Modeled microgravity alters lipopolysaccharide and outer membrane vesicle production of the beneficial symbiont Vibrio fischeri

Reduced gravity, or microgravity, can have a pronounced impact on the physiology of animals, but the effects on their associated microbiomes are not well understood. Here, the impact of modeled microgravity on the shedding of Gram-negative lipopolysaccharides (LPS) by the symbiotic bacterium Vibrio fischeri was examined using high-aspect ratio vessels. LPS from V. fischeri is known to induce developmental apoptosis within its symbiotic tissues, which is accelerated under modeled microgravity conditions. In this study, we provide evidence that exposure to modeled microgravity increases the amount of LPS released by the bacterial symbiont in vitro. The higher rates of shedding under modeled microgravity conditions are associated with increased production of outer-membrane vesicles (OMV), which has been previously correlated to flagellar motility. Mutants of V. fischeri defective in the production and rotation of their flagella show significant decreases in LPS shedding in all treatments, but levels of LPS are higher under modeled microgravity despite loss of motility. Modeled microgravity also appears to affect the outer-membrane integrity of V. fischeri, as cells incubated under modeled microgravity conditions are more susceptible to cell-membrane-disrupting agents. These results suggest that, like their animal hosts, the physiology of symbiotic microbes can be altered under microgravity-like conditions, which may have important implications for host health during spaceflight.

ACTIN CYTOSKELETON ORGANIZATION IN OOCYTES AT VARIOUS STAGES OF DROSOPHILA MELANOGASTER OOGENESIS AFTER EXPOSURE IN A MICROGRAVITY SIMULATOR OVER THE COMPLETE GAMETOGENESIS CYCLE

The paper deals with the effects of modeled microgravity on actin cytoskeleton in oocytes at various stages of Drosophila melanogaster oogenesis over the complete gametogenesis cycle. Total actin content and F-actin singly was determined using immunohistochemical analysis. The results point to the growth of both total beta-actin and its polymer recognized by phalloidin. This finding can have key implications for evaluation of risks for the reproductive potential from the spaceflight factors.

STROME-DEPENDENT EXPANSION OF HEMOPOIETIC PRECURSORS DURING 7-DAY MODELING OF THE EFFECTS OF MICROGRAVITY

It is well known that long-term space missions influence various myeloid shoots of hemopoiesis in humans and animals. In this investigation, we used several optimized models of co-culturing mesenchymal stromal cells (MSCs) and hemopoietic stem and progenitor cells (HSPCs) to analyze the HSPCs suspension fractions following 7-day modeling of the effects of microgravity. We determined the number of developing HSPCs, presence of colony-forming units (CFU) of various hemopoietic shoots and phenotype spectrum of cells in colonies. We observed an increased number of HSPCs, decreased number of CFUs, and changes in the structure of CFU population after the exposure to modeled microgravity.

Blockade of IL-6 alleviates bone loss induced by modeled microgravity in mice

This study investigated the effects of blockade of IL-6 on bone loss induced by modeled microgravity (MG). Adult male mice were exposed to hind-limb suspension (HLS) and treated with IL-6-neutralizing antibody (IL-6 nAb) for 4 weeks. HLS in mice led to upregulation of IL-6 expression in both sera and femurs. IL-6 nAb treatment in HLS mice significantly alleviated bone loss, evidenced by increased bone mineral density of whole tibia, trabecular thickness and number, bone volume fraction of proximal tibiae, and ultimate load and stiffness of femoral diaphysis. IL-6 nAb treatment in HLS mice significantly enhanced levels of osteocalcin in sera and reduced levels of deoxypyridinoline. In MC3T3-E1 cells exposed to MG in vitro, IL-6 nAb treatment increased mRNA expression and activity of alkaline phosphatase, mRNA expression of osteopontin and runt-related transcription factor 2, and protein levels of osteoprotegerin and decreased protein levels of receptor activator of the NF-κB ligand. In RAW254.7 cells exposed to MG, IL-6 nAb treatment downregulated mRNA expression of cathepsin K and tartrate-resistant acid phosphatase (TRAP) and reduced numbers of TRAP-positive multinucleated osteoclasts. In conclusion, blockade of IL-6 alleviated the bone loss induced by MG.

