Laboratoire de Bactériologie, Université Victor Segalen Bordeaux 2 and Hôpital Pellegrin, Place Amélie Raba-Léon, 33076 Bordeaux Cedex, France1
AstraZeneca R and D, Boston, Waltham, MA, USA2
Author for correspondence: Francis Mégraud. Tel: +33 5 56 79 59 10. Fax: +33 5 56 79 60 18. e-mail: francis.megraud{at}chu-bordeaux.fr
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ABSTRACT |
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Keywords: diversity, genome, pathogenicity
b The GenBank accession numbers for the H. pylori sequences reported in this paper are AF326599AF326607 for region A, AF326608AF326616 for region B, AF326617AF326625 for region C, AF326626AF326634 for region D, AF327212AF327220 for region E, AF328909AF328916 and AF328924 for region F, and AF32917AF32923 for region G.
a Present address: Laboratoire de Virologie, Institut de Biologie Végétale Moléculaire, Institut National de la Recherche Agronomique, Bordeaux, France.
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INTRODUCTION |
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The discovery of new strain-dependent factors potentially involved in the clinical outcome of infection with H. pylori has ensued from the genomic and post-genomic eras. The total genome sequence of two H. pylori strains has been available since 1999 (Alm et al., 1999 ; Tomb et al., 1997
), and the availability of these complete genome sequences was truly the beginning of comparative genomics for H. pylori. The overall genome organization of the two sequenced strains of H. pylori differs by 10 inverted or transposed regions. Genes conserved in both strains and so-called strain-specific genes (Alm & Trust, 1999
; Doig et al., 1999
) have been identified by comparison of the gene content of the two strains. Strain-specific genes were originally defined as being present in only one of the two completely sequenced H. pylori genomes, although subsequent analysis has led to some of these genes being identified in other H. pylori isolates (Occhialini et al., 2000
; Salama et al., 2000
). Although the functions of the putative encoded proteins are unknown for most of the strain-specific genes (70%), these genes may play a role in the virulence capacities of H. pylori strains by encoding factors that contribute to a different disease outcome. Concerning their location, almost half of the strain-specific genes are clustered in a single hypervariable region, the so-called strain-specific plasticity zone described by Alm et al. (1999)
. A study by Occhialini et al. (2000
) involved the analysis of the diversity of the plasticity zone in 43 H. pylori strains and showed that this region appears highly mosaic in nature.
The goal of the present study was to measure the genetic diversity of H. pylori strains by analysing the loci that contain the J99 or 26695 strain-specific genes (65 in strain 26695 and 47 in strain J99) located outside the plasticity zone. Although these strain-specific genes were not clustered into one locus, their location did not seem to be random. Indeed, it was noted that in 17 corresponding loci both reference strains contained strain-specific genes, suggesting a limit in the flexibility of the genome to strain-specific content (Alm & Trust, 1999 ; Alm et al., 1999
). Hence, we proposed the hypothesis that other H. pylori strains contain their own set of specific genes located in similar loci. Seven strain-specific loci among the 17 loci common to both reference strains were selected; the genetic composition of these loci in nine H. pylori strains isolated from patients suffering from the principal diseases caused by H. pylori was analysed.
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METHODS |
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In preparation for DNA extraction, the strains were subcultured on the same medium as described above for 48 h, harvested in 1 ml Brucella broth (BBL Microbiology Systems) and centrifuged for 15 min at 3000 g; the resulting pellets were stored in sterile vials at -80 °C until use.
Total DNA extraction.
The cells were resuspended in 1 ml extraction buffer [20 mM Tris/HCl (pH 8), 0·5% Tween 20] and treated with 10% SDS and proteinase K (100 µg ml-1). After at least 1 h at 56 °C, the proteins were eliminated from the lysate by solvent extraction using a standard protocol (Sambrook et al., 1989 ). Nucleic acids were precipitated from the lysate in the presence of 70% ethanol and 0·3 M sodium acetate (pH 5·2) at -80 °C for 30 min. After centrifugation and washing of the DNA with 70% ethanol, it was dissolved in an appropriate volume of sterile water and stored at -20 °C. The DNA concentration was determined at 260 nm.
Amplification of strain-specific loci.
