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The medium is the message: Manipulating salmonella in spaceflight curtails infectiousness

December 12, 2008

Infectious pathogens like Salmonella typhimurium employ a startling array of techniques to skillfully outwit the body's defense mechanisms and produce illness. Through their expression of genes-the fundamental building blocks of cellular physiology-such microbes ingeniously adapt to varied environments, modifying their disease-causing potential or virulence.

Although the study of a broad range of microbial virulence factors is now well advanced, many pieces of the puzzle are still missing. Cheryl Nickerson, a researcher at Arizona State University's Biodesign Institute, has explored the novel environment of space to investigate the cellular and molecular machinery of virulence. There, the space shuttle crew grow the bacteria in triple-enclosed containers under conditions of minimized gravity (or microgravity). Nickerson's spaceflight experiments have shown that Salmonella gene expression and virulence are profoundly altered by microgravity, with the pathogenic cells undergoing a significant increase in their infectious disease potential.

Nickerson's latest findings, published in the journal PLoS ONE, are derived from experiments aboard NASA space shuttle mission STS-123, launched in March, 2008. This research validated results and broadened the scope of spaceflight experiments from STS-115, conducted two years earlier.

In addition to confirming the effects of microgravity observed in the STS-115 experiments (known as MICROBE), the new study homed in on the importance of the microbial growth medium to gene expression and virulence during spaceflight. "Pathogenic cells are smart," Nickerson stresses, pointing to their remarkable ability to fine-tune virulence factors in response to subtle environmental cues.

S. typhimurium, Nickerson's pathogen of choice, is a rod-shaped, motile bacterium and occasional unwelcome visitor to the human gastrointestinal tract, where it is a leading cause of food poisoning and related illnesses.

In both spaceflights, bacteria cultured in a nutrient-rich medium known as Lennox Broth (LB) consistently displayed a heightened virulence and exhibited differential expression of 167 distinct genes. These results were largely consistent with previous earthbound experiments in the laboratory, in which microgravitational conditions were simulated using a rotating wall vessel bioreactor-a device designed by NASA engineers to replicate elements of spaceflight.

Nickerson was able to examine the activity of genes in fine-grained detail through a technique known as microarray analysis, which allowed for a complete profile of gene expression across the entire 4.8 million DNA base pairs that make up the circular Salmonella chromosome. The 167 changes in gene levels produce a tremendous diversity of protein products, pointing to a global transformation in response to microgravity.

Interestingly, many of the 167 differentially expressed genes observed in the space-traveling microbes coded for an assortment of ionic response pathways. To Nickerson, these compelling results now suggested a possible means of limiting or eliminating the enhanced virulence imparted by spaceflight, through manipulation of the ionic content of the bacterium's surrounding environment.

In both the STS-115 and STS-123 missions, Nickerson compared the spaceflight response of Salmonella grown in Lennox Broth to the same bacteria grown in a minimal medium-one requiring the cells to synthesize most of their metabolic needs from scratch. This alternate growth medium, dubbed M9, contained high concentrations of five critical ions. The effects of this medium were dramatic, with the M9 cultures exhibiting a decrease in virulence in response to microgravity, despite exhibiting altered expression of many of the same genes and gene families that were observed in the LB cultures, where virulence under microgravity was intensified.

To test the hypothesis that ionic concentrations present in the M9 medium played a role in virulence reduction, a hybridized culture media known as LB-M9 was prepared for the March 2008 mission, consisting of the LB formula supplemented with five ions occurring in the M9 medium, but which were found to be at lower concentrations in LB. Bacteria cultured with LB-M9 again displayed a decreased virulence in response to microgravity. Subsequent bioreactor studies conducted by Nickerson's team on earth have hinted that phosphate ions may be a principle component of the observed virulence reduction.

One of Nickerson's most intriguing findings involves a specific RNA-binding protein known as Hfq, which appears to regulate central aspects of S. typhimurium's response to the spaceflight environment, acting as a "global molecular master switch." Hfq is known to regulate one third of the 167 differentially expressed genes in the spaceflight LB cultures. Interestingly, a large number of Hfq-regulated genes were also found to be differentially expressed in the M9 flight samples. In addition to Hfq's known properties as a virulence factor, the protein also acts to regulate ion response pathways,and has been associated with phosphate regulation. Moreover, Hfq appears to be an evolutionarily conserved regulatory factor, and may serve to globally modify bacterial responses to microgravity, regardless of the phenotypic outcome-a decrease in virulence for M9 cultures grown in microgravity environments and an increase for bacteria steeped in the LB medium.

But what was causing Salmonella to undergo such a dramatic transformation under conditions of microgravity? At least part of the answer, Nickerson believes, is related to the mechanical forces exerted upon the bacterial cell's membrane by the growth conditions-a property known as fluid shear. Specifically, the microgravity conditions aboard the space shuttle produce a condition of reduced fluid shear, an effect that appears to trigger an intensification of virulence in Salmonella grown in LB medium. As Nickerson points out, "No one had thought to look at a mechanical force like fluid shear on the disease-causing properties of a microorganism."

If a rolling stone gathers no moss, a bacterium like S. typhimurium appears to gather virulence when its movement is slowed down and fluid shear across its surface is minimized. Nickerson speculates that Salmonella encounters just such conditions not only during spaceflight but also in vivo in an infected individual when the bacterium makes contact with an intestinal host cell and becomes ensnared in the fingerlike projections known as microvilli.

Thus, space travel may trick the microbes into behaving as though they were in an environment hospitable to cell infection, thereby switching on an increased virulence response, given appropriate environmental preconditions. "They're responding to an environmental signal that they're used to seeing right here on earth, during the natural course of the infectious disease process," Nickerson states, emphasizing that this response is masked in traditional microbial studies performed using lab cell cultures, which fail to replicate the low fluid shear conditions found in vivo, particularly in the gastrointestinal tract-Salmonella's favored site of infection.

One result of spaceflight not replicated in the earthly bioreactor simulations was the formation of what appear to be biofilms-conglomerations of bacterial cells associated with infectious virulence. Nickerson emphasizes the potential importance, should such findings be confirmed. Up to 70 percent of bacterial infections in humans may be associated with the formation of such biofilms, which seem to arm bacterial pathogens with formidable resistance to the host's immune system as well as to antibiotics.

How do the disparate variables-extracellular phosphate concentration, mechanical forces like fluid shear and genetic regulation of pathogenic virulence-combine and interact during the infection process? While the current research provides tantalizing hints, a full understanding of the complex interplay of forces and the in vivo mechanisms of Salmonella pathogenesis await further research.

Fortunately, new opportunities for study are opening up, which may illuminate these issues. NASA is one of the primary partners in the construction and operation of the International Space Station (ISS), a semi-permanent research platform allowing for further investigations into microbial responses to low fluid shear environments. Because cells cultured in microgravity exhibit biomedically relevant phenotypes that can not be observed using traditional experimental approaches, Nickerson believes the therapeutic benefits of such research will extend beyond infectious pathogens like S. typhimurium, eventually inspiring new clinical approaches to cancer, aging, bone and muscle wasting diseases, among other earthly afflictions.

"We can use the innovative research platform of the ISS to contribute to these new translational advances for the development of new strategies to globally advance human health."

Arizona State University