Effect of Spaceflight on Microbial Gene Expression and Virulence (Microbe) - 07.28.16
The Microbe experiment will investigate the effects of the space flight environment on virulence (ability to infect) of three model microbial pathogens: Salmonella typhimurium, Pseudomonas aeruginosa, and Candida albicans, that have been identified as potential threats to crew health based upon previous space flight missions. Science Results for Everyone
Microbes, like humans, change in space. This investigation examined changes in the virulence and gene expression of three microbes that can make astronauts and the general public sick. Mice infected with Salmonella typhimurium cultured in space died in greater numbers and sooner than those infected with the same microbe on Earth. Analysis of S. typhimurium and Pseudomonas aeruginosa bacteria showed differences in expression of multiple genes and an apparent role of a regulatory protein, Hfq, in that alteration. Data indicate that P. aeruginosa virulence may increase in space as well. Experiment Details
Cheryl A. Nickerson, Ph.D., Arizona State University, Tempe, AZ, United States
Kent Buchanan, Ph.D., Tulane University, New Orleans, LA, United States
Michael J. Schurr, Ph.D., University of Colorado, Denver, CO, United States
Charlie Mark Ott, Ph.D., Johnson Space Center, Houston, TX, United States
Duane L. Pierson, Ph.D., Johnson Space Center, Houston, TX, United States
NASA Ames Research Center, Moffett Field, CA, United States
Sponsoring Space Agency
National Aeronautics and Space Administration (NASA)
Human Exploration and Operations Mission Directorate (HEOMD)
ISS Expedition Duration
April 2006 - September 2006
Yeast-GAP, a similiar investigation to Microbe was initially performed on ISS Expedition 8.
- Microorganisms are carried to space on the human body and in water or food. Many organisms that have been identified aboard spacecraft can cause crewmembers to become sick and put a long duration mission at risk.
- The virulence (disease causing potential) of these microbes can be significantly altered in space due to radiation and microgravity.
- The Microbe experiment will study three prevalent microbes, Salmonella typhimurium, Pseudomonas aeruginosa and Candida albicans that have been identified as potential threats to crew health based upon previous space flight missions.
- This evaluation of the effect of space flight on the gene expression and disease causing potential of these microorganisms will identify whether the risk of infection increases as a result of long-duration space flight which is important for the safety and performance during our future Lunar and Martian missions.
A human presence in space, whether permanent or temporary, is accompanied by the presence of microbes. The extent of changes to microorganisms in response to space flight conditions is not completely understood. Because the length of human space missions is increasing, there is an increased risk to orbiting humans of infectious disease events occurring inflight. Previous studies have indicated that space flight weakens the immune system in both humans and animals. As astronauts and cosmonauts live for longer periods in a closed environment and using recycled water and air, there is an increase in the potential for negative impacts of microbial contamination upon the health, safety, and performance of crewmembers. Therefore, understanding how the space environment affects microorganisms and their disease causing potential is critically important for space flight missions and requires further study.
The Microbe experiment employed three model microbes, Salmonella typhimurium, Pseudomonas aeruginosa, and Candida albicans to examine the global effects of space flight on microbial gene expression and virulence attributes. These represent different types of bacteria and yeast. Salmonella is the most common agent for gastroenteritis in humans. Sanitation procedures are used to eliminate Salmonella from food sent to orbit, but if some were missed, the impact on crew health could be significant. Pseudomonas has been detected as a contaminant in the water system of spacecraft, and was once a cause of crewmember infection during the Apollo era. Candida is a yeast that is present as part of the natural human flora, but has the potential for harmful overgrowth if microbial communities were to change over time in space.
The experiment was flown inside self-contained culture chambers which can be activated manually by a crewmember turning a hand crank to release growth media into the cell chamber. After 24 hours of growth at ambient temperatures, the growth was stopped by a crewmember turning the hand crank once more. Upon landing, one third of the samples were recovered as soon as possible and the live cells will be used immediately for the virulence studies while the remaining stabilized samples were frozen at minus 80 degrees C. Ground analysis focused on identifying differences in growth rates and patterns, changes in gene expression, and changes in virulence of the microbes in space compared to Earth.
By understanding the changes that microorganisms undergo in the space environment, these studies may lead to the development of vaccines and other novel countermeasures for the treatment and prevention of infectious diseases occurring during space flight and on Earth.