Oligonucleotides used as primers to amplify strain-specific loci present in H. pylori DNA were designed on the basis of the published sequences of H. pylori strains J99 and 26695 (Alm et al., 1999 ; Tomb et al., 1997
) [available at the Helicobacter pylori Genome Database web site (http://scriabin.astrazeneca-boston.com/hpylori) and the Institute for Genomic Research web site (http://www.tigr.org), respectively] and are listed in Table 1
. Primers which annealed to conserved flanking genes within the sequences of J99 and 26695 were used, to allow the amplification of the intervening sequences; the sizes of the amplicons produced by these primers are shown for both reference strains in Table 1
. In addition to the aforementioned primers, primers which annealed within the six strain-specific genes of interest have been described (Table 2
); these were used to screen for the presence of these genes in the larger panel of strains.
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Sequencing of amplified fragments.
DNA sequencing was performed by using the dideoxynucleotide chain termination method (Sanger et al., 1977 ) with the dRhodamine Termination Cycle Sequencing Kit (Perkin-Elmer). Before sequencing, the amplicons were purified by using Wizard PCR preps (Promega). The same primers as used for PCR were employed for sequencing (Table 1
), as well as internal primers (not shown). According to the manufacturers protocol, reagent mixtures containing 15 µl of purified PCR product were placed in the thermal cycler and cycling was carried out under the following conditions: 25 cycles at 96 °C for 10 s, 50 °C for 5 s and 60 °C for 4 min. The resulting sequences were analysed through a polyacrylamide (4·25%) urea (7 M) gel in TBE buffer [89 mM Tris/HCl (pH 8·3), 89 mM boric acid, 2 mM EDTA] at 51 °C in an ABI PRISM 377 Genetic Analyser (Perkin-Elmer). For each sample, both strands of the PCR product were sequenced.
Sequence analysis and comparisons.
Nucleotide sequences were analysed by using the programs SEQUENCE NAVIGATOR and AUTOASSEMBLER 2.0 (Perkin-Elmer). Predicted coding regions were defined by searching for ORFs longer than 50 codons that had a ribosome binding consensus site upstream of a potential start codon. The sequences were compared with those within the GenBank databases by using the BLAST (basic local alignment search tool) and PSI-BLAST (position-specific iterative BLAST) programs (Altshul et al., 1997 ) at the National Center for Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov/). Particular motifs were identified using the PFSCAN software at the Swiss Institute for Experimental Cancer Research (ISREC; http://www-isrec.unil.ch/).
Nucleotide sequence accession numbers.
DNA sequences generated in this study were deposited in the GenBank database with the following accession numbers: AF326599AF326607 for region A; AF326608AF326616 for region B; AF326617AF326625 for region C; AF326626AF326634 for region D; AF327212AF327220 for region E; AF328909AF328916 and AF328924 for region F; AF32917AF32923 for region G.
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RESULTS |
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On the basis of the above criterion, among the candidate loci of H. pylori with no assigned function, seven regions of the genomes of strains J99 and 26695 that contained strain-specific genes were chosen for further investigation (Table 3). All of these regions contained at least one gene with an unknown or hypothetical function, depending on whether they belonged to the H. pylori-specific with no known function group or the conserved with no known function group, respectively. The latter group indicates that orthologous genes have been identified in other species, but these orthologues have no known function. Three of the regions (B, D and G) of the H. pylori genome identified here encode only genes that fall into the two aforementioned categories; the four remaining regions (A, C, E and F) contain genes with assigned functions and putative genes with no homologues (Table 3
).
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Fig. 1 shows the location of the seven strain-specific loci within the J99 genome, and their distribution around the chromosome.
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The most-conserved regions of the H. pylori genome, in terms of the size of the amplified fragment, were regions A and E (Table 4). With respect to region E, the nine clinical isolates resembled strain J99 more than they resembled strain 26695. The ORF present in region E shared significant homology with ORF JHP540, which is located between the same flanking genes in the J99 genome as region E is in the nine clinical strains (Tables 3
and 4
). Despite the similar sizes of the amplified fragments from the nine H. pylori clinical strains, region A does not encode the same proteins in all of these strains. Indeed, from the amplification products of four of the strains (2C, 15U, 35g and 38g), ORFs similar to the IceA1 protein of strain 26695 were predicted (Table 4
). Although no ORF was predicted in strains 4C, 9C, 14U and 29g that was similar to iceA1, sequence similarity was detected between the region A sequences of these strains and this gene. The lack of predicted ORFs for region A of these four strains seemed to be due to the accumulation of mutations in this region that led to the creation of stop codons. Region A of strain 16U was found to encode an ORF homologous to ORF JHP1132 of J99. In strain J99, this ORF encodes the IceA2 protein. In contrast to region E, region A generally seemed to be more closely related to the corresponding locus in strain 26695 than the one in strain J99.
