During the STS-115 and STS-123 space shuttle missions, our Institute-led research team studied three common microorganisms -- Salmonella typhimurium, Pseudomonas aeruginosa and Candida albicans -- that were chosen because of their potential threats to crew health. Sending these microbes into space allowed us to investigate the microbes’ genetic adaptation and ability to cause infectious disease in microgravity, and to better understand the astronauts’ space environment.

In the STS-115 ‘Microbe’ experiment, we sent specially contained tubes of microbes in an experimental payload aboard the Space Shuttle Atlantis. The tubes of bacteria were placed in triple containment for safety and posed no threat to the health and safety of the crew during or after the mission.

After the bacteria returned to Earth, we performed the first global analysis of Salmonella, using microarray analysis to measure gene expression at the mRNA level, and evaluate the effects of space flight on pathogen gene and protein expression and virulence. By measuring the gene and protein patterns, we could hone in on the key molecular players necessary for virulence from among thousands of potential candidates.

Compared to bacteria that remained on earth, the space-traveling Salmonella had changed expression of 167 genes. After the flight, animal virulence studies showed that bacteria that were flown in space were almost three times as likely to cause disease when compared with control bacteria grown on the ground.

Our results revealed a key role for a master regulator, called Hfq, in triggering the genetic changes that show an increase in the virulence of Salmonella as a result of space flight. Hfq is a protein that binds to and regulates a number of regulatory RNAs, which in turn, control gene expression. Our studies suggest that there may be a role for these regulatory RNAs in the cellular response to the physical and mechanical forces found in space flight, which are relevant to conditions that cells encounter here on Earth during the normal course of their lifecycles.

These results have important implications for human health since Salmonella (and other gut-related bacterial pathogens) are a leading cause of food-borne illness and infectious disease, especially in the developing world. Hfq is a potential therapeutic target, since no vaccine currently exists for Salmonella food-borne infections in humans. In addition, the space flight studies may shed new light on why Salmonella has become increasingly resistant to antibiotic treatment.

We also studied the morphology of the bacteria in response to space flight, and the change that we observed is consistent with what looks like formation of a biofilm. The ground grown samples did not show biofilm formation. Biofilms are associated with increased pathogenicity because the immune system can’t clear the bacteria effectively and antibiotics don’t treat them effectively.

The results of NASA space shuttle mission STS-123, launched in March, 2008, both validated results and broadened the scope of spaceflight experiments from STS-115. In addition to confirming the effects of microgravity observed in the STS-115 MICROBE experiments, our latest study homed in on the importance of the microbial growth medium to gene expression and virulence during spaceflight. During both of our spaceflights, bacteria cultured in a nutrient-rich Lennox Broth (LB) medium 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 microgravity conditions were simulated using a rotating wall vessel bioreactor—a device designed by NASA engineers to replicate elements of spaceflight.

Interestingly, many of the 167 differentially expressed genes observed in the space-traveling microbes coded for an assortment of ionic response pathways. For our team, 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, our group 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 our team on earth have hinted that phosphate ions may be a principle component of the observed virulence reduction.

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 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, we believe, 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. No one had thought to look at a mechanical force like fluid shear on the disease-causing properties of a microorganism. We speculate 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. 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.

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.

We will now use the innovative research platform of the International Space Station to contribute to these new translational advances for the development of new strategies to globally advance human health.