ( (1) and the Akaike information criterion (AIC). Unrooted phylogenetic trees depicting relationships among caga nucleotide sequences or CagA amino acid sequences were built using MrBayes in Geneious. MLST analysis. Among the strains included in this study, 35 had been previously analyzed by MLST, and whole genome sequences were available from 8 other strains. MLST analysis was performed as previously described (18, 37). In brief, partial nucleotide sequences of seven housekeeping genes (atpa, efp, muty, ppa, trpc, urei, and yphc) were compared to the corresponding sequences of 490 reference H. pylori strains that were previously classified into distinct groups (20, 35, 39). Nucleotide sequences of the concatenated MLST loci were aligned using the MUSCLE algorithm within MEGA5 (55). Phylogenetic relationships were analyzed using MEGA5 with the Kimura 2-parameter model of nucleotide substitution and neighborjoining clustering. Analysis of polymorphisms in caga. Full-length caga sequences or deduced CagA protein sequences were aligned using ClustalW (58) and the resulting multiple sequence alignment was visualized in Jalview, version (62). The presence or absence of EPIYA-C and EPIYA-D 7

8 motifs was evaluated using previously published criteria (26). Informative sites that allowed differentiation among CagA groups were identified by inspecting the CagA multiple sequence alignment and identifying polymorphisms of interest by eye. Polymorphisms were considered informative if >95% of the residues at a particular position in one group were different from all residues at that site in a second group. Analysis of caga in H. pylori strains from the United States or Europe. Twenty-one cagapositive H. pylori strains isolated from patients in the United States or Europe were selected for study. Eleven of the selected strains contained a type m2 vaca allele and ten contained a type m1 vaca allele, as determined based on previously described methods (5). A caga fragment was amplified from each strain using either 5'-TTTTATGGAAMATAYCATACAACCCCC-3' or 5'- CKCYAATAAAGCGATCAAAAATCCTRCC-3' as forward primers and 5'- AAATTAGAATAATYAACAAACATCACGCC-3' or 5'- ATCTAAAGCTTTATCTKSTTTTTTCCC-3' as reverse primers. The nucleotide sequences of the amplified products were then determined using Sanger sequencing. Analysis of mean nucleotide diversity and positive selection. A sliding window analysis of intragroup nucleotide diversity at synonymous sites (π s with Jukes and Cantor correction) was performed using an alignment of 129 caga sequences (2940 nucleotides analyzed after removal of indels and hypervariable regions) with the program DnaSP ( (47). Parameters used in the sliding window analysis included a window size of 50 bases and a step size of 10 bases. For further analysis, caga was subdivided into three domains (Fig. 1). Domain 1 includes nucleotides of caga from strain 26695, Domain 2 includes nucleotides (corresponding to a region encoding EPIYA domains) and Domain 3 includes nucleotides Parameters used for sliding window analysis of Domain 1 (~2200 nucleotides after 8

9 indel removal), Domain 2 (~700 nucleotides) and Domain 3 (~500 nucleotides) included a window size of 9 bases and a step size of 3 bases. P-values were calculated using the Mann- Whitney U test. For analysis of Ka/Ks, we selected representative caga DNA sequences from Group 1, Group 2, and Group 3. Sequences were aligned, indels were removed, and subsequently pairwise comparisons were conducted using DnaSP (window size of 50 bases and step size of 10 bases). McDonald Kreitman analysis was conducted as previously described (25). Results Phylogeny of caga. Phylogenetic analysis of 129 full-length caga nucleotide sequences retrieved from Genbank revealed that nearly all of the sequences clustered into four main groups, denoted Groups 1-4 (Fig. 2A, Supplemental Table S1 and Supplemental Fig. S1A). These four groups were also observed in an analysis of the corresponding CagA amino acid sequences (Fig. 2B and Supplemental Fig. S1B). The phylogenetic analysis of caga nucleotide sequences revealed several subgroups within Groups 2 and 3 (Fig. 2A). As a first step in analyzing the four main caga groups, we examined the global geographic locations where the corresponding H. pylori strains were isolated (Supplemental Fig. S2 and Supplemental Tables S1, S2 and S3). Strains containing Group 1 caga sequences were isolated from a wide variety of geographic sites throughout the world, whereas nearly all strains that contained Group 2 caga sequences were isolated from East Asia (mostly Japan) or Southeast Asia (Thailand, Vietnam and the Philippines) (Supplemental Fig. S2B). The majority of strains containing Group 3 caga sequences were isolated from East Asia (Okinawa, Japan), but several of these strains were isolated from other geographic regions (Supplemental Fig. S2B). Strains 9