Results from this single flight experiment will provide important information on the threat of pathogens in the space environment, which will assist with development of diagnostic tools to monitor the atmosphere, water and surfaces for the presence of these microbes. Understanding the molecular responses of these organisms to space flight is a necessary step that will significantly contribute to development of systems that meet requirements for supplying and storing potable water that is free of microbial contaminants. Furthermore, identification of the changes caused by space flight to genes and proteins will provide novel targets for pharmacological intervention to prevent and control infectious disease, which will ultimately facilitate safe and productive long-term exploration of the Moon and Mars.
By understanding the unique spectrum of microbial genetic and virulence changes induced by space flight, this experiment will yield valuable knowledge leading to advances in vaccine development and other therapeutics for treatment, prevention and control of infectious diseases on Earth as well as in space.
Operational Requirements and Protocols
The microbes will be contained in the glass barrel of a Fluid Processing Apparatus (FPA). The FPA is a tube that contains 2 or 3 separate liquids in addition to the sample. The liquid can be introduced to the sample in a controlled order. The FPA is contained in a Group Activation Pack (GAP). The GAP will hold up to 8 FPAs that can be processed simultaneously. For this experiment, a total of 12 GAPs will be used, 6 for Salmonella and 3 each for Pseudomonas and Candida. For activation and termination, the crew will turn a hand crank that has been inserted onto the top of the GAP. Growth of the samples will last 24 hours before the experiment is terminated. Once the samples are on the ground and have been stabilized, they will be frozen at minus 80 degrees C then shipped to the PI laboratory. The growth of some samples will not be terminated and will be maintained as viable cultures at ambient temperature for infection studies.
The microbes, Salmonella typhimurium, Pseudomonas aeruginosa, and Candida albicans, will be contained in a Fluid Processing Apparatus (FPA). In order to activate the sample, the crew will turn a hand crank that has been inserted onto the top of the GAP which contains the FPAs. This will release the growth media into the samples initiating growth. The samples will grow for 24 hours in ambient conditions. The crew will then turn the hand crank again to introduce another media to terminate the growth. Once the samples have returned to ground, the live cells will be used in virulence studies while the stabilized samples will be frozen to minus 80 degrees C and shipped to the PI laboratory for gene expression studies.
Decadal Survey Recommendations
Plant and Microbial Biology P1
A human presence in space, whether permanent or temporary, is accompanied by the presence of microbes. However, the response of microorganisms to growth during a spaceflight mission is not completely understood. While several spaceflight studies have investigated changes in microbial characteristics when cultured during spaceflight, the Microbe experiment was the first to investigate changes in virulence and gene expression in several microbes that are pertinent to both astronauts and the general public on Earth. The organisms investigated were ,i>Salmonella enterica, Salmonella Typhimurium, Pseudomonas aeruginosa, and Candida albicans.
Within a few hours after return to Earth aboard STS-115, the S. Typhimurium grown in space was used to infect mice to determine the disease causing potential (virulence) of the organism. Mice infected with bacteria cultured in space displayed a decreased time to death and increased percent mortality compared with those infected with ground controls (Wilson et al 2007). To better understand why the spaceflight-grown cultures were more virulent, an analysis of the gene expression was performed. The S. Typhimurium grown in space expressed a total of 167 genes differently compared to the identically grown cultures on Earth. Surprisingly, many genes that are commonly associated with increased virulence were not differentially regulated. Perhaps the most interesting finding was that a regulatory protein, Hfq, appeared to play a role in the alteration in gene expression in response to spaceflight culture. This finding was the first to identify a potential mechanism by which a microorganism was being altered when grown in space. One additional finding was alterations in microbial morphology.
