Assessing the behavior and virulence potential of obligate and opportunistic pathogens aboard spacecraft and the International Space Station (ISS) is of central importance to evaluate the risk for infectious disease in the context of long-term manned missions. Furthermore, since bacteria encounter microgravity analogue low-fluid-shear forces in the host during their natural course of infection, bacterial spaceflight research can provide novel insights into the in vivo
infection process. Indeed, spaceflight increased the virulence of S.
Typhimurium, while global gene expression profiling revealed a general downregulation of key virulence genes in this pathogen (46
). The present study demonstrated for the first time that the opportunistic pathogen P. aeruginosa
responded to culture in the microgravity environment of spaceflight through differential regulation of 167 genes and 28 proteins. A significant part of the spaceflight stimulon was under the control of the RNA binding protein Hfq. Hfq is important for the virulence and stress resistance of several (opportunistic) pathogens, including P. aeruginosa
), by modulating the function and stability of small regulatory RNAs (sRNAs) and interfering with their interactions with mRNAs (reviewed in references 31
). Interestingly, Hfq was also found to be an important regulator in the responses of (i) P. aeruginosa
to microgravity analogue low-fluid-shear conditions (LSMMG, using the RWV bioreactor) and (ii) S.
Typhimurium to actual spaceflight and LSMMG conditions (13
). Hence, Hfq is the first transcriptional regulator ever shown to be commonly involved in the spaceflight and LSMMG responses of two bacterial species.
Among the P. aeruginosa
genes with the highest fold inductions under spaceflight conditions were the genes encoding the lectins LecA and LecB. Lectins bind galactosides, play a role in the bacterial adhesion process to eukaryotic cells, and are thus important virulence factors in P. aeruginosa
). P. aeruginosa
lectins have cytotoxic effects in human peripheral lymphocytes and respiratory epithelial cells in vitro
and increase alveolar barrier permeability in vivo
). Lectin production in P. aeruginosa
is regulated through the N
-homoserine lactone (C4
-HSL) quorum-sensing system (50
), which has been previously reviewed (45
). However, the downregulation of rhlI
, the gene encoding the C4
-HSL synthase, under spaceflight conditions was unexpected. Nevertheless, rhlA
, which is dependent on C4
-HSL quorum-sensing regulation and encodes the rhamnosyltransferase I involved in rhamnolipid surfactant biosynthesis, was induced during spaceflight culture. Rhamnolipids are glycolipidic surface-active molecules that have cytotoxic and immunomodulatory effects in eukaryotic cells (5
). Interestingly, rhamnolipids and rhlA
transcripts were also found in P. aeruginosa
in larger amounts under low-fluid-shear compared to higher-fluid-shear growth conditions, using the RWV bioreactor (12
). These data indicate that rhamnolipid production could be induced upon sensing of low fluid shear.
Gene expression profiles of P. aeruginosa
grown under spaceflight conditions also revealed the differential regulation of a significant fraction of genes involved in growth under oxygen-limiting conditions. Spaceflight induced mainly genes involved in anaerobic metabolism, which was reinforced by a lower expression in spaceflight samples of CcoP2, a cytochrome with high affinity for oxygen that is typically induced under microaerophilic conditions (2
). At the time of measurement, the most prominent way to cope with the apparent oxygen shortage under spaceflight conditions seemed to occur through denitrification and not through fermentation. Indeed, under oxygen-limiting conditions, P. aeruginosa
switches to anaerobic respiration in the presence of the alternative electron acceptor nitrate or nitrite (16
). The downregulation of ArcA, a protein involved in arginine fermentation, accentuates that fermentation was presumably not activated in spaceflight-grown bacteria.