10 containing Group 4 caga sequences were isolated from Amerindian populations in South America (Supplemental Fig. S2B). As an additional approach for characterizing the four main caga groups, we analyzed 43 strains for which both caga sequences and MLST data were available. Based on MLST analysis, these strains represent multiple populations or subpopulations of H. pylori (Fig. 3, Supplemental Tables S1, S4). Based on our analyses, combined with a review of previous publications describing various sequences in each group, Group 1 of the caga tree contains caga sequences that have previously been described as a Western type (26, 27), and these are primarily from H. pylori strains that are classified as hpeurope, based on MLST analysis (Fig. 4). Group 2 corresponds to caga sequences previously described as an East Asian type (26), and these are from strains classified as hspeasia, hspasia2, hspmaori, or hpsahul (Fig. 4). Group 4 corresponds to Amerindian caga sequences (34, 36, 53), and these are from strains classified as hspamerind (Fig. 4). Group 3 of the caga tree includes caga sequences from strains classified as hpeurope based on MLST analysis (Fig. 4) and also a large number of sequences from strains originating from Okinawa, Japan; the latter sequences have previously been designated as a Japanese caga subtype, known as J-Western (60, 63). Among 17 strains with an hpeurope MLST type, the majority contained caga sequences classified as Group 1, and four strains contained caga sequences classified as Group 3 (Supplemental Fig. S3). All strains with an hspeasia MLST type contained caga sequences classified as Group 2 (Supplemental Fig. S3). Three of four strains classified as hspwafrica contained caga sequences classified as Group 1. Strains with an hspamerind MLST type contained caga sequences that were classified as Group 4. 10

11 Although there are correlations between groups of the caga phylogeny and groups in the phylogeny of housekeeping gene sequences, there are some features of the caga phylogeny that do not correlate with MLST classification. For example, strains classified as hpeurope contain caga sequences classified as Group 1 or Group 3 rather than a single well-defined group (Supplemental Fig. S3). Additionally, MLST analysis of H. pylori strains consistently identifies well-defined African groups designated hpafrica1 (including subgroups hspwafrica and hspsafrica) and hpafrica2 (Fig. 3); however, distinct African groups are not observed in the caga tree (Fig. 2). This discrepancy could potentially reflect horizontal transfer of the cag PAI into H. pylori at a timepoint subsequent to the period when housekeeping genes of African strains and non-african strains diverged. Our phylogenetic analysis suggests that Group 3 (J-Western) caga sequences are found not only in strains from Japan, but also in strains from other parts of the world, including North America and Europe (Supplemental Fig. S2). Notably, this group of caga sequences has not been analyzed previously in any detail. Therefore, we undertook analyses designed to further investigate the group of caga sequences classified as Group 3 or J-Western. Sequence analysis of CagA EPIYA motifs. Previous studies have shown that East Asian and Western forms of CagA can be differentiated by analysis of EPIYA-C and EPIYA-D motifs (26). These motifs, as well as other indels and hypervariable regions, were excluded from the initial phylogenetic analyses (Fig. 2). To evaluate which forms of EPIYA motifs are present in each of the four groups, we analyzed an alignment of CagA sequences in which indels or hypervariable regions were not removed, and examined each sequence to determine whether EPIYA-C or EPIYA-D motifs were present. 11

12 Consistent with previous analyses of EPIYA motifs in various CagA sequences used in the present study, nearly all CagA sequences in Groups 1 and 3 contain at least one EPIYA-C motif, and nearly all sequences in Group 2 contain a single EPIYA-D motif (Table 1). As previously reported, the EPIYA motifs in CagA sequences of Group 4 (classified as hspamerind) are quite different from EPIYA-C or EPIYA-D motifs (34, 36, 53). These Amerindian EPIYA motifs contain a 5-amino-acid segment (DDLGG) found in EPIYA-C motifs, as well as a 12-amino-acid segment (GVGXFSGXGXXD) that is not present in EPIYA- C motifs (Fig. 5). Multiple residues within this latter segment (GVGXFSGXGXX) match a segment found in EPIYA-D motifs (Fig. 5). Therefore, the Amerindian EPIYA motifs can be considered chimeras or hybrids that contain features characteristic of both EPIYA-C and EPIYA- D motifs, and we designate these as EPIYA-DC motifs. Since Group 1 and Group 3 CagA sequences each contain EPIYA-C motifs, we examined these regions in more detail to identify possible differences between the two groups (Fig. 5). We detected a higher level of heterogeneity in this region among Group 3 sequences than among Group 1 sequences (see positions 2, 5, 7, 20, and 21 in Fig. 5, indicated by asterisks), but we did not identify any fixed differences when comparing these two groups. In summary, Group 3 CagA sequences contain EPIYA-C motifs that are indistinguishable from those found in Group 1 CagA sequences and are very different from the EPIYA-D motifs characteristic of Group 2 (East Asian) sequences or EPIYA-DC motifs characteristic of Group 4 (Amerindian) sequences. Identification of a unique insertion in Group 3 CagA sequences. Since Group 1 and Group 3 sequences could not be readily distinguished based on analysis of EPIYA-C motifs, we sought to identify other indels or hypervariable regions that might allow differentiation of the two groups. This analysis revealed a four-amino-acid insertion (PNGD or STGE or PTGE) near the CagA 12

