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,1
* Division of Infectious Diseases, Vanderbilt University School of Medicine, and VA Medical Center, Nashville, Tennessee, USA;
Division of Gastroenterology, Zurich University School of Medicine, Zurich, Switzerland;
Departments of Medicine and Microbiology, New York University School of Medicine, and VA Medical Center, New York, New York, USA;
Departments of Molecular Microbiology and of Genetics, Washington University School of Medicine, St. Louis, Missouri, USA; and
|| Laboratory of Gastrointestinal and Liver Studies, Digestive Diseases Division, Department of Medicine, Uniformed Services of the Health Sciences, Bethesda, Maryland, USA
1Correspondence: Department of Medicine, New York University School of Medicine, 550 First Ave., New York, NY 10016, USA. E-mail: martin.blaser{at}med.nyu.edu
SPECIFIC AIMS
Both human cells and H. pylori populations are polymorphic for the expression of the fucosylated Lewis antigens. In an animal model using rhesus monkeys that differed in the major Lewis phenotypes, we asked whether H. pylori populations adapt their Lewis phenotypes to those of the host. Because H. pylori cells have hypermutable homopolymeric tracts in genes that regulate Lewis expression, we sought to determine whether variation was generated in vivo that could explain the phenotypic changes.
PRINCIPAL FINDINGS
1. Rhesus monkeys represent a model system to study Lewis phenotypic variation in the stomach
Humans express both monofucosylated (including Lea and Lex) and difucosylated (Leb and Ley) Lewis antigens on surfaces of many cell types, including erythrocytes and gastric epithelial cells, as well as on the highly glycosylated proteins (mucins) comprising the mucus layer. Conversely, 
90% of H. pylori isolates express human-type Le antigens in their LPS, preferentially the type 2 antigens (Lex and Ley). H. pylori strains are highly diverse, in part reflecting continued evolution during persistent colonization of individual hosts. Even within a single gastric biopsy, the H. pylori cells present can show extensive diversity in Lewis expression, because of the existence of antigenic variants within single clones.
We have proposed that H. pylori populations contain cells with differing Lewis phenotypes and that host phenotypes select the predominant populations. Rhesus monkeys are human-like in polymorphism for, and expression of, Lewis antigens, and gastric colonization by H. pylori is highly similar in the two primate species. When monkeys were challenged with mixtures of H. pylori strains isolated from humans, one or a few strains eventually predominated. Testing four rhesus monkeys (designated A–D) for Lea or Leb antigens in saliva and gastric juice, by a hemagglutination inhibition method, we found that two (A and B) were Lea+b– and two (C and D) were Lea–b+. Therefore, in the former monkeys, the predominant gastric epithelial expression was Lea and Lex, whereas in the latter monkeys, Leb and Ley were predominantly expressed. The four monkeys were inoculated with seven individual H. pylori strains, with a broad range of Le phenotypes, but only four strains could be recovered over the next 40 wk from the test animals. Although in the short term, strain J238 was predominant in the mixed bacterial populations, eventually all strains in each animal were outcompeted by J166. Thus, these experimental conditions permitted exploration of the evolution of H. pylori Le phenotype in a strain (J166) that was successful in colonizing animals that varied in their predominant Le phenotype.
2. Predominant Lewis phenotypes in the H. pylori population resemble those of the host by 40 wk
In the sample of preinoculation strain J166, Ley expression was variable but predominated, whereas some Lex-expressing cells were present in the population (Fig. 1
A). In Lea+b– monkey E6C (animal A), by 14 wk, Ley expression was highly variable, and Lex expression was substantially increased (B). By week 40, Lex expression predominated (C). Despite the same initial J166 inoculum, by 40 wk of colonization of the two Lea+b– monkeys, Lex expression had become significantly higher, whereas in the two Lea–b+ monkeys, Ley expression remained higher (Fig. 2
). In total, these results indicate that within 40 wk H. pylori populations can undergo substantial changes in Le phenotype in vivo and that this variation is related to predominant host Lewis phenotype in the predicted pattern.
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3. Genetic mechanisms for altered H. pylori Lewis expression
H. pylori Lex/y expression reflects the action of products of at least four genes, including the ßbeta;-(1,4) galactosyltransferase (ßbeta;-(1,4) galT), two 
-(1,3) fucosyltransferases (futA/futB) and the -(1,2) fucosyltransferase (futC). Most of these genes are metastable due to repetitive sequences with high frequency frame shifts. We examined genotypes in strain J166 preinoculation and in strain 98–169, a J166 descendant, isolated from Lex-expressing monkey B at 40 wk; the Lex/Ley phenotype of this strain was 1990/10 compared with 480/1180 ODU for the population in a single colony of J166 tested preinoculation. In futC of J166, there was a poly-C tract with 9 cytosines and the full gene was in frame (Fig. 3
). In contrast, for futC of 98–169, the poly-C tract contained 10 cytosines, and the ORF was truncated. The alleles of the other three genes tested showed no frame shifts. Examination of futC in two other isolates (98–149 and 99–171) that had lost Ley expression showed the identical frame shift as in 98–169, but 98–208, a single colony recovered from monkey D, which did not have a changed phenotype, did not have the frame shift. In total, these data provide evidence that a +1 frame shift in the poly-C tract of futC resulted in the in vivo change in phenotype with loss of Ley expression.