When comparing the gene expression profiles of P. aeruginosa grown in spaceflight and P. aeruginosa grown in LSMMG, a limited but significant overlap was found. Besides the role of Hfq and its regulon in the response of P. aeruginosa PAO1 to both spaceflight and LSMMG (see above), a significant fraction of genes involved in both microaerophilic and anaerobic metabolism were commonly induced. In contrast to P. aeruginosa grown under spaceflight conditions, LSMMG-grown P. aeruginosa induced genes involved in arginine and pyruvate fermentation, while denitrification did not appear to play a role in the LSMMG response of this bacterium. The observation that spaceflight samples were presumably more deprived of oxygen than LSMMG-grown bacteria, compared to their respective controls, could be explained by the fact that actual spaceflight conditions are characterized by even lower fluid shear levels than LSMMG conditions. Indeed, due to the absence of convection currents in microgravity, oxygen limitation will be more pronounced in space than in LSMMG. Furthermore, the role of the experimental setup needs to be considered. As depicted in Fig. , cells grown in the bioreactors used for growth of P. aeruginosa in LSMMG and spaceflight have different oxygen availabilities. While the bioreactors have a gas-permeable membrane, the membrane surface-to-volume ratio of FPA bioreactors (used in spaceflight) is 12 times lower than that of the RWVs (LSMMG) [based on the formula πr2/(πr2 × h) or 1/h, with r = radius and h = height]. Hence, oxygen availability overall will be higher in RWVs than in the FPA devices. It also needs to be mentioned that despite differences in aeration and fluid shear between the spaceflight and LSMMG studies, the RWV mimics only certain aspects of the spaceflight environment. Indeed, enhanced irradiation and vibration or potential direct effects of microgravity (such as effects on the cell or cellular components instead of on the extracellular environment) during spaceflight could lead to differences in gene and protein expression profiles between spaceflight and LSMMG-grown P. aeruginosa. Accordingly, the RWV bioreactor was unable to mimic the complete repertoire of spaceflight-induced alterations in P. aeruginosa.
FIG. 2. Schematic representation of the different hardware used for cultivation of P. aeruginosa under microgravity analogue conditions (LSMMG versus control, using RWV bioreactors) and under spaceflight conditions (spaceflight versus ground, using FPA devices). (more ...)
Since the present study was conducted by growing P. aeruginosa
in a liquid environment under spaceflight conditions, our results are relevant mainly to the assessment of bacterial virulence in fluid niches of the spacecraft. Indeed, astronauts are in regular contact with water-containing sources that could be contaminated with P. aeruginosa
, such as drinking water, rinseless shampoo, toothpaste, mouthwash, and water for laundry. Similarly, water-related sites in the hospital environment are most likely to harbor P. aeruginosa
(e.g., faucets, showers, medication, disinfectants, mouthwash, and other hygiene products) and are at the origin of a significant number of nosocomial infections (28
). Furthermore, P. aeruginosa
is occasionally part of the normal human flora of the mouth, pharynx, anterior urethra, and lower gastrointestinal tract. In these regions of the human body, P. aeruginosa
is present in a fluid environment, which will be affected by microgravity and will presumably result in the exposure of P. aeruginosa
to lower-fluid-shear conditions than on Earth.
This study was the first to characterize the comprehensive transcriptional and translational responses of an opportunistic pathogen that is frequently found in the space habitat. We demonstrated that spaceflight conditions activated pathways in P. aeruginosa that have been shown previously to be involved in the in vivo infection process. However, the regulation of several of these pathways appears to be differentially controlled during spaceflight compared to conventional culture. Hfq was put forward as a main transcriptional regulator in the spaceflight response of P. aeruginosa, therefore representing the first transcriptional regulator commonly involved in the spaceflight responses of different bacterial species. We also identified interesting similarities and differences between P. aeruginosa grown in spaceflight and under the LSMMG conditions of the RWV. Despite the limited overlap of identical genes between spaceflight- and LSMMG-grown P. aeruginosa, it was observed that different genes of the same regulon or stimulon could be induced or downregulated in spaceflight and LSMMG. The experimental setup was proposed as one of the putative factors at the origin of the oxygen-related transcriptional differences between LSMMG culture in the RWV bioreactor and spaceflight-cultured P. aeruginosa in the FPAs. These data emphasize the importance of using identical hardware for spaceflight experiments and ground simulations, especially when oxygen is a limiting factor. In addition, differences in fluid shear and other environmental conditions (such as irradiation) between actual microgravity and LSMMG need to be considered when comparing bacterial responses to the two test conditions. This study represents an important step in understanding the response of bacterial opportunistic pathogens to the unique spaceflight environment. Furthermore, it allows assessment of the role that low-fluid-shear regions found in the human body play in the regulation of bacterial virulence. It remains to be determined whether the phenotype of P. aeruginosa acquired under spaceflight conditions will effectively lead to increased pathogenicity, as was observed for S. Typhimurium. This will be an important consideration and key area of future study in order to further assess the risk for infectious disease during long-term missions.