13 amino-terminus present in all Group 3 CagA sequences, but absent from Group 1 and all the other groups (Fig. 6). We did not identify any additional indels or hypervariable regions that were restricted to particular CagA groups. Group 3 caga genes in H. pylori strains from the United States and Europe. Thus far, most of the H. pylori strains containing Group 3 caga sequences have been isolated from persons in Okinawa, Japan, and the corresponding caga sequences were previously designated as a Japanese subtype of Western caga (J-Western) (26, 60, 63). Our current analysis reveals that closely related caga sequences are present in H. pylori strains from sites outside Japan (Fig. 2, Supplemental Fig. S2, Supplemental Tables S2 and S3). The Group 3 sequences from Western origins and East Asian origins can be differentiated by their DNA sequences (compare subgroups 3a and 3b in Fig. 2, Supplemental Tables S2 and S3). Furthermore, all of the Group 3 sequences from strains isolated in Europe and North America contain a PNGD or PNGE insertion whereas the Group 3 sequences from strains isolated from East Asia harbor either a STGE or PTGE insertion (Fig. 6). We hypothesized that Group 3 caga sequences might be present in Western countries at a higher frequency than previously recognized. To test this, we analyzed caga in 21 H. pylori strains that were isolated from patients in the United States or Europe, as described in Methods. This analysis revealed that 9 strains contained caga sequences encoding a four-amino acid insertion characteristic of Group 3 sequences, and the remainder lacked this insertion. In each case, the four-amino-acid insertion (PNGD or PNGE) was characteristic of Western Group 3 CagA sequences. Based on the presence or absence of this insertion, along with additional phylogenetic analysis (data not shown), we classified 12 of the 21 CagA sequences as Group 1 and 9 sequences as Group 3 (Supplemental Table S5). These results provide further evidence 13

14 that Group 3 caga sequences are not found exclusively in Okinawa, Japan, but have a broader geographic distribution. Association of Group 3 CagA with type m2 VacA. In a previous study, we noted that there were similarities in the topology of VacA and CagA trees, and Group 3 forms of caga were found mainly in strains containing type m2 forms of vaca (25). This previous analysis was limited by the availability of relatively few strains for which both complete VacA and CagA sequences were available, and all of the Group 3 forms of CagA were in H. pylori strains from Okinawa, Japan. To test whether there was any relationship between VacA and CagA in H. pylori strains from the United States and Europe, we compared the VacA and CagA types of the 21 strains described above. Among 10 strains with type m1 vaca, 9 contained caga alleles classified as Group 1 and one contained a caga allele classified as Group 3 (Supplemental Table S5). Among 11 strains with type m2 vaca, 3 contained Group 1 caga alleles and 8 contained Group 3 caga alleles. Thus, in this population of strains from the United States and Europe, Group 3 caga alleles are present more commonly in strains with type m2 vaca than in strains with type m1 vaca (p = , Fisher s exact test). Analysis of sites that allow discrimination of Group 3 and other CagA groups. To gain further insight into relationships between Group 3 and the other CagA groups, we undertook a detailed analysis of CagA sequence variation. Examination of the aligned amino acid sequences revealed numerous sites in which at least one of the 129 sequences harbored a polymorphism different from the consensus sequence; however, we focused on a limited number of sequence differences that would allow differentiation of the three main groups (Groups 1, 2, and 3). Group 4 sequences were not included in this analysis due to the small number of available sequences. By analyzing alignments of CagA sequences (without removal of indels), we 14

15 identified 140 informative sites (defined as described in Methods) at which there were fixed or nearly fixed substitutions between particular groups (Fig. 7). Pairwise comparisons of CagA sequences (including indels) among the three main groups revealed 126 informative sites when comparing sequences from Group 1 and Group 2, 112 informative sites when comparing sequences from Group 2 and Group 3, and 45 informative sites when comparing sequences from Group 1 and Group 3. As shown in Supplemental Fig. S4, the amino acids found at many of the informative sites correspond to polar or charged residues, which are predicted to be surfaceexposed. Therefore, the observed polymorphisms at these sites are predicted to result in variations in the surface features of CagA (44). At present, the only region of CagA for which three-dimensional structural data are available is a 14-amino acid region of the CagA CM motif bound to PAR1b/MARK2 (41). In the future, as additional CagA structural data become available, it will be important to map the sites of these polymorphisms onto a CagA crystal structure and analyze the structural correlates of group-specific diversity. The phylogenetic analyses shown in Fig. 2 indicate that Groups 1 and 2 are the most highly divergent groups and Groups 3 and 4 arise from branches positioned between Groups 1 and 2. Correspondingly, as described above, the highest number of informative differences was detected when comparing Groups 1 and 2. To further study the features of Group 3 that are distinct from Groups 1 and 2, we used Groups 1 and 2 as references, and analyzed the positions in Fig. 7 at which residues in Group 3 CagA sequences were identical to residues in either Group 1 or Group 2 CagA sequences. This analysis revealed a non-random pattern of relatedness. In particular, we identified a large region (corresponding to amino acid positions in Fig. 7, which encompasses the EPIYA motifs) where Group 3 was nearly identical to Group 1 at informative sites. This is consistent with the presence of EPIYA-C motifs in Groups 1 and 3, 15