Both during the preparation for spaceflight experiments and to fully understand the results after the experiment, scientists used spaceflight analogues, like the rotating wall vessel (RWV) and the random position machine (RPM) to mimic spaceflight growth conditions in order to gain insight into the potential behavior of P. aeruginosa in microgravity (Nickerson et al., 2004). For MICROBE, microarray analysis samples of P. aeruginosa grown in a RWV were compared to samples grown in normal gravity controls. The results revealed an alteration in a regulatory role of the sigma factor AlgU, which consequently led to an increase in production of the extracellular substance alginate. This change in gene regulation and increased production of alginate resulted in an increase in heat and oxidative stress resistance. Perhaps most interesting was the involvement of Hfq in response to culture in the RWV, consistent with MICROBE spaceflight findings in S. Typhimurium and P. aeruginosa. Also aboard grown aboard STS-115 were cultures of P. aeruginosa. A comparison of spaceflight grown cultures to those grown identically on Earth indicated that 167 genes were differentially regulated, with many being different than those seen with S. Typhimurium. However, one key similarity was that when analysis of the data was performed, Hfq was again identified as a key regulator for many of the differentially regulated genes. This finding reinforced the role for Hfq in microbial response to spaceflight and also suggested that this response may be evolutionarily conserved between species. The differentially regulated gene expression data also indicated that many P. aeruginosa virulence characteristics may increase in response to spaceflight culture. Cultured samples of Candida albicans, an infectious fungus, grown in spaceflight conditions revealed that short-term spaceflight caused changes in more than 8% of analyzed genes than ground-grown organism. Among the affected genes are those involved in cell aggregation (lumping together), antifungal agent, and stress resistance, including enhanced activity of cellular protein transporters and facilitators. Reduced activity of genes involved in actin (microfilaments) cytoskeleton was observed, and spaceflight-cultured fungus showed more random (abnormal) budding as opposed to bipolar (180-degree opposite) budding patterns for ground samples. Interestingly, the regulator gene CAP1, a gene with key role for virulence of C. albicans, plus over 30% of the CAP1 group genes was differentially expressed (reproduced) in spaceflight-cultures. Although C. albicans in microgravity showed a global change in gene expression that could potentially make the them more dangerous, no evidence for this was observed when mice were infected with the space-grown fungus. The data collectively represent an important basis for the assessment of the risk microorganisms pose during human spaceflight missions. The study also reinforces the role that physical forces in the human body, such as low fluid-shear in the body, could play in the infection process; insights that hold promise to fundamentally advance our understanding of infectious disease on Earth (Crabbé et al., 2010, 2011, 2013).
The Microbe experiment had far reaching implications. It clarified the mechanisms behind the observations of microbial spaceflight experiments over the past 40 years and initiated studies to understand how these findings impact risk assessment to crew health. In addition, the knowledge gained from space flight research has been the focus of commercial and academic entities toward the discovery of novel therapeutic and vaccine approaches leading to the implementation of new strategies for translation of this research into health benefits for the general public (Sarker et al 2010).^ back to top
Crabbe A, Schurr MJ, Monsieurs P, Morici L, Schurr J, Wilson JW, Ott CM, Tsaprailis G, Pierson DL, Stefanyshyn-Piper HM, Nickerson CA. Transcriptional and Proteomic Responses of Pseudomonas aeruginosa PAO1 to Spaceflight Conditions Involve Hfq Regulation and Reveal a Role for Oxygen. Applied and Environmental Microbiology. 2011; 77(4): 1221-1230. DOI: 10.1128/AEM.01582-10. PMID: 21169425.
Crabbe A, Nielson-Preiss S, Woolley CM, Barrila J, Buchanan K, McCracken J, Inglis DO, Searles SC, Nelman-Gonzalez MA, Ott CM, Wilson JW, Pierson DL, Stefanyshyn-Piper HM, Hyman LE, Nickerson CA. Spaceflight enhances cell aggregation and random budding in Candida albicans. PLOS ONE. 2013 December 4; 8(12): e80677. DOI: 10.1371/journal.pone.0080677.
Sittka A, Pfeiffer V, Tedin K, Vogel J. The RNA chaperone Hfq is essential for the virulence of Salmonella typhimurium. Molecular Microbiology. 2007; 63(1): 193-217. DOI: 10.1111/j.1365-2958.2006.05489.x. PMID: 17163975.
Wilson JW, Ott CM, Honer zu Bentrup K, Ramamurthy R, Quick L, Porwollik S, Cheng P, McClelland M, Tsaprailis G, Radabaugh T, Hunt A, Fernandez D, Richter E, Shah M, Kilcoyne M, Joshi L, Nelman-Gonzalez MA, Hing SM, Parra MP, Dumars PM, Norwood KL, Devich J, Bober R, Ruggles AD, Goulart C, Rupert M, Stodieck LS, Stafford P, Catella LA, Schurr MJ, Buchanan K, Morici L, McCracken J, Allen PL, Baker-Coleman C, Hammond TG, Vogel J, Nelson R, Pierson DL, Stefanyshyn-Piper HM, Nickerson CA. Space flight alters bacterial gene expression and virulence and reveals a role for global regulator Hfq. Proceedings of the National Academy of Sciences of the United States of America. 2007; 104(41): 16299-16304. DOI: 10.1073/pnas.0707155104. PMID: 17901201.
