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:This study presents the first global transcriptional profiling and phenotypic characterization of the major human opportunistic fungal pathogen, Candida albicans, grown in spaceflight conditions. Microarray analysis revealed that C. albicans subjected to short-term spaceflight culture differentially regulated 454 genes compared to synchronous ground controls, which represented 8.4% of the analyzed ORFs. Spaceflight-cultured C. albicans induced genes involved in cell aggregation (similar to flocculation), which was validated by microscopic and flow cytometry analysis. We also observed enhanced random budding of spaceflight-cultured cells as opposed to more normal bipolar budding patterns for ground samples, in accordance with the gene expression data. Furthermore, genes involved in antifungal agent and stress resistance were differentially regulated in spaceflight, including induction of ABC transporters and members of the major facilitator family, downregulation of ergosterol-encoding genes, and upregulation of genes involved in oxidative stress resistance. Finally, downregulation of genes involved in the actin cytoskeleton was observed. Interestingly, the transcriptional regulator Cap1 and over 30% of the Cap1 regulon was differentially expressed in spaceflight-cultured C. albicans. A potential role for Cap1 in the spaceflight response of C. albicans is suggested, as this regulator is involved in random budding, cell aggregation, actin cytoskeleton, and oxidative stress resistance; all related to observed spaceflight-associated changes of C. albicans. While culture of C. albicans in microgravity potentiates a global change in gene expression that could induce a virulence-related phenotype, no increased virulence in a murine intraperitoneal (i.p.) infection model was observed. This study represents an important basis for the assessment of the risk that commensal flora could play during spaceflight missions. Furthermore, since the low fluid-shear environment of microgravity is relevant to physical forces encountered by pathogens during the infection process, insights gained from this study could identify novel infectious disease mechanisms, with downstream benefits for the general public. Cells were grown for 24 hours on the space shuttle or as ground-based controls, preserved in RNALater, and stored at -80C. Four samples of each flight- and ground-based controls were harvested for microarray analysis. GAP is Group Activation Pack and each GAP contains 8 FPAs. The numbers represent the # assigned to the particular GAP and the number assigned to the specific FPA (1-8) within the indicated GAP. The same hardware is used for the flight samples and the ground samples.

Project description:This study presents the first global transcriptional profiling and phenotypic characterization of the major human opportunistic fungal pathogen, Candida albicans, grown in spaceflight conditions. Microarray analysis revealed that C. albicans subjected to short-term spaceflight culture differentially regulated 454 genes compared to synchronous ground controls, which represented 8.4% of the analyzed ORFs. Spaceflight-cultured C. albicans induced genes involved in cell aggregation (similar to flocculation), which was validated by microscopic and flow cytometry analysis. We also observed enhanced random budding of spaceflight-cultured cells as opposed to more normal bipolar budding patterns for ground samples, in accordance with the gene expression data. Furthermore, genes involved in antifungal agent and stress resistance were differentially regulated in spaceflight, including induction of ABC transporters and members of the major facilitator family, downregulation of ergosterol-encoding genes, and upregulation of genes involved in oxidative stress resistance. Finally, downregulation of genes involved in the actin cytoskeleton was observed. Interestingly, the transcriptional regulator Cap1 and over 30% of the Cap1 regulon was differentially expressed in spaceflight-cultured C. albicans. A potential role for Cap1 in the spaceflight response of C. albicans is suggested, as this regulator is involved in random budding, cell aggregation, actin cytoskeleton, and oxidative stress resistance; all related to observed spaceflight-associated changes of C. albicans. While culture of C. albicans in microgravity potentiates a global change in gene expression that could induce a virulence-related phenotype, no increased virulence in a murine intraperitoneal (i.p.) infection model was observed. This study represents an important basis for the assessment of the risk that commensal flora could play during spaceflight missions. Furthermore, since the low fluid-shear environment of microgravity is relevant to physical forces encountered by pathogens during the infection process, insights gained from this study could identify novel infectious disease mechanisms, with downstream benefits for the general public.

Project description:Physical forces associated with spaceflight and spaceflight analogue culture regulate a wide range of physiological responses by both bacterial and mammalian cells that can impact infection. However, our mechanistic understanding of how these environments regulate host-pathogen interactions in humans is poorly understood. Using a spaceflight analogue low fluid shear culture system, we investigated the effect of Low Shear Modeled Microgravity (LSMMG) culture on the colonization of Salmonella Typhimurium in a 3-D biomimetic model of human colonic epithelium containing macrophages. RNA-seq profiling of stationary phase wild type and hfq mutant bacteria alone indicated that LSMMG culture induced global changes in gene expression in both strains and that the RNA-binding protein Hfq played a significant role in regulating the transcriptional response of the pathogen to LSMMG culture. However, a core set of genes important for adhesion, invasion, and motility were commonly induced in both strains. LSMMG culture enhanced the colonization (adherence, invasion and intracellular survival) of Salmonella in this advanced model of intestinal epithelium using a mechanism that was independent of Hfq. Although S. Typhimurium hfq mutants are normally defective for invasion when grown as conventional shaking cultures, LSMMG conditions unexpectedly enabled high levels of colonization by an isogenic hfq mutant. In response to infection with either the wild type or mutant, host cells upregulated transcripts involved in inflammation, tissue remodeling, and wound healing during intracellular survival. Interestingly, infection by the hfq mutant led to fewer transcriptional differences between LSMMG- and control-infected host cells relative to infection with the wild type strain. This is the first study to investigate the effect of LSMMG culture on the interaction between S. Typhimurium and a 3-D model of human intestinal tissue. These findings advance our understanding of how physical forces can impact the early stages of human enteric salmonellosis.

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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Transcriptional and proteomic response of Pseudomonas aeruginosa PAO1 to spaceflight conditions involves Hfq regulation and reveals a role for oxygen

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