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Experiments May Lead to Ways of Neutralizing Virulent Microbes
09.09.02
 
Getting From Wild to Mild - Experts believe that as many as 4 million cases of food poisoning in the United States are caused annually by salmonella bacteria. The results of ground-based and in-space studies to learn about the ways bacteria multiply and conquer the immune system could lead to a better understanding of preventing or lessening the severity of bacterial infections.

Spacefarers can remain in a closed system for weeks, sometimes months, and for proposed long-duration flights, maybe even years, breathing recycled air and drinking recycled water. Given that some virulent microbes appear to thrive in microgravity, that's not a promising scenario for health, according to Cheryl Nickerson, assistant professor of microbiology and immunology at Tulane University Health Sciences Center's program in molecular pathogenesis and immunity. Nickerson says that spacegoers already appear to have a higher risk of falling ill.

"Disease is related, first, to the host's immune status. The second part is microbial virulence, the ability of a microbe to cause disease," she explains. "When humans are in space, there appears to be some compromise to their immune systems. This suggests a higher risk of infection could occur in flight."

Photo of Cheryl Nickerson working with her laboratory staff. Image to right: Cheryl Nickerson (at far right) works with her laboratory staff (from left to right): Carly LeBlanc, Rajee Ramamurthy, Kerstin Honer zu Bentrup, and Jim Wilson.

In ground-based studies simulating microgravity, Nickerson and her research team have found that a common strain of bacteria known as Salmonella typhimurium can alter its genetic profile, upping the production of certain self-protecting proteins that may enhance virulence. That could be unwelcome news for future astronauts. Given that microgravity may also reduce antibiotic effectiveness - because of an as-yet poorly defined interaction between the body and pharmaceutical measures, certain studies have suggested - and absent any new pharmacological approach, the difficult task of in-space treatment would be made even more challenging.

In the course of their investigation, Nickerson and her colleagues found that more than 100 salmonella genes, or about 3 percent of the salmonella genome, altered genetic expression. The changes made the bacteria far more lethal: mice injected with the strains grown in modeled microgravity died, on average, three days earlier than expected, from shock and from large-organ failure. At autopsy, Nickerson's research team found greater-than-anticipated numbers of the microbes within the livers and spleens of the experimental animals. "Clearly, there's something going on that is able to defeat the host's immune system," she says.

Nickerson's original studies in simulated microgravity involved the use of a device known as a rotating wall bioreactor, a vessel designed by NASA that mimics reduced gravity. Cells of S. typhimurium were placed in a culture within the bioreactor chamber. When the bioreactor spun, it maintained the cells in close approximation of freefall, which astronauts experience as up to one-millionth of Earth's normal gravity. The researchers also cultured S. typhimurium under normal-gravity conditions.

NASA - photo of Cheryl Nickerson working with her laboratory staff. Image to right: Nickerson's research focuses on a well-known pathogen, Salmonella typhimurium, whose genetic response to gravity's near absence could provide clues to infection protection.

In addition, to study how S. typhimurium causes infection in people, Nickerson and her colleagues used the bioreactor to culture three-dimensional human intestinal epithelial cells, which more accurately model the physiology of human intestinal tissue than does conventional tissue culture. In response to the microbial invasion, the cells produced higher levels of substances called anti-inflammatory cytokines, which may help limit damage to the epithelial tissue following salmonella infection. The three-dimensional intestinal cells also showed less damage and cell death following salmonella infection as compared to other types of cells, known as monolayers. These observations are consistent with the self-limiting nature of salmonella infection, according to Nickerson, which can damage or kill epithelial cells in otherwise healthy individuals before being destroyed by immune reaction.

Treating and Neutralizing
The salmonella family of microbes that colonize uncooked or undercooked meat and poultry and nonpasteurized dairy products cause an estimated two to four million cases of gastrointestinal illness in the United States annually, costing up to $2 billion in yearly losses due to lost time at work and the need for medical treatments.

According to the Centers for Disease Control, salmonella-related maladies are among the most common intestinal infections in the United States, with 40,000 cases reported yearly. However, scientists estimate that because only 3 to 5 percent of salmonella cases are actually reported nationwide, and many milder cases are never diagnosed, the true incidence is much higher, likely in the millions. As many as 1,000 Americans die annually from salmonella infections.

Bacteria are not premeditated killers. Their goals, like all organisms, are to survive, thrive, and reproduce. To do so, they release certain proteins. In natural environments, these proteins neutralize substances harmful to the bacterium. When ingested into a human digestive tract, the same mechanisms are engaged. Although the strong acids found in the stomach kill up to 99 percent of the would-be bacterial colonizers, the 1 percent that do survive are able to "express," or release, the protective proteins that cause so much upset to their human hosts. The immunologic battle between host and pathogen can be fierce. Most of the time, the immune system wins, containing the infection, but sometimes the bacteria can overcome all defenses, and death can result.