Ground Based Results Publications
Nauman EA, Ott CM, Sander E, Tucker DL, Pierson DL, Wilson JW, Nickerson CA. A Novel Quantitative Biosystem to Model Physiological Fluid Shear Stress on Cells. Applied and Environmental Microbiology. 2007 Feb; 73(3): 699-705. DOI: 10.1128/AEM.02428-06.
Crabbe A, Pycke B, Van Houdt R, Monsieurs P, Nickerson CA, Leys N, Cornelis P. Response of Pseudomonas aeruginosa PAO1 to low shear modeled microgravity involves AlgU regulation. Environmental Microbiology. 2010; 12(6): 1545-1564. DOI: 10.1111/j.1462-2920.2010.02184.x.
Nickerson CA, Ott CM, Wilson JW, Ramamurthy R, Pierson DL. Microbial Responses to Microgravity and Other Low-Shear Environments. Microbiology and Molecular Biology Reviews. 2004 June; 68(2): 345-361. DOI: 10.1128/MMBR.68.2.345-361.2004. PMID: 15187188.
Sarker SF, Ott CM, Barrila J, Nickerson CA. Discovery of Spaceflight-Related virulence Mechanisms in Salmonella and Other Microbial Pathogens: Novel Approaches to Commercial Vaccine Development. Gravitational and Space Biology. 2010; 23(2): 75-78.
Wilson JW, Ramamurthy R, Porwollik S, McClelland M, Hammond TG, Allen PL, Ott CM, Pierson DL, Nickerson CA. Microarray Analysis Identifies Salmonella Genes Belonging to Low-Shear Modeled Microgravity Regulon. Proceedings of the National Academy of Sciences of the United States of America. 2002; 99(21): 13807-11382. DOI: 10.1073/pnas.212387899.
Wilson JW, Ott CM, Ramamurthy R, Porwollik S, McClelland M, Pierson DL, Nickerson CA. Low-Shear modeled microgravity alters the Salmonella enterica serovar typhimurium stress response in an RpoS-independent manner. Applied and Environmental Microbiology. 2002; 68(11): 5408-5416. DOI: 10.1128/AEM.68.11.5408-5416.2002.
Wilson JW, Schurr MJ, LeBlanc CL, Ramamurthy R, Buchanan K, Nickerson CA. Mechanisms of bacterial pathogenicity. Journal of Postgraduate Medicine. 2002; 78(918): 216-224.
Ott CM, Marshburn TH, Nickerson CA. The International Space Station: an extreme environment for key host-microbe discoveries: Microgravity encountered during spaceflight helps to determine how various forces influence microbes as they interact with hosts and environments. Microbe Magazine. 2016 June 1; 11(6): 253-261. DOI: 10.1128/microbe.11.253.1.
Nickerson CA, Ott CM, Mister SJ, Morrow BJ, Burns-Keliher L, Pierson DL. Microgravity as a Novel Environmental Signal Affecting Salmonella enterica Serovar Typhimurium Virulence. Infection and Immunity. 2000; 68(6): 3147-3152.
The Biodesign Institute - Arizona State University
Ames Research Center - Microbe Hardware
Image of Candida albicans.This is one of three microbial pathogens examined for the Microbe investigation. Image courtsey from Arizonia State University.
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Image of gram negative Salmonella typhimurium. This is one of three microbial pathogens examined for the Microbe investigation. Image courtsey from Arizonia State University.
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Scanning Electron Micrograph (SEM) of gram negative Pseudomonas aeruginosa. This is one of three microbial pathogens examined for the Microbe investigation. Image courtsey from Arizonia State University.
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NASA Image: S115e07274 - Astronaut Heidemarie M. Stefanyshyn-Piper, Mission Specialist holding the Microbe Group Activation Pack containing eight Fluid Processing Apparatuses in the middeck of the Space Shuttle Atlantis during Expedition 13 and STS-115 joint operations.
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The Group Activation Pack (GAP), left, is a triple-enclosed containment vessel used to house microbiology experiments in microgravity. The Ground-based bioreactor, right, is used in ground studies to simulate microgravity in the laboratory. (Photo courtesy of Arizona State University)
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Researcher Hami Teal, Ph.D. examines hardware for Microbe experiment. Image courtesy of Ames Research Center.
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Researcher Hami Teal, Ph.D. examines and activates the hardware for Microbe experiment. Image courtesy of Ames Research Center.
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Principal Investigator Cheryl Nickerson, Ph.D. Image courtesy of the Biodesign Institute, Arizona State University.
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