Even though the size of the amplified fragments varied extensively (8591556 bp; Table 4), region B was highly conserved in the nine clinical isolates. Indeed, this region was either homologous to the JHP318 gene of strain J99, or similar to the JHP1024 gene of strain J99, a paralogue of JHP318 (Table 4
).
With respect to region F, three groups of strains were distinguished depending on the composition of this region (Table 4). The first region F group of strains was composed of the three gastritis strains and strain 2C, in which this region was the largest (5000 bp). Several ORFs were predicted from the sequence of region F in these four strains. The first two ORFs had similarity with ORFs JHP46 and JHP45 of strain J99; the other predicted ORFs resembled chimeric ORFs of genes found in strains 26695 (ORFs HP52 and HP51) and J99 (ORF JHP44). Indeed, ORF1 (361 codons) of strains 2C, 29g, 35g and 38g was found to be homologous to JHP44 of strain J99 in the NH2 part (first 72 codons) and to HP51 of strain 26695 in the remaining part (289 codons). The same situation was observed for ORF2 (408 codons), whose first 292 codons were homologous to HP52 of 26695 (90% identity) and whose remaining 116 codons were homologous to JHP44 of strain J99 (85% identity). The second region F group of strains (4C, 9C and 14U) was related to strain J99. The size of region F and the three homologous ORFs encoded by these strains were similar to those found in strain J99. Finally, the third group of region F strains comprised strains 15U and 16U. These strains had a deleted form of region F compared to that of strain J99. Indeed, region F of 15U and 16U contained only one ORF, which was similar to ORF JHP44 of strain J99, instead of the three J99 ORFs JHP44, JPH45 and JHP46.
Regions C, D and G were of particular interest to us. In some strains these regions were found to contain genes defined as strain-specific due to their absence from the genomes of strains J99 and 26695 and their presence in only one of our nine clinical isolates. Region D of strains 2C and 16U contained strain-specific ORFs (Table 4). Region D of the seven other strains contained an ORF similar to ORF JHP1437 of strain J99 (region D). Of the strain-specific loci studied here, regions C and G presented the greatest diversity five combinations for region C and six for region G (Table 3
). Moreover, both regions contained strain-specific genes in strains 14U and 2C. In the other seven clinical strains, regions C and G contained either ORFs homologous to those expected in the reference strains J99 or 26695 or ORFs encoded by the J99 or 26695 genomes but present in another locus (e.g. ORF JHP1044 in region G of strain 35g; Table 4
). After examining the composition of region C in more detail, it was noted that the genes present at this locus belonged to the same paralogous gene family, i.e. the ghp type I restriction enzyme, specificity subunit family. The gene homologous to HP848 (hsdS_2) found in region C of strains 35g and 9C was a paralogue of the HP790 (hsdS_5) gene contained in strains 2C, 4C, 15U and 38g, and reference strain 26695 (Table 4
); these genes displayed 56% identity in their amino-acid sequences. Moreover, the HP848 gene of strain 26695 corresponded to the JHP785 (hsdS_2) gene of strain J99, which was also found in region C of strain 16U (92·6% identity). Finally, the ORF homologous to JHP1422 of strain J99 predicted in region C of strain 14U (hsdS_3a) was a paralogue of ORF JHP785 of J99 (22% identity). The same observation could be made for the ORFs present in region G.
Characterization of the six newly identified strain-specific genes of H. pylori
The characterization of seven strain-specific loci (detailed above) of the nine clinical isolates of H. pylori studied here allowed the discovery of six strain-specific genes that were not present in reference strains J99 and 26695. Only three of the seven strain-specific regions contained strain-specific genes regions C, D and G. Not all of the clinical strains contained these genes. Region C of strain 14U encoded a strain-specific ORF of 282 codons. Strain 2C contained two strain-specific ORFs in regions D and G of 227 and 50 codons, respectively. Finally, region D of strain 16U presented three strain-specific ORFs with sizes of 52, 55 and 102 codons.