16 and the presence of highly divergent EPIYA-D motifs in Group 2. The corresponding DNA sequences (encoding the residues depicted in Fig. 7, beginning at position 799 and ending at position 1020) from Group 1 and Group 3 were nearly indistinguishable. Within the N-terminal portion of CagA, amino acids in informative sites from Group 3 are identical to amino acids from either Group 1 or Group 2 (for example, residues at positions , and and residues , and , respectively, depicted in Fig. 7). Similar to what was observed in the EPIYA region, the regions of relatedness often comprised contiguous amino acids, and the corresponding DNA sequences of different groups were identical in the regions listed above. In summary, there are numerous sites at which Group 3 is distinct from either Group 1 or Group 2. Group 3 sequences are closely related to Group 1 sequences in several regions (particularly the region encompassing EPIYA motifs, corresponding to residues at positions in Fig. 7), and are related to Group 2 sequences within several short tracts in the N-terminal portion of CagA. These relationships suggest that the evolution of these three groups was shaped by a series of recombination events. Analysis of synonymous site diversity and positive selection. Prior to the current study, Group 3 forms of CagA had been detected almost exclusively in H. pylori strains from Okinawa, Japan (26, 60, 63). One hypothesis is that these forms of CagA arose recently, perhaps in association with the presence of American troops in Okinawa following World War II (26). However, the current results, demonstrating a geographic distribution of Group 3 sequences in several continents (North America, Europe, and East Asia), suggest that these sequences may represent a form of caga that has been in existence for a relatively long period of time. To compare the effective population sizes of the three main caga groups, we calculated the mean intragroup 16

17 synonymous site diversity (π s ) for caga sequences within Group 1, Group 2, and Group 3 (Table 2). Although the whole-gene π s values were similar (0.086, 0.083, and 0.066, respectively), the Group 3 π s was significantly lower than π s of Groups 1 and 2 (p<0.001 and p<0.01, respectively) (Table 2). This indicates that Group 3 has a slightly smaller effective population size than Groups 1 or 2. Nevertheless, the level of synonymous site diversity observed within Group 3 suggests that this group has existed for a sufficiently long period of time to accumulate a substantial number of synonymous mutations. These results, along with the observed global distribution of Group 3 sequences, provide evidence that Group 3 forms of CagA have not arisen recently. To determine if the reduction in Group 3 π s is gene-wide or domain specific, we analyzed π s in the three domains of CagA depicted in Fig. 1. Domain 1 includes the N-terminal portion of caga (nucleotides in caga from strain 26695), Domain 2 includes the EPIYA region (nucleotides in caga from strain 26695), and Domain 3 includes the C-terminal portion of caga. As shown in Table 2, π s is significantly reduced in Domain 1 of Group 3 compared to Domain 1 of Groups 1 and 2 (p<0.001). In contrast, π s values in the other two domains of Group 3 are not significantly reduced compared to the corresponding domains of Groups 1 and 2. Despite evidence that the three groups have fairly similar effective population sizes (whole gene π s ranging from to 0.086), Groups 1 and 3 are closely related within the region encompassing EPIYA motifs (positions in strain 26695) and Group 2 is highly divergent in this region (Fig. 5 and Fig. 7). One potential explanation for this pattern of relatedness could be the occurrence of a recombination event in which the ancestral sequence from Group 1 or Group 3 was replaced with a corresponding sequence from the other group. 17

18 Alternatively, there could have been a relatively rapid genetic change in the EPIYA region of Group 2. To differentiate between these two possibilities, we analyzed intragroup synonymous nucleotide diversity in the region of caga that encodes the EPIYA-A, B, C or D motifs, denoted Domain 2. This region corresponds to the large region in Fig. 7 where the amino acids in informative sites of Group 3 are identical to those in Group 1. As shown in Table 2, π s values for Groups 1 and 3 within Domain 2 are similar to what is observed for the entire gene for these groups. In contrast, π s in Domain 2 of Group 2 is significantly lower than π s of Domain 1, Domain 3 or the entire gene within Group 2 (Table 2). Moreover, π s in Domain 2 of Group 2 is significantly lower than that of Domain 2 within Groups 1 or 3 (p<0.001 and p<0.01, respectively). These results suggest that Group 2, Domain 2 specifically had a marked reduction in effective population size compared to the corresponding domains of Groups 1 and 3. To corroborate these results and further define the regions exhibiting a reduction in π s, we conducted a sliding window analysis of π s within Domain 2. This revealed two specific regions of Group 2, Domain 2 that have lower π s than the corresponding regions of Domain 2 in Groups 1 and 3 (Fig. 8). These two regions correspond to amino acid positions and positions in Fig. 7, where Groups 1 and 3 are closely related to each other and Group 2 is highly divergent. Reductions in π s can be due to bottleneck events or a recent bout of selection that purged genetic diversity. As a first step in differentiating between these possibilities and investigating whether adaptive evolution in Domain 2 is driving the divergence of this group, we used a McDonald Kreitman test (MKT) to compare full-length caga sequences from Groups 1, 2, and 3. The MKT analyzes the neutral theory prediction that the ratio of synonymous-tononsynonymous polymorphism (Ps/Pn) within groups should be the same as the ratio of 18

