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Rheumatic Fever Bacterium Sequenced
DNA evidence links two epidemics in Utah twelve years apart

By Edward R. Winstead

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Infectious Diseases

In the last fifteen years Salt Lake City has experienced two outbreaks of rheumatic fever. Despite its reputation as a disease gone-by, acute rheumatic fever remains the leading cause of heart disease among children worldwide. Both outbreaks in Utah were serious enough to qualify as epidemics and were caused by genetically similar strains of ‘group A’ Streptococcus (GAS) bacteria. The pathogen had apparently remained in the population at very low levels for twelve years before mounting a remarkable resurgence.

Two M18 strains from the study on blood agar plates. T18P is from Great Lakes Naval Training Center during WWII, and T18G is from the Lowry Air Force Base in 1968.

Scientists made this discovery by analyzing the genomes of bacteria associated with both outbreaks. The work is part of a larger investigation of hundreds of pathogenic strains isolated from children and young adults during both epidemics. The researchers are using genomic tools to pinpoint key genes that allow some Streptococci strains to cause rheumatic fever in humans.

The researchers have recently sequenced the genome of one of the Salt Lake strains. James M. Musser, chief of the Laboratory of Human Bacterial Pathogenesis at US National Institute of Allergy and Infectious Diseases in Hamilton, Montana, led the project and says the sequencing was a necessary first step in the research.

“We sequenced the organism in order to get through a particular bottleneck in the research and into the molecular biology and pathogenesis of the organism,” says Musser. “We view sequencing as a means of getting to a far more interesting set of endeavors, such as generating new vaccines and therapeutics.”

Group A Streptococci are relatively common and cause a range of conditions from strep throat and rheumatic fever to toxic shock syndrome and ‘flesh-eating’ disease. This study was a novel opportunity for researchers to try to learn what makes strep infections so dangerous to the heart.

Once inside a person, the bacteria often subvert the immune system, allowing them to attack heart tissue. In severe cases, surgery is required to replace damaged heart valves. Researchers do not know which genes transform a harmless bacterium into a pathogen or how the disease progresses from a throat infection into heart disease.

“We need to know once and for all how the disease occurs, and the genome opens a whole new area of research on rheumatic fever,” says Patrick Schlievert, a microbiologist who studies GAS bacteria at the University of Minnesota-Minneapolis. “We can now identify genes that are required to cause severe strep infections and proteins that are candidates for vaccines. To do this you need the genome.”

Thousands of researchers have tried to make the connection between bacterial infection and heart disease, and none has tied it down in a persuasive way, according to Richard M. Krause, the former director of the US National Institute of Allergy and Infectious Diseases.

Three-dimensional structure of SPE C protein.

“For decades we have tried to understand how streptococcal infections can cause serious heart disease, but the answer has escaped us,” says Krause. Now, he adds, “we have an opportunity to possibly make the connection by studying the genes of pathogens responsible for specific outbreaks.”

The new study, which appears in Proceedings of the National Academy of Sciences, is dedicated to Krause, a pioneer in research on Streptococcus and other infectious bacteria. He participated in the first successful efforts to use penicillin to treat streptococcal infection.

There are more than a hundred varieties of group A Streptococcus, each of which is distinguished by the type of ‘M’ protein it has. Musser’s group sequenced a type called M18. Last year, Vincent A. Fischetti, of The Rockefeller University in New York, and colleagues sequenced an M1 strain, also known as Streptococcus pyogenes.

Musser says his team has now isolated and purified most of the surface proteins that are likely to mediate interactions between the bacteria and host. Surface proteins can be recognized by DNA patterns in their genes and are potential vaccine targets.

Two M18 proteins in particular caught the attention of the researchers. They look like another M18 protein, called SPE C, which triggers a massive immune reaction in host cells and leads to streptococcal toxic shock syndrome. Schlievert’s laboratory investigates SPE C and is now studying the newly identified proteins. These types of protein are called superantigens.