The six strain-specific ORFs identified in this study shared no similarity among themselves, even when found in the same locus in different strains, i.e. region D in strains 2C and 16U. A comparison of the amino-acid sequences of the six newly identified strain-specific ORFs with the sequences contained within the databases showed that five of the six ORFs had no orthologue and hence should be classified as H. pylori-specific the majority of the strain-specific genes found in the genomes of J99 and 26695 already have this classification (Doig et al., 1999 ). The ORF found in region D of strain 2C showed weak similarity (E-value of 10-8) with a transposase of Thermotoga maritima (32% similarity, 23% identity). When searching for particular motifs in this ORF, a slight similarity was detected with an N-glycosylation site. Slight similarities with protein kinase C phosphorylation sites were also found in the ORFs in region C of strain 14U, in region G of strain 2C and in the ORFs of 52 and 102 codons in region D of strain 16U (data not shown).
Finally, screening for the presence of the six newly identified strain-specific ORFs in a panel of strains from the same geographical origin was performed, but no association with disease outcome was found for these genes (Table 5).
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DISCUSSION |
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The observation that 17 strain-specific loci were common in the two reference strains J99 and 26695 (Alm et al., 1999 ) led us to develop a classical approach for the study of these loci in nine clinical isolates of H. pylori. This approach consisted of (i) amplification of the loci containing the strain-specific genes and (ii) the subsequent identification of the genes contained in the amplified fragments by sequencing and comparison of the resulting gene sequences with those contained in the databases. The results partially verified the hypothesis of a similar location of strain-specific genes in different strains of H. pylori, in that six new strain-specific genes not present in the genomes of the two reference strains were identified. Five are putative genes and, hence, are specific to H. pylori, like the majority of the strain-specific genes of J99 and 26695 (Alm et al., 1999
). It should be noted that three predicted ORFs, two in region D of strain 16U and one in region G of strain 2C, are very small in size (52, 55 and 50 codons, respectively) (Table 4
) and, therefore, may not be genes. Nevertheless, we found consensus ribosome-binding sites upstream of initiation codons in these small predicted ORFs. The remaining strain-specific gene identified in region D of strain 2C showed homology with a transposase of T. maritima. This finding of a gene involved in DNA exchange and which may promote genetic diversity in H. pylori is not surprising. Indeed, many of the strain-specific genes of the two H. pylori reference strains belong to putative restrictionmodification systems (10%) and 4% share similarities with genes encoding transposases (Salama et al., 2000
). Nevertheless, Kong et al. (2000)
found that <30% of the potential type II restrictionmodification systems in H. pylori J99 were fully functional. Another H. pylori strain, J166, has been shown to contain 18 specific genes when compared by subtractive hybridization to strain 26695, seven of which show homology to restrictionmodification systems (Akopyants et al., 1998
). Kersulyte et al. (2000)
identified a transposable element called IS607 in H. pylori, located on a fragment present in only certain strains of this organism, which was also discovered by subtractive hybridization. Strain-specific genes involved in such systems have been identified in other bacterial species, e.g. Klebsiella pneumoniae (Lai et al., 2000
), Neisseria meningitidis (Bart et al., 2000
; Claus et al., 2000
) and Aeromonas hydrophila (Zhang et al., 2000
). Using representational difference analysis, Bart et al. (2000)
and Claus et al. (2000)
showed that restrictionmodification systems were specifically present in lineage III meningococci. Suppression subtractive hybridization was used to identify genetic differences between virulent and avirulent strains of A. hydrophila isolated from diseased fish (Zhang et al., 2000
). Among the 69 genomic regions present only in the virulent strain of A. hydrophila, two-thirds encoded genes specific to A. hydrophila and one ORF belonged to a type II restrictionmodification system. Using the same methodology as Akopyants et al. (1998)
, Lai et al. (2000)
identified genes specifically present in a virulent strain of K. pneumoniae; among the 25 subtracted DNA clones, one encoded the transposase of Tn3926.
Besides the identification of the six new strain-specific genes in the H. pylori clinical isolates, we detected the presence of ORFs homologous to those found in either J99 or 26695 in the same loci. These results confirm that the gene order is highly conserved among isolates of H. pylori (Alm et al., 1999 ; Bereswill et al., 2000
; Doig et al., 1999
), despite the extreme genetic diversity displayed by this bacterium, as shown by studies on genetic variability and population structure (Achtman et al., 1999
; Suerbaum, 2000
; Suerbaum et al., 1998
). Overall, the nine clinical isolates are more closely related to strain J99 than to strain 26695, especially with regard to the plasticity zone (Occhialini et al., 2000
). However, in region A, eight of the nine strains contained a DNA fragment homologous to that present in 26695, i.e. the iceA1 gene (Table 4
). The remaining strain (16U) harboured the unrelated gene iceA2, found in J99. As in the study by Figueiredo et al. (2000)
, who analysed the iceA locus in 321 H. pylori strains from 24 different countries, we confirmed the presence of these two gene families (i.e. iceA and iceA2) at this locus. Figueiredo et al. (2000
) found that the majority of strains (14/19) did not encode the full-length homologue of NlaIII, a restriction endonuclease from Neisseria lactamica (Morgan et al., 1996
; Peek et al., 1998
). In our study, four of the eight strains studied contained an ORF of 228 codons that potentially encodes a full-length IceA1 protein (Table 4
). Nevertheless, the association between the presence of iceA1-positive strains and the development of peptic ulcers, as described by Peek et al. (1998)
and van Doorn et al. (1998)
, was not verified in our study. A recent study by Solcà et al. (2001)
also showed that the iceA1 allele was more frequent than the iceA2 allele in H. pylori (59% versus 41%).