19 synonymous-to-nonsynonymous divergence (Ds/Dn) between groups. Excess nonsynonymous fixation, one signature of adaptive protein evolution, causes the Neutrality Index (NI) in the MKT to be less than 1. A significant deviation from neutrality was indicated when comparing Group 1 with Group 2 (NI= 0.592, p=0.007) and Group 2 with Group 3 (N=0.556, p=0.009), but not when comparing Group 1 with Group 3 (NI=0.686, p=0.100). These results indicate that caga is under strong positive selection, and are in agreement with previous reports (25, 44, 59). To test the hypothesis that a reduction in effective population size of Group 2, Domain 2 is associated with positive selection events and to identify specific regions of Domain 2 that might be under positive selection, we compared representative sequences of each group using a sliding window Ka/Ks analysis (Fig. 9). Strikingly, in this analysis, one of the regions with a very high Ka/Ks value (located near nucleotide 2800 in Fig. 9) corresponds to one of the regions of Group 2, Domain 2 with a low π s (starting around nucleotide 475 in Fig. 8B) and corresponds to amino acid positions in Fig. 7. This region encodes a portion of the EPIYA-D motif illustrated in Fig. 5. As expected, strong positive selection in this region of caga was only detected in the Ka/Ks sliding window analyses when comparing Group 2 to Groups 1 or 3 (subgroups a and b) (Fig. 9), but not when comparing Group 1 to Group 3 (Fig. 9 and Supplemental Fig. S5). Similar results were observed when comparing Groups 1 and 3 to representative sequences from Group 2 subgroups b-d (subgroups shown in Fig. 2a) (data not shown). Discussion The current phylogenetic analysis of 129 full-length caga sequences from H. pylori strains isolated throughout the world provides a comprehensive view of caga sequence diversity, 19

20 and reveals the existence of four main groups of caga sequences. The caga sequences classified as Group 3 or J-Western have not been studied previously in any detail, and there are unresolved questions about the origin and evolution of this group. Therefore, we conducted an in-depth analysis of this group and its relationship to the other major groups of caga sequences. Prior to the current study, Group 3 or J-Western caga sequences had been detected almost exclusively in H. pylori strains from Okinawa, Japan, and these were considered to be a Japanese subtype of Western caga (26, 60, 63). In the current study, we show for the first time that J-Western caga sequences are not simply a Japanese subtype of Western caga (as had been previously proposed) (14, 24, 26, 60), but in fact, comprise a major caga group with a much broader geographic distribution than previously recognized. Consistent with a broad geographic distribution, J-Western caga sequences occur in strains with multiple different ancestral origins, based on MLST analysis. Since J-Western caga sequences were previously identified mainly in Okinawa, Japan but contained a Western-type EPIYA-C motif, it was suggested that these caga alleles might have arisen recently as a result of recombination between Western and East Asian forms of caga, possibly related to the presence of American troops in Okinawa at the end of World War II (26). Our current analysis shows that there are 45 fixed or nearly fixed amino acid differences that distinguish J-Western CagA sequences from the Western CagA sequences, and 112 differences that distinguish J-Western CagA from East Asian CagA. This level of diversification, combined with data showing that J-Western, Western, and East Asian caga groups have similar effective population sizes, provides evidence that J-Western forms of caga did not arise recently. Groups 1 and 3 have very similar EPIYA-C motifs (Fig. 5), and are also closely related at numerous other sites within this region of CagA (designated Domain 2 in the current study) (Fig. 20