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CONCLUSIONS AND SIGNIFICANCE
Using rhesus monkeys, we provide direct evidence supporting the hypothesis that host Le phenotype is a determinant for particular Le phenotypes within an H. pylori population. Monkeys are the only animals other than humans that have the natural occurrence of H. pylori, natural expression of human-like gastric Le antigens, and they are suitable for periodic upper gastrointestinal endoscopy.
Colonization of monkeys for 40 wk can be considered relatively brief, compared with adult humans, who usually have carried their H. pylori strains for decades. Whether the predominant Le phenotypes we observed would be maintained indefinitely or not is not known and could be the subject of future studies. In any event, the fact that the overall Le phenotype of the J166 strain changed substantially within 40 wk, and that it out-competed the other inoculated strains in every monkey implies that this early period is important for study. The variation we observed parallels the changes detected in expression of H. pylori outer membrane proteins, which are involved in host ligand binding. Among a small number of paired H. pylori isolates obtained from humans 7 to 10 yr apart, Ley levels decreased with time, whereas Lex levels remained constant, paralleling the expected changes in human gastric Le expression with age-dependent increases in gastric intestinal metaplasia and confirming observations of Le expression changes after our inoculation of monkeys (Fig. 2)
.
When hosts are exposed to several H. pylori strains, both bacterial and host characteristics may influence which strain is most successful. Whereas short-term J238 dominance clearly seemed strain-determined and independent of the host colonized, long-term dominance by J166 appeared to be both strain- and host-related, as reflected by the variation in bacterial Le phenotypes in the different animals. That the dichotomy of bacterial Le phenotypes observed at 40 wk could have developed at random is improbable, but it could have been subject to periodic selection. If random, then mixtures of different Le phenotypes also would have been expected, rather than the relatively homogeneous populations observed at 40 wk. The consistency of the Le changes in each of the 4 animals and the intermediate state of J166 Le expression in animal A at week 14 suggests a relatively ordered selection process, as modeled mathematically.
Insights into the possible selective pressures behind such host-adaptation by H. pylori are provided by the recent demonstration that H. pylori colonization induces dynamic changes in host mucosal sialyl-Lex and Leb expression, consequently affecting binding site availability. In contrast, Le variation occurring in vivo at similar frequency to that demonstrated in vitro probably would not be sufficient to explain the magnitude of changes seen in our experiment, in the absence of selection. Because Le expression diversity already was present in J166 preinoculation, the uniformity of Le epitopes of bacteria recovered from each animal late after challenge is consistent with selection for better-adapted phenotypes.
A frame shift in the -(1,2) fucosyltransferase gene (futC) in all three Lex-expressing isolates tested is sufficient to explain both the observed loss of Ley expression and the increase in Lex expression, as Lex is no longer the substrate for the inactive FutC; changes in the three other genes that affect upstream steps in Le antigen synthesis are not required. These genotypic studies suggest that the capability for host-dependent Lewis phenotypic adaptation has been selected and maintained at particular loci in H. pylori. This model is both stochastic, with the host selecting for differential fitness of variants in the population, yet "programmed" in that the genes with hotspots for variation are present in highly specific loci, not randomly distributed in the genome. Consistent with the known heterogeneity within H. pylori populations colonizing an individual host, the initial J166 inoculum likely included 10-cytosine futC cells; however, with the strong overall Ley expression observed in that culture, such 10-c cells likely represented a minor constituent of the overall population. Nevertheless, they would represent the "founding" cells that ultimately had fitness advantage in the Lea–b+ monkeys.
In summary, these results support the hypothesis that H. pylori populations are selected on the basis of their Le phenotype, which is dependent on that of the host colonized. We propose that the ability of H. pylori populations to adapt their predominant Le expression to that of the host contributes to their persistence in the gastric niche. That Le expression is not necessary for successful long-term H. pylori colonization of mice illustrates the desirability of primate models for mirroring human conditions. That local gastric Lewis phenotypes change in response to H. pylori indicates the dynamic and complex interactions between persistent H. pylori and its host.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5529fje
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  J. I. Keenan, K. A. Davis, C. R. Beaugie, J. J. McGovern, and A. P. Moran Alterations in Helicobacter pylori outer membrane and outer membrane vesicle-associated lipopolysaccharides under iron-limiting growth conditions Innate Immunity, October 1, 2008; 14(5): 279 - 290. [Abstract] [PDF]
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