Bacterial gene essentiality under modeled microgravity

ABSTRACTThe health of eukaryotic hosts is tightly connected to relationships with symbiotic microorganisms, yet how these relationships develop and evolve during long-duration spaceflight is not well understood. In this study, we asked what bacterial genes are required for growth under modeled, or simulated, microgravity conditions compared to normal gravity controls. To conduct this study, we focused on the marine bacterium Vibrio fischeri, which forms a monospecific symbiosis with the Hawaiian bobtail squid, Euprymna scolopes. The symbiosis has been studied during spaceflight and in ground-based modeled microgravity conditions. We employed a library of over 40,000 V. fischeri transposon mutants and compared the fitness of mutants in modeled microgravity compared to the gravity controls using transposon insertion sequencing (INSeq). We identified dozens of genes that exhibited fitness defects under both conditions, likely due to the controlled anaerobic environment, yet we identified relatively few genes with differential effects under modeled microgravity or gravity specifically: only mutants in rodA were more depleted under modeled microgravity, and mutants in 12 genes exhibited greater depletion under gravity conditions. We additionally compared RNA-seq and INSeq data and determined that expression under microgravity was not predictive of the essentiality of a given gene. In summary, empirical determination of conditional gene essentiality identifies few microgravity-specific genes for environmental growth of V. fischeri, suggesting that the condition of microgravity has a minimal impact on symbiont gene requirement.IMPORTANCEThere is substantial evidence that both the host immune system and microbial physiology are altered during space travel. It is difficult to discern the molecular mechanisms of these processes in a complex microbial consortium and during the short durations of experiments in space. By using a model organism that is amenable to high-throughput genetic approaches, we have determined that V. fischeri does not require a separate genetic repertoire for media growth in modeled microgravity versus gravity conditions. Our results argue that future studies on how this organism forms a specific and stable association with its animal host will not be confounded by growth effects in the environment. The identification of similar genetic requirements under modeled microgravity and gravity suggest that fitness pressures on microbiome growth in space may be similar to those on Earth and may not negatively impact their animal hosts during long-duration spaceflight.

Effects of Microgravity and Clinorotation on the Virulence of Klebsiella, Streptococcus, Proteus, and Pseudomonas

To evaluate effects of microgravity on virulence, we studied the ability of four common clinical pathogens—Klebsiella, Streptococcus, Proteus, and Pseudomonas—to kill wild type Caenorhabditis elegans (C. elegans) nematodes at the larval and adult stages. Simultaneous studies were performed utilizing spaceflight, rotation in a 2D clinorotation device, and static ground controls. Nematodes, microbes, and growth media were separated until exposed to true or modeled microgravity, then mixed and grown for 48 hours. Experiments were terminated by paraformaldehyde fixation, and optical density measurements were used to assay residual microorganisms. Spaceflight was associated with reduced virulence for Klebsiella and Streptococcus, but had negligible effect on Enterococcus and Pseudomonas. Clinorotation generated very different results with all four organisms showing significantly reduced virulence. We conclude that clinorotation is not a consistent model of the changes that actually occur under microgravity conditions. Further, bacteria virulence is unchanged or reduced, not increased during spaceflight.

Treatment with hydrogen sulfide donor attenuates bone loss induced by modeled microgravity

The present study was undertaken to explore the therapeutic potential of hydrogen sulfide against bone loss induced by modeled microgravity. Hindlimb suspension (HLS) and rotary wall vessel bioreactor were applied to model microgravity in vivo and in vitro, respectively. Treatment of rats with GYY4137 (a water soluble donor of hydrogen sulfide, 25 mg/kg per day, i.p.) attenuated HLS-induced reduction of bone mineral density in tibiae, and preserved bone structure in tibiae and mechanical strength in femurs. In HLS group, GYY4137 treatment significantly increased levels of osteocalcin in sera. Interestingly, treatment of HLS rats with GYY4137 enhanced osteoblast surface, but had no significant effect on osteoclast surface of proximal tibiae. In MC3T3-E1 cells exposed to modeled microgravity, GYY4137 stimulated transcriptional levels of runt-related transcription factor 2 and enhanced osteoblastic differentiation, as evidenced by increased mRNA expression and activity of alkaline phosphatase. HLS in rats led to enhanced levels of interleukin 6 in sera, skeletal muscle, and tibiae, which could be attenuated by GYY4137 treatment. Our study showed that GYY4137 preserved bone structure in rats exposed to HLS and promoted osteoblastic differentiation in MC3T3-E1 cells under modeled microgravity.