Project description:Characterization of bacterial behavior in the microgravity environment of spaceflight is of importance towards risk assessment and prevention of infectious disease during long-term missions. Further, this research field unveils new insights into connections between low fluid-shear regions encountered by pathogens during their natural infection process in vivo, and bacterial virulence. This study is the first to characterize the global transcriptomic and proteomic response of an opportunistic pathogen that is actually found in the space habitat, Pseudomonas aeruginosa. Overall, P. aeruginosa responded to spaceflight conditions through differential regulation of 167 genes and 28 proteins, with Hfq identified as a global transcriptional regulator in the response to this environment. Since Hfq was also induced in spaceflight-grown Salmonella typhimurium, Hfq represents the first spaceflight-induced regulator across the bacterial species border. The major P. aeruginosa virulence-related genes induced in spaceflight conditions were the lecA and lecB lectins and the rhamnosyltransferase (rhlA), involved in the production of rhamnolipids. The transcriptional response of spaceflight-grown P. aeruginosa was compared with our previous data of this organism grown in microgravity-analogue conditions using the rotating wall vessel (RWV) bioreactor technology. Interesting similarities were observed, among others with regard to Hfq regulation and oxygen utilization. While LSMMG-grown P. aeruginosa mainly induced genes involved in microaerophilic metabolism, P. aeruginosa cultured in spaceflight adopted an anaerobic mode of growth, in which denitrification was presumably most prominent. Differences in hardware between spaceflight and LSMMG experiments, in combination with more pronounced low fluid shear and mixing in spaceflight when compared to LSMMG conditions, were hypothesized to be at the origin of these observations. Collectively, our data suggest that spaceflight conditions could induce the transition of P. aeruginosa from an opportunistic organism to potential pathogen, results that are of importance for infectious disease risk assessment and prevention, both during spaceflight missions and in the clinic. This study describes the transcriptional response of P. aeruginosa PAO1 to low-Earth orbit environmental conditions. Our aim was to assess whether the microgravity environment of spaceflight could induce virulence traits in P. aeruginosa. To this end, P. aeruginosa cultures were grown in space, and the expression profile was compared with ground control samples (both in biological triplicate). Two RWV samples also examined (did not re-analyze them, only compared the outputs).

Project description:Anticipating the risk for infectious disease during space exploration and habitation is a critical factor to ensure safety, health and performance of the crewmembers. As a ubiquitous environmental organism that is occasionally part of the human flora, Pseudomonas aeruginosa could pose a health hazard for the immuno-compromised astronauts. In order to gain insights in the behavior of P. aeruginosa in spaceflight conditions, two spaceflight-analogue culture systems, i.e. the rotating wall vessel (RWV) and the random position machine (RPM), were used. Microarray analysis of P. aeruginosa PAO1 grown in the low shear modeled microgravity (LSMMG) environment of the RWV compared to the normal gravity control (NG), revealed a regulatory role for AlgU (RpoE). Specifically, P. aeruginosa cultured in LSMMG exhibited increased alginate production and up-regulation of AlgU-controlled transcripts, including those encoding stress-related proteins. This study also shows the involvement of Hfq in the LSMMG response, consistent with its previously identified role in the Salmonella LSMMG- and spaceflight response. Furthermore, cultivation in LSMMG increased heat- and oxidative stress resistance and caused a decrease in the culture oxygen transfer rate. Interestingly, the global transcriptional response of P. aeruginosa grown in the RPM was similar to that in NG. The possible role of differences in fluid mixing between the RWV and RPM is discussed, with the overall collective data favoring the RWV as the optimal model to study the LSMMG-response of suspended cells. This study represents a first step towards the identification of specific virulence mechanisms of P. aeruginosa activated in response to spaceflight-analogue conditions, and could direct future research regarding the risk assessment and prevention of Pseudomonas infections for the crew in flight and the general public. The wild type P. aeruginosa PAO1 strain (ATCC 15692) was used in this study and all cultures were grown in Lennox L Broth Base (LB) (Life Technologies) at 28 Â°C. An overnight shaking culture (125 r.p.m.) of P. aeruginosa in LB was washed and diluted in 0.85% NaCl solution to an OD600 of 1. This bacterial suspension was used to inoculate fresh LB medium at a final concentration of 10-4 CFU/ml. Synthecon Rotating Wall Vessel bioreactors (RWV) (50 ml or 10 ml) were filled with inoculated medium so that no headspace (i.e. no bubbles) was present. Other than for stress resistance assays, for which 10 ml capacity bioreactors were used, RWV bioreactors with a capacity of 50 ml were adopted for all experiments. Identical bioreactors were mounted in triplicate on (i) a RWV device in vertical position (LSMMG) (Cellon), (ii) a RWV device in horizontal position (NG) and (iii) the center of the inner Random Positioning Machine (RPM) frame (RG) (Fokker Space), and placed in a large humidified (70%-80% relative humidity) culture chamber, to avoid evaporation of culture medium through the gas-permeable membrane at the back of each vessel (Figure 1). A 25 r.p.m. rotation speed was adopted for the RWV cultures, while RPM-cultures were randomly rotated at 10 r.p.m. (60Â°/s). Bacteria were grown in the three described test conditions for 24 hours. After 24 hours of cultivation, the contents of every bioreactor was gently mixed by pipetting and divided into several aliquots. Ten millilitres of culture from each growth condition was immediately fixed with RNA Protect Reagent (Qiagen), following the manufacturer's instructions, and fixed cell pellets were frozen at -20 Â°C until RNA extraction. Samples were immediately exposed to different stresses.

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Pseudomonas aeruginosa PAO1

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