Analysis of Pseudomonas aeruginosa virulence in modeled microgravity conditions

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Montana State University - Bozeman, College of Letters & Science


Spaceflight conditions may enhance bacterial growth, alter antimicrobial susceptibility and possibly affect virulence. Since spaceflight causes astronauts to experience secondary immunosuppression, increased virulence would increase the risk of infection. Limited opportunities for spaceflight experiments necessitate ground-based simulations. Clinostats and rotating wall vessel bioreactors, e.g. High Aspect Ratio Vessels (HARVs) are used to simulate microgravity effects at 1xg. Clinostat and HARV rotation on the horizontal axis results in the g-vector being time averaged to near zero. In controls rotated on the vertical axis, the g-vector acts on cells, as in static cultures. Salmonella enterica serovar Typhimurium virulence genes are up-regulated in modeled microgravity (MMG); a MMG "regulon" has been postulated. We hypothesize that the virulence of P. aeruginosa (PA) may also be affected by microgravity, which could be observed in MMG. This study focused on the regulation of Exotoxin A (ETA) in PA during growth in MMG. PA103 was grown in ETA medium at 37oC in Clinostat-MMG syringes incubated and sampled at intervals up to 24 h at 15 RPM; and also in HARV MMG bioreactors incubated for time zero, 12 h and 24 h at 15 RPM.
MMG samples were rotated on the horizontal axis and gravity controls were rotated on the vertical axis or static position. Agar plate counts (PC) were done on R2A. ETA was monitored by ELISA. Protein expression in clinostat and HARV samples was assayed by 2-dimensional gel analyses. MMG did not affect ETA production in the Clinostat or HARV (horizontal vs. vertical rotation), whereas, PC showed growth in MMG was comparable to controls. Clinostat and HARV experiments indicated that MMG had negligible effects on ETA production. However, HARV cultures gave much lower ETA production in either MMG or horizontal control rotation compared to static controls. In addition, formulation of the growth medium (presence/absence of nitrate) and culture aeration had dramatic effects on ETA production. These differences should be taken into account in future ground and flight experiments. Analysis of 2-D gels from preliminary experiments was inconclusive. This study indicates that MMG has a minor effect on the growth of P. aeruginosa PA103 under these conditions.




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