21 ). Analyses of nucleotide diversity suggest that the similarity of Groups 1 and 3 in the region of EPIYA-C motifs is not primarily attributable to a recombination event between these groups, but instead, this region of Group 2 (East Asian caga) appears to have undergone a recent and substantial diversification compared to the ancestral sequence from which Groups 1 and 3 arose (see Table 2 and Fig. 8). The divergence in this region of Group 2 was driven by strong positive selection, whereas in contrast, there is relatively strong conservation of Domain 2 in Groups 1 and 3 (see Fig. 9 and Supplemental Fig. S5). Furthermore, positive selection within Domain 2 of Group 2 resulted in a purging of genetic diversity (Fig. 8). Since a loss of genetic diversity is observed in a specific region of caga rather than throughout the entire gene, this suggests that some form of recombination was coupled with the selection event, so that the region under positive selection recombined into multiple ancestral haplotypes. A different pattern of relationships among the groups is observed within the N-terminal region of CagA (corresponding to Domain 1 in the current study). In this N-terminal domain, there are small tracts of sequence where Group 3 is closely related to Group 1 and other small tracts of sequence where Group 3 is closely related to Group 2, a pattern which suggests that a series of recombination events may have occurred (see Fig. 7). In support of this view, analyses of synonymous nucleotide diversity indicate that the N-terminal portion of Group 3 had a recent purging of genetic diversity compared to the corresponding regions of either Group 1 or Group 2 (Table 2). We speculate that there were ancestral recombination events among the groups within the N-terminal region and a sufficient amount of time subsequently to generate silent site mutations within each group. Previous studies have shown that there are detectable differences in the activities of Western, East Asian, and Amerindian forms of CagA in cell-based assays (3, 31, 36, 40, 41, 53). 21

22 Potentially the different forms of CagA have varying activities in vivo that may influence the risk for development of diseases such as gastric cancer or peptic ulcer disease (6, 32, 40). In future studies, it will be important to investigate the relative activity of Group 3 (or J-Western) forms of CagA in cell-based assays, and to determine whether these forms of CagA are associated with a relatively high risk or low risk of gastric disease. Finally, we show that in an analysis of H. pylori strains from the United States, J-Western caga sequences are found mainly in strains producing m2 forms of the secreted VacA toxin. This association is biologically relevant because CagA and VacA are known to be functionally interacting proteins that inhibit each other s activities (3, 45, 57, 64). Nearly all of the studies of VacA-CagA functional interactions thus far have been performed using strains that express type m1 forms of VacA. It will be important in future studies to analyze further the functional relationships between different forms of VacA and CagA. Since Group 3 CagA sequences are found mainly in strains producing m2 forms of the secreted VacA toxin (25)(and this study), we propose that these functionally interacting proteins have co-evolved to optimize the gastric colonization capacity of H. pylori. Acknowledgments We thank Mark McClain and John Loh for advice on the preparation of figures. This work was supported by National Institutes of Health grants AI068009, AI039657, P01 CA116087, GM and by funding from the Department of Veterans Affairs. 22

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25 Katzowitsch, X. Wang, M. Achtman, and S. Suerbaum Traces of human migrations in Helicobacter pylori populations. Science 299: Figueiredo, C., J. C. Machado, P. Pharoah, R. Seruca, S. Sousa, R. Carvalho, A. F. Capelinha, W. Quint, C. Caldas, L. J. van Doorn, F. Carneiro, and M. Sobrinho-Simoes Helicobacter pylori and interleukin 1 genotyping: an opportunity to identify highrisk individuals for gastric carcinoma. J Natl Cancer Inst 94: Fischer, W Assembly and molecular mode of action of the Helicobacter pylori Cag type IV secretion apparatus. FEBS J 278: Franco, A. T., E. Johnston, U. Krishna, Y. Yamaoka, D. A. Israel, T. A. Nagy, L. E. Wroblewski, M. B. Piazuelo, P. Correa, and R. M. Peek, Jr Regulation of gastric carcinogenesis by Helicobacter pylori virulence factors. Cancer Res 68: Furuta, Y., K. Yahara, M. Hatakeyama, and I. Kobayashi Evolution of caga oncogene of Helicobacter pylori through recombination. PLoS One 6:e Gangwer, K. A., C. L. Shaffer, S. Suerbaum, D. B. Lacy, T. L. Cover, and S. R. Bordenstein Molecular evolution of the Helicobacter pylori vacuolating toxin gene vaca. J Bacteriol 192: Hatakeyama, M Anthropological and clinical implications for the structural diversity of the Helicobacter pylori CagA oncoprotein. Cancer Sci 102: Hatakeyama, M Oncogenic mechanisms of the Helicobacter pylori CagA protein. Nat Rev Cancer 4: Hatakeyama, M SagA of CagA in Helicobacter pylori pathogenesis. Curr Opin Microbiol 11: Hatakeyama, M., and H. Higashi Helicobacter pylori CagA: a new paradigm for bacterial carcinogenesis. Cancer Sci 96: Higashi, H., R. Tsutsumi, A. Fujita, S. Yamazaki, M. Asaka, T. Azuma, and M. Hatakeyama Biological activity of the Helicobacter pylori virulence factor CagA is determined by variation in the tyrosine phosphorylation sites. Proc Natl Acad Sci U S A 99:

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28 Schneider, T. D., and R. M. Stephens Sequence logos: a new way to display consensus sequences. Nucleic Acids Res 18: Stein, M., R. Rappuoli, and A. Covacci Tyrosine phosphorylation of the Helicobacter pylori CagA antigen after cag-driven host cell translocation. Proc Natl Acad Sci U S A 97: Suerbaum, S., and C. Josenhans Helicobacter pylori evolution and phenotypic diversification in a changing host. Nat Rev Microbiol 5: Suerbaum, S., and P. Michetti Helicobacter pylori infection. N Engl J Med 347: Suerbaum, S., J. M. Smith, K. Bapumia, G. Morelli, N. H. Smith, E. Kunstmann, I. Dyrek, and M. Achtman Free recombination within Helicobacter pylori. Proc Natl Acad Sci U S A 95: Suzuki, M., K. Kiga, D. Kersulyte, J. Cok, C. C. Hooper, H. Mimuro, T. Sanada, S. Suzuki, M. Oyama, H. Kozuka-Hata, S. Kamiya, Q. M. Zou, R. H. Gilman, D. E. Berg, and C. Sasakawa Attenuated CagA oncoprotein in Helicobacter pylori from Amerindians in Peruvian Amazon. J Biol Chem. 54. Suzuki, M., H. Mimuro, K. Kiga, M. Fukumatsu, N. Ishijima, H. Morikawa, S. Nagai, S. Koyasu, R. H. Gilman, D. Kersulyte, D. E. Berg, and C. Sasakawa Helicobacter pylori CagA phosphorylation-independent function in epithelial proliferation and inflammation. Cell Host Microbe 5: Tamura, K., J. Dudley, M. Nei, and S. Kumar MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24: Tegtmeyer, N., S. Wessler, and S. Backert Role of the cag-pathogenicity island encoded type IV secretion system in Helicobacter pylori pathogenesis. FEBS J 278: Tegtmeyer, N., D. Zabler, D. Schmidt, R. Hartig, S. Brandt, and S. Backert Importance of EGF receptor, HER2/Neu and Erk1/2 kinase signalling for host cell elongation and scattering induced by the Helicobacter pylori CagA protein: antagonistic effects of the vacuolating cytotoxin VacA. Cell Microbiol 11:

29 Thompson, J. D., D. G. Higgins, and T. J. Gibson CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22: Torres-Morquecho, A., S. Giono-Cerezo, M. Camorlinga-Ponce, C. F. Vargas-Mendoza, and J. Torres Evolution of bacterial genes: evidences of positive Darwinian selection and fixation of base substitutions in virulence genes of Helicobacter pylori. Infect Genet Evol 10: Truong, B. X., V. T. Mai, H. Tanaka, T. Ly le, T. M. Thong, H. H. Hai, D. Van Long, K. Furumatsu, M. Yoshida, H. Kutsumi, and T. Azuma Diverse characteristics of the CagA gene of Helicobacter pylori strains collected from patients from southern vietnam with gastric cancer and peptic ulcer. J Clin Microbiol 47: Tummuru, M. K., T. L. Cover, and M. J. Blaser Cloning and expression of a highmolecular-mass major antigen of Helicobacter pylori: evidence of linkage to cytotoxin production. Infect Immun 61: Waterhouse, A. M., J. B. Procter, D. M. Martin, M. Clamp, and G. J. Barton Jalview Version 2--a multiple sequence alignment editor and analysis workbench. Bioinformatics 25: Yamazaki, S., A. Yamakawa, T. Okuda, M. Ohtani, H. Suto, Y. Ito, Y. Yamazaki, Y. Keida, H. Higashi, M. Hatakeyama, and T. Azuma Distinct diversity of vaca, caga, and cage genes of Helicobacter pylori associated with peptic ulcer in Japan. J Clin Microbiol 43: Yokoyama, K., H. Higashi, S. Ishikawa, Y. Fujii, S. Kondo, H. Kato, T. Azuma, A. Wada, T. Hirayama, H. Aburatani, and M. Hatakeyama Functional antagonism between Helicobacter pylori CagA and vacuolating toxin VacA in control of the NFAT signaling pathway in gastric epithelial cells. Proc Natl Acad Sci U S A 102:

30 Figure Legends Fig. 1. Schematic representation of CagA and EPIYA motifs. A) Schematic diagram of CagA (not drawn to scale). The indicated domains correspond to regions analyzed in Table 2. EPIYA motifs are sites of tyrosine phosphorylation. EPIYA-C motifs are found commonly in non-asian CagA sequences and EPIYA-D motifs are found commonly in East Asian CagA sequences. B) Representative EPIYA-C and EPIYA-D motifs (26). Fig. 2. Phylogenetic analysis of A) full-length caga DNA sequences and B) CagA amino acid sequences. Four main groups are present. Group 2 and 3 are divided into smaller subgroups with distinct geographic distributions (see Supplemental Tables S2 and S3). Most Group 3a sequences were found in strains from North America or Europe, whereas all Group 3b sequences were found in strains isolated in East Asia (specifically Okinawa, Japan). PNGhigh85 is a strain classified as hpsahul based on MLST analysis. The CagA sequence from strain F80 did not cluster with any of the main groups. Fig. 3. MLST analysis of strains analyzed in this study. Closed circles indicate 38 strains analyzed in this study for which both CagA sequences and housekeeping gene sequences were available. Five additional strains analyzed in the current study were previously classified by MLST analysis, but are not depicted in this figure because the sequences are not publically available. The other taxa represent reference strains that were previously classified into distinct population groups by MLST analysis. Colors indicate distinct groups of strains identified by MLST. 30