The results of the study by Figueiredo et al. (2000) suggested that the organization of the iceA2 locus is very complex, with the presence of a variable number of tandem repeats (VNTRs) of an 8 bp sequence in the intergenic region upstream of the initiation codon of the IceA2 ORF. Moreover, iceA2 was shown to encode proteins of various sizes, consisting of two conserved domains of 14 and 10 aa in length and a variable number of a 35 aa cassette, which was made up of domains of 13, 16 and 6 aa in length. This classification allowed Figueiredo et al. (2000)
to distinguish five iceA2 variants. Therefore, the iceA2 variant present in strain 16U should be defined as being of the iceA2B form, as it could encode a protein of 59 residues that includes the 14 and 10 aa cassettes, flanking three internal peptide domains of 13, 16 and 6 aa, respectively. Only one VNTR was located in the intergenic region between iceA2 and JHP1133/HP1210. The same proportion of iceA2-positive strains (from Costa Rica) was found in this study as in the study by Figueiredo et al. (2000)
(5/34 strains). A relationship between the cassette structure of iceA2 and expression was shown by Peek et al. (1998)
. In vitro expression of iceA2 in strain 16U was confirmed by RT-PCR (data not shown). Neither the role of iceA2 in H. pylori nor the relevance of the conserved genetic organization of this gene is understood, as yet.
Six of the nine clinical isolates of H. pylori included in our study were among the 43 strains whose plasticity zones have been analysed and for whom the compositions of the cag pathogenicity island have been determined (Occhialini et al., 2000 , 2001
). Therefore, we attempted to find a correlation between the organization of the plasticity zone and the cag pathogenicity island, and between the organization of the plasticity zone and the strain-specific loci. All nine of the clinical strains studied here were found to contain an intact cag pathogenicity island (Occhialini et al., 2000
). Four patterns for the plasticity zone were distinguished among the nine strains A1, A2, B1 and B2 (Occhialini et al., 2001
). No association was found between any one of these plasticity-zone groups and the composition of the strain-specific loci, which is consistent with the high level of DNA diversity seen within strains of H. pylori.
Finally, the identification of new strain-specific genes in our study supports the idea that H. pylori strains contain other strain-specific genes that are not present in the J99 and 26695 sequences (Salama et al., 2000 ). Indeed, the study by Salama et al. (2000)
was conducted to characterize the genetic diversity of H. pylori by examining the genomic content of 15 clinical isolates of this organism, using a whole-genome H. pylori DNA micro-array. These authors found that at least 1218% of the genome of each strain was composed of strain-specific genes that were not present in all of the strains surveyed (i.e. they lay outside of the core set of genes). Micro-array technology is a particularly powerful tool for quantifying differential levels of expression of each gene for cells grown under different conditions (Nierman et al., 2000
); however, for genetic variability studies, the experimental system itself leads to an underestimation of the number of strain-specific genes, as a micro-array contains only genes present in sequenced genomes. Alternative strategies for the identification of new strain-specific genes are promising, such as subtractive hybridization (Akopyants et al., 1998
; Kersulyte et al., 2000
; Lai et al., 2000
; Zhang et al., 2000
) or the classical methodology used in this study, which was made possible by the previous identification of candidate loci.
Although the discovery of the strain-specific genes described in this study adds to our knowledge of the H. pylori genome, none of these genes seems to be clinically relevant, based on the small survey performed here. The inclusion of these newly identified genes on H. pylori DNA micro-arrays will confirm their distribution and a functional approach to identifying their specific functions will contribute to assessing their role in H. pylori.
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ACKNOWLEDGEMENTS |
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Received 1 February 2002;
revised 17 May 2002;
accepted 17 July 2002.