Although most S. typhimurium-caused infections in the United States don't require hospitalization or serious medical intervention, at least in healthy people, they are potentially fatal if untreated in people with weakened immune systems (in developing countries, S. typhimurium is a leading cause of death, especially in children, due to dehydration). Deciphering the bacteria's molecular responses could lead - with new drugs and vaccines - to a means to treat or even neutralize salmonella infections, quickly lessening or eliminating the characteristic nausea, vomiting, intestinal inflammation, and diarrhea that they cause.

As humans work for longer periods in space, they may bring with them preexisting infections. Moreover, despite precautions, foods brought on board could conceivably harbor salmonella bacteria. Depending on severity, a salmonella-induced illness could pose serious dangers.

"We've all had it," Nickerson says. "You suffer for three to four days. You can't go to work. If you're on a space mission, it's even worse. Something like food poisoning could put a mission at risk, or in the worst case, threaten crew survival."

Drawing a Genetic Map
Because S. typhimurium is a well-known pathogen, investigation of the strain's genetic response to gravity's near absence could provide clues to infection protection from it or related microorganisms - perhaps even other, distantly related strains. "Salmonella is the best characterized of all the bacterial pathogens," Nickerson points out. "It's very closely related to E[scherichia] coli, which is the most commonly studied bacterium known.

"We're going to find out if spaceflight gives similar results to what we've found on the ground. We want to build a detailed molecular road map of how salmonella senses and responds to microgravity. It will be an incredibly complex map, but one we hope will guide us to effective remediation strategies." Nickerson should be able to draw a more complete map once results are in from a salmonella-virulence experiment that is scheduled to fly on an upcoming space shuttle mission.

Once on board, the experimental apparatus that contains the bacteria will pose no danger to the space shuttle crew. The experiment is entirely isolated from human contact and designed to easily withstand the rigors of takeoff and landing. The system is automated and will engage roughly one hour into the mission.

Eight test tubes made from a hardened plastic will be carried in a boxlike apparatus known as an isothermal containment module, or ICM. The tubes make up the Automated Group Activation Pack, or auto-GAP, which fits into the ICM. The auto-GAP uses a direct-current motor drive to autonomously start and stop experiments based on preprogrammed instructions. Each of the tubes allows controlled, sequential mixing of up to three fluids, which are isolated into three sections by rubber partitions, or septa. A bypass in the tubes permits fluid mixing in an adjacent chamber as plungers push the septa forward.

Three separated liquid media are contained within the tubes. One fluid will hold the bacteria, the second is a medium in which the bacteria will be grown and cultured, and the third is a fixing solution that essentially halts in place all biological activity, preserving the sample for later study. The entire apparatus is temperature-controlled so that bacteria will grow at 98.1ºF, or 37 ºC.

Just after launch, as the first plungers are engaged, the bacteria will be pushed into the second chambers' growth media. Two days later, after bacterial multiplication, the second plunger will engage, and all eight tubes will be fixed and ready for on-ground analysis once the shuttle lands. These samples will then be subjected to microarray analysis to analyze global changes in salmonella gene expression in response to spaceflight, as compared to on-ground controls.

"A dangerous thing in science is speculation about research you haven't yet conducted," says Nickerson. "I don't know what to expect. We do know what we've found on the ground. In space, we're dealing with a complete unknown. We're going to have to wait and see what the experiment tells us."

Resources

SpaceResearch, Volume 1, Number 4, September 2002 - This story appeared as the Fundamental Space Biology Research Update in the Fall 2002 issue.

More information about Nickerson's experiment - At the SYNTHECON, Inc. Web site, Home of the Rotary Cell Culture System

Three-Dimensional Tissue Assemblies: Novel Models for the Study of Salmonella enterica Serovar Typhimurium Pathogenesis - The paper written by Cheryl A. Nickerson, Thomas J. Goodwin, Jacqueline Terlonge, C. Mark Ott, Kent L. Buchanan, William C. Uicker, Kamal Emami, Carly L. LeBlanc, Rajee Ramamurthy, Mark S. Clarke, Charles R. Vanderburg, Timothy Hammond, and Duane L. Pierson published in Infection and Immunity, November 2001, p. 7106-7120, Vol. 69, No. 11

More information on the hardware that will house the experiment - The hardware developer is BioServe Space Technologies
 
 
Author: James Schultz
Editor: SpaceResearch Editorial Board