31 Fig. 4. Relationships between CagA type and MLST classification of H. pylori strains. This figure analyzes 43 strains for which both full-length CagA sequences and housekeeping gene sequence data to permit MLST classification were available. The figure illustrates the MLST classification of strains that contained specific caga types (defined as shown in Fig. 2) Fig. 5. Analysis of EPIYA-C and EPIYA-D motifs. Consensus sequences from CagA proteins within specific groups are indicated, with variation displayed in WebLogo format (17, 48). Asterisks indicate sites where there is a higher level of diversity among Group 3 EPIYA-C sequences than among Group 1 EPIYA-C sequences. EPIYA sequences in strains of Amerindian origin (Group 4) contain segments that match EPIYA-C motifs at the C-terminus and EPIYA-D motifs at the N-terminus (as indicated by boxes). Fig. 6. Analysis of a 4-amino-acid insertion in Group 3 CagA sequences. The CagA sequence from strain (Group 1 CagA) corresponds to amino acids 187 to 226. Strains OK160 and OK107 were isolated in Okinawa, Japan. Strains J223, J123, and J174 were isolated in the United States, and the CagA sequences of these strains were analyzed as part of the current study. STGE or PTGE insertions are present in Group 3 strains from Japan, and PNGD and PNGE insertions are present in strains isolated in non-asian geographic regions. Fig. 7. Analysis of informative sites that differentiate CagA sequences in Group 1, 2, and 3. The figure compares Group 1, Group 2, and Group 3 CagA sequences, and shows sites at which amino acid residues from one group are different from corresponding residues in a second group in >95% of comparisons. Consensus sequences are shown. Gray shading indicates identity between residues found in Group 3 and the corresponding residues in either Group 1 or Group 2. Numbers represent the location of each informative site, mapped to the corresponding amino 31

32 acid position in the CagA sequence of a prototype reference strain, EPIYA motifs in Group 1 and Group 3 are located at positions (EPIYA-A), (EPIYA-B) and (EPIYA-C). The EPIYA-D motif found in Group 2 is located at positions Domain 1 (illustrated in Fig. 1) includes amino acid positions , Domain 2 includes positions and Domain 3 includes positions A crystal structure is available for the residues located at positions (41). Fig. 8. Analysis of mean nucleotide diversity of synonymous sites within Domain 2 of CagA. Sliding window analysis was used to analyze mean nucleotide diversity of synonymous sites within a region of caga that encodes the EPIYA-A, B, C or D motifs, denoted Domain 2 (corresponding to nucleotides in caga from strain 26695). This region corresponds to the large region in Fig. 7 where the amino acids in informative sites of Group 3 are identical to those in Group 1. Panels A, B, and C depict analyses of this region in caga sequences from Groups 1 (46 sequences), 2 (54 sequences), and 3 (16 sequences), respectively. Amino acid sequences are shown for the regions in Group 2 with the lowest π s values and the corresponding regions from Groups 1 and 3. Fig. 9. Analysis of positive selection within caga. Sliding window analysis was performed using representative caga sequences from Groups 1, 2, 3a, and 3b (26695, 98-10, B8 and OK155, respectively). Sequences were aligned and Ka/Ks ratios were calculated using DnaSP. Parameters for the sliding window analysis were set at 50 bases (window size) and a step size of 10 bp. A Ka/Ks value of >1 indicates positive selection. When comparing Group 2 with either Group 1 or Group 3 (subgroup a or b), a region of strong positive selection (peak denoted by *, 32

33 located near nucleotide 2800) was identified. This corresponds to one of the regions with a very low π s value (Fig. 8). The deduced amino acid sequences corresponding to the peak (*) in three sliding window analyses are shown in the bottom right part of the figure. 33

34 766 Table 1. Correlation between caga classification and presence of EPIYA-C or EPIYA-D motifs EPIYA Motif 768 caga classification No. of strains EPIYA-C EPIYA-D EPIYA-CD EPIYA-DC ΔEPIYA-C/D Group Group 2 Group Group 4 F Table 2. Analysis of mean nucleotide diversity of synonymous sites Mean nucleotide diversity (π s ) caga classification Domain 1 Domain 2 Domain 3 Whole gene Group Group a Group b c Groups 1,2, For this analysis, Group 1 contained 49 sequences, Group 2 contained 55 sequences and Group 3 contained 21 sequences. a p<0.001 compared to Group1, p<0.01 compared to Group 3 b p<0.001 compared to Group 1 and Group 2 c p<0.001 compared to Group1, p<0.01 compared to Group 2 34

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