“Superantigens are interesting molecules because they allow the bacterium to survive in the host for an extended period of time,” says Schlievert. His laboratory, in collaboration with Musser’s, will knock out the proteins in M18 strains and test the virulence of modified strains in rabbits or mice to see whether something relevant can be learned about the mechanism of strep infection in the heart.

The researchers also constructed DNA microarrays containing genes from both the M18 strain and the M1 strain. The arrays were used to compare the genomes of 36 M18 strains. The samples included isolates taken from patients at the Great Lakes Naval Training Center during World War II and the Lowry Air Force Base in Colorado in 1968. Rheumatic fever outbreaks tend to occur in environments where there is crowding, such as military barracks, schools, and large households, where bacteria and other infectious agents spread easily.

The microarray analysis showed that the genomes were remarkably similar among the strains. Differences tended to occur in regions containing DNA from viruses that had infected the bacteria, and these will be the focus of future studies.

The microarrays also showed that GAS bacteria isolated from patients during the 1998-1999 Salt Lake City epidemic were nearly identical to those from the 1986-1987 epidemic in the same area. “The isolates that caused the disease twelve years apart are essentially identical twins,” says Musser. “There was remarkably little variation.”

Detail from subset of 223 strain-specific ORFs identified by DNA microarray analysis of 36 serotype M18 strains of GAS.

In a companion paper to be published in the coming months, Musser’s group will report the results of a large-scale targeted gene sequencing on more than 900 strains collected in Salt Lake City from 1984 to the present. They found that during peaks of rheumatic fever outbreaks the M18 strains were dominant.

“The M18 strains more or less took over during the peaks,” says Musser, noting that in between the outbreaks, the M18 organism was present in the population but in low numbers relative to all other strains. “It was hanging out, if you will, until it reached a critical mass.” One explanation is that the bacteria can survive in persons for long periods of time without causing symptoms.

“This is really the first time anyone has used DNA microarrays to analyze two epidemics of bacterial disease,” says Musser. Using microarrays to explore a testable hypothesis about an outbreak of human disease is something that Musser would like to see happen more often.

“There is very little true meshing of genomic information with pathogenesis research,” he says. Major human pathogens have been sequenced, but “the amount of downstream work using those sequences is relatively small.”

Rheumatic fever is no longer on the front pages of newspapers in the U.S., but it remains a major health problem in the developing world. Richard Krause has recently returned from a trip to India where he visited a hospital near New Delhi whose surgeons had operated on some 1,000 children to replace heart valves that were inflamed beyond repair. “These operations are expensive,” he says. “This was in just one large hospital, so you can imagine what the burden is for the entire country.”

Giving countries like India penicillin does not necessarily solve the problem because children do not see doctors regularly and antibiotics are ineffective against the bacterial toxins. A prophylactic vaccine based on the studies involving the sequenced M18 strain is next on the research agenda. “If we had a vaccine with no side effects that was cheap, we’d use it tomorrow,” says Krause.

Detail from atlas of the chromosome of serotype M18 strain MGAS8232.

“We entered the 1950s in this country with two major diseases to prevent—polio and rheumatic fever,” says Krause, now 77. “And we have learned how to prevent both of them.”

“I hope the new research helps us discover why the bacterium has remained susceptible to penicillin,” says Krause. “There has to be a genetic explanation for this, and I would dearly love to know what it is. If we knew why the bacterium does not develop resistance, we might learn how other organisms develop resistance.”

See related GNN article: Genome sequence of Streptococcus pyogenes, the flesh-eating bacterium

Smoot, J.C. et al. Genome sequence and comparative microarray analysis of serotype M18 group A Streptococcus strains associated with acute rheumatic fever outbreaks. Proc Natl Acad Sci USA 99, 4668-4673 (April 2, 2002). Published online March 26, 2002.
Ferretti, J.J. et al. Complete genome sequence of an M1 strain of Streptococcus pyogenes. Proc Natl Acad Sci USA 98, 4658-4663 (April 10, 2001).

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