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Surveying the diversity of H. pylori genomes in one man’s stomach
Edward R. Winstead

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In 1994, doctors in Tennessee treated an ulcer patient who was infected with the bacterium Helicobacter pylori. As is standard practice with this infection, the patient began taking a combination of antibiotics to kill the bacteria. But the side effects were so unpleasant that the man went off the medicine, and, fortunately for scientists, the bacteria in his stomach were never eradicated.

Image of Helicobacter pylori by Carl Löfman.

Six years later, 'Ned Wilson' returned to Vanderbilt University Medical Center in Nashville, Tennessee, complaining of stomach pain. By then, a unique piece of information had been added to his medical records: The genome sequence of his pathogen. It turned out that doctors had isolated H. pylori from Wilson's stomach during his initial visit, and a few years later, researchers in Cambridge, Massachusetts, sequenced his organism.

Bacteria that exist ‘in a continuous state of genetic flux.’

Wilson's strain, known as J99, was the second H. pylori genome sequence published in the late 1990s. The pathogen is well studied because half the world's population is infected with H. pylori. Many of these individuals experience no symptoms, while others develop ulcers. The bacteria remain in the body for life and are a risk factor for stomach cancer.

Wilson's unexpected return to the hospital allowed researchers to document changes in the bacteria over time. For at least six years, his stomach had been a repository of H. pylori. With the patient's consent, doctors took additional biopsies and isolated 30 strains from different regions of his stomach. Those strains have now been analyzed and compared to one another—and to the two completely sequenced strains of H. pylori.

The comparison revealed a related but genetically diverse population. DNA fingerprinting, which profiles a portion of the genome, showed that all 30 strains were relatives of the J99 strain. But when the researchers used DNA microarrays to survey the content of genes in each genome, they found significant differences among the strains.

"The coexisting strains were all closely related, but they did not have the same complement of genes," says Dawn A. Israel, of Vanderbilt University Medical Center, who led the study. It has been known for some time that H. pylori have plastic genomes—like other bacteria, they pick up and delete genes. And strains isolated from different individuals are relatively diverse.

Image of Helicobacter pylori by Carl Löfman.

The same traits—diversity and plasticity—characterized the bacteria in Wilson’s stomach, a relatively homogenous population. While a core group of genes was present in all strains, others were found only in some. These H. pylori appeared to exist in "a continuous state of genetic flux," the researchers write in Proceedings of the National Academy of Sciences.

The pathogen's ability to transform its genome may help it survive in the stomach environment. "It is hard to understand why there would be this degree of genetic difference unless the diversity provides some evolutionary advantage," says Israel.

Israel's team screened the 30 strains for genes also present in either of the sequenced strains. This led to a puzzling result: Some newly identified strains contained genes that were not in the original J99 strain. How did they get there?

"That is the two-million-dollar question," says Israel. She offers two hypotheses. Wilson might have been infected by additional strains in the interim years. Or, more likely, the researchers simply missed bacteria the first time around. Strains other than J99 may have been in Wilson's stomach but were not picked up by the original biopsy.

In 1994, Wilson's doctors had no reason to analyze several pieces of infected tissue. At the time no genome of any kind had been sequenced, and the idea of comparing sequenced genomes was a distant hope. Moreover, it was assumed that the patient would take the antibiotics and rid himself of the bacteria.

But as it happened, Wilson remained infected and technology came along that made the new study possible. DNA microarrays, developed for widespread use in recent years, offered Israel's team an affordable means of comparing the content of many genomes. Microarrays are glass slides—or microchips—that contain thousands of genes and can detect the presence of these genes in tissue.

Detail from schematic of sequenced H. pylori genomes. View larger

"The microarrays allowed us to assay every single gene that was in the original sequence strain for its presence or absence in the other strains," says Israel. "That is something we couldn't do before, and we never would have picked up the diversity without the microarrays."

The first sequenced H. pylori strain, called 26695, was completed by The Institute for Genomic Research in Rockville in 1997. Two years later, the J99 strain was sequenced by Astra Research Center Boston (now AstraZeneca R&D Boston) in collaboration with Genome Therapeutics Corporation, Waltham, Massachusetts. Both sequences were published in Nature and the second paper included one of the first back-to-back comparisons of two sequenced genomes.

A second recent study—also involving one infected individual—provides evidence that genetic variation influences the pathogenicity of H. pylori. The researchers isolated two related but genetically diverse strains of H. pylori from a 90-year-old patient with gastric ulcer disease. They then infected mice with the strains and found different outcomes, which they determined were related to genetic differences.

One H. pylori strain did not have a 'pathogenicity island' and infected only some mice; the other strain, which had pathogenicity islands, infected all the mice. These islands are groups of genes that increase a pathogen's ability to cause disease. The term originated with E. coli researchers, and the mechanism that confers increased virulence is still being worked out. Why these genes are present in only some bacteria is not known.

"In animal experiments, we found that the two strains differed greatly in their virulence potential," says Britta Björkholm, of the Swedish Institute for Infectious Disease Control in Solna, who led the research. "The experiments showed that the isolate without the pathogenicity island was unable to cause a persistent infection."

The researchers determined that the two strains were descendents of the same parent strain but had undergone considerable diversification during the course of infection. For a human or a mouse, the outcome of an H. pylori infection is likely to reflect a number of factors, including the "net virulence of the entire bacterial population" and the interplay between bacteria and host, says Björkholm. The findings appeared in Infection and Immunity.

Nina Salama, of the Fred Hutchison Cancer Research Center in Seattle, Washington, was a member of both research teams. "The two recent H. pylori studies demonstrate that the bacterium can acquire and lose genes over time, and that a patient can be infected with a mixed population," she says.

Electron micrograph of Helicobacter pylori.

"Before this experience people didn't realize there was such diversity within a single host, so this will have a big impact on how we study H. pylori within a single host," says Salama. By taking just one strain from a patient, as is common practice, researchers stand to miss genes in other strains that are important in disease. These genes could lead to antibiotics with fewer side effects.

New drugs would be good news for people like Ned Wilson who do not respond well to the existing therapies. According to Israel, he is still living with H. pylori. "In the end, it turns out that the patient refused treatment," she says.

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Israel, D.A. et al. Helicobacter pylori genetic diversity within the gastric niche of a single human host. Proc Natl Acad Sci USA 98, 14625-14630 (December 4, 2001).
Björkholm, B. et al. Comparison of genetic divergence and fitness between two subclones of Helicobacter pylori. Infect Immun 69, 7832-7838 (December 2001).
Alm, R.A. et al. Genomic-sequence comparison of two unrelated isolates of the human gastric pathogen Helicobacter pylori. Nature 397, 176-180 (January 14, 1999).
Tomb, J.F. et al. The complete genome sequence of the gastric pathogen Helicobacter pylori. Nature 388, 539-547 (August 7, 1997).


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