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MICROBIOLOGY Unique Genes Found in 7th Pandemic Cholera StrainResearchers have identified 22 specific genes in a particularly nasty strain of cholera bacteria that has evicted classic varieties from Asia and established the deadly diarrheal disease in Africa and Latin America. The genes may give the bacteria an edge as a human pathogen or environmental organism or both.
Using the completed Vibrio cholerae genome, researchers in the lab of John Mekalanos identified groups of genes that may eventually reveal how the bug becomes a global health threat. Photo by Graham Ramsay Using the powerful postgenomic tool of microarray technology, a study in the Feb. 5 Proceedings of the National Academy of Sciences also found genes unique to other strains responsible for most local outbreaks and global pandemics of cholera. Until 1817, when people began tracking the disease globally, Vibrio cholerae seemed to confine its destruction mostly to Asia. Since then, some of the many pathogenic strains lurking in the brackish waters along the coasts of Asia, Africa, Europe, and the Americas have flared into six pandemics. Waves of cholera afflicted U.S. populations in 1832, 1848, and 1866. By 1900, cholera had disappeared from the Americas and most of Africa and Europe. By 1950, it again was found primarily in Asia. Then came the seventh pandemic in 1961, when the El Tor 01 biotype apparently spread out of Indonesia to other countries in Asia and the Middle East. In the 1970s, carried in the ballast water of ships or by zooplankton in ocean currents, El Tor invaded Africa, southern Europe, and several Pacific islands. In 1991 it gained a foothold in South America. El Tor may live longer in aquatic ecosystems than previous pandemic strains, and it appears to have developed antibiotic resistance. Scientists want to discover and disable the secret of El Tor's success in the seventh pandemic, which continues today. Like other bacteria, Vibrio cholerae is notoriously promiscuous with its genes, swapping base pairs after not much more than a wink and a nudge. Not all Vibrio cholerae causes disease. The virulence of the pathogenic strains comes from the bacteria's easy acceptance of two pieces of foreign DNA, one a virus carrying the cholera toxin gene and another segment that enables the organism to adhere to the intestinal wall. The bacteria's genome might contain other genes that help El Tor survive in its aquatic environment or infect and multiply in its human hosts. The El Tor BlueprintTwo years ago, a definitive version of the dominant El Tor cholera genome sequence was completed by The Institute for Genomic Research in Washington D.C. (better known as TIGR) and a group of researchers including John Mekalanos, the Higgins professor of microbiology and molecular genetics and chair of that department, who is a senior author of the latest study. That gave HMS research associate Michelle Dziejman, technician Emmy Balon, and their colleagues a starting point."We wanted to use microarray technology to tell us the genetic differences between strains of Vibrio cholerae," said Dziejman, first author of the paper. "Then, based on those differences, we could go on to use other, more traditional biochemical or genetic methods to see what the role of the genes is or to see if we can attribute known properties and differences to any genes we've identified."
Microarray results. Compared to the El Tor N16961 cholera strain (bottom row), researchers found only about a 1 percent difference in genes among nine strains of vibrio cholerae, including two El Tor strains that did not become pandemic (NCTC 8457, MAK 757), an infectious El Tor strain that does not cause disease (2740-80), three strains of classic cholera from the sixth pandemic (569B, O395, NIH 41), two strains of seventh pandemic El Tor (HK1, C6709), and the closely watched 0139 El Tor strain (MO10) that emerged in 1992 in South Asia and that some people believe may herald an eighth pandemic. Courtesy of Michelle Dziejman and PNAS The researchers assembled a microarray from scratch using all 3,890 genes identified in the sequenced El Tor strain N16961. Strain by strain, they compared genes from nine distinct strains. The one-sided microarray identification only finds genes the test strains lack in comparison to the sequenced El Tor strain. Dziejman was surprised to find only about a 1 percent variation in genetic difference among the strains. In contrast, other research groups using similar techniques have found 6 to 12 percent differences in strains of staphylococcus and helicobacter. Bobbing for Bad ApplesAmong the few differences, they found overlapping groups of genes shared by El Tor strains but not classic strains, genes shared by strains able to cause epidemic disease, and 22 genes present only in seventh pandemic strains. Most of the 22 genes unique to the seventh pandemic are clustered in two islands on the larger of Vibrio's two chromosomes, suggesting horizontal transfer from another organism."We certainly don't claim to have found every genetic difference responsible for the behavior of some of these strains," Dziejman said, "or even if these genes are responsible for traits giving El Tor the ability to spread globally. We don't know what functions even make a pandemic strain." Mekalanos's lab plans a battery of tests, including creating knockout versions of the cholera bacteria to determine the function of these genes. They also are using microarray technology to compare gene expression patterns. "After interrogating a lot of strains, comparative genomics told us, 'Yes, some genes are different,'" Mekalanos said. "It gives us a direction to find out if these are key genes in the success of the clone." Microarray techniques also miss other categories of genetic variation, such as single nucleotide substitutions affecting the function of a protein or expression of genes, pointed out infectious disease specialist Gary Schoolnik, professor of medicine and microbiology at Stanford University. Nor can the technique identify duplicate genes, distinguish among homologous genes, or look at genes partly present or slightly rearranged. Nonetheless, Schoolnik is using microarray technology to examine Vibrio cholerae's lifestyle outside the host. Despite the limitations, microarray comparisons provide interesting and important information for understanding the organism, said Rita Colwell, head of the National Science Foundation. Colwell's lab also is using microarray analysis to probe the ecological role of cholera, with a working hypothesis that the same mechanism cholera uses in osmotic regulation in host crustaceans may wreak havoc in the human intestines. Schoolnik and Colwell see a paradigm organism for looking at climate change and infectious disease, but Mekalanos studies the Vibrio cholerae as a model for host- pathogen interactions. "The evolutionary success of the seventh pandemic clone of V. cholerae as an endemic and pandemic pathogen may be more related to its improved interaction with the human host than to its improved fitness within environmental reservoirs," the paper concludes. --Carol Cruzan Morton |
