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Low-shear modeled microgravity alters virulence of Serratia marcescens in Drosophila melanogaster

Victor H. Quach1,2, William Wade3, Sharmila Bhattacharya3 1SLSTP Research Associate, Space Biosciences Division, NASA Ames Research Center, 2University of California, Santa Barbara – Department of Chemistry and Biochemistry

3Space Biosciences Research Branch, NASA Ames Research Center

Abstract Astronauts are exposed to prolonged durations of microgravity during spaceflight, leading to a wide variety of metabolic changes, bone loss, and decline in immune function. Additionally microgravity induces a variety of phenotypic changes in bacterial pathogens ranging from enhanced virulence to altered biofilm production. Given the limitations of healthcare that astronauts are able to receive in space, it is extremely important to understand these phenotypic changes in pathogens in order to improve crew health and ensure mission success. Serratia marcescens is an opportunistic entomopathogenic bacterium to humans that has been previously shown to be lethal to Drosophila melanogaster. The Rotating Wall Vessel (RWV) is a ground based low-shear model of microgravity (LSMMG), which has previously been used to mimic changes in bacteria phenotype observed during spaceflight. The goal of this project is to assess the impact of LSMMG on the virulence of Serratia marcescens on the model organism Drosophila melanogaster. S. marcescens will be grown in the RWV, then injected directly into the coelom of D. melanogaster via entrance through the terminal ventral abdomen near abdominal sternite 5 & 6. D.melanogaster survival will then be monitored in static intervals following the injection event along with bacterial load of the flies over time. Mutant fly lines deficient in important immune pathway genes (relish, spatzle) will also be examined to both determine important virulence pathways for S. marcescens in Drosophila and mimic the potentially weakened immune system of the host as seen in microgravity. This project aims to further the understanding of host pathogen interactions by studying a bacterial and model host combination that has yet to be studied under altered gravity conditions. Strategies can then be developed to counteract these virulence-enhancing mechanisms, providing novel treatments and helping to prevent bacterial disease both in space and here on Earth.

ABSTRACT

METHODS

RESULTS

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Rotating Wall Vessel (RWV)

Figure 1: Bacteria is grown for 12 or 24 hours inside the rotating wall vessel.

Figure 2: Drosophila are injected in the area near abdominal sternite 5 & 6 with a bolus diamter of 5 µm as measured using a micrometer.

1.  Wilson, et. al (2007) Space flight alters bacterial gene expression and virulence and reveals a role for global regulator Hfq.

2.  Nehme (2007) A model of Bacterial Intestinal Infection in Drosophila melanogaster. 3.  Kurz (2003) Virulence factors of the human opportunistic pathogen Serratia marcescens

identified by in vivo screening. 4.  Wilson, et. Al (2008) Meidia Ion Composition Controls Regulatory and Virulence

Response of Salmonella in Spaceflight.

Figure 5, Survival of Drosophila: Drosophila were injected with a bolus diameter of 5µm with bacteria grown in the RWV for 12 hours. Survival was then monitored post-injection. 3 replicates were performed, each with a sample size of n=20. PBS injections were used as a negative control.

Figure 6, Bacterial Load: 3 Drosophila were homogenized in 100µL PBS for each time point and plated on LB.

Figure 3, Survival of Drosophila: Drosophila were injected with a bolus diameter of 5µm with bacteria grown in the RWV for 12 hours. Survival was then monitored post-injection. 3 replicates were performed, each with a sample size of n=20. PBS injections were used as a negative control.

Figure 7, Survival of Drosophila: Drosophila were injected with a bolus diameter of 5µm with bacteria grown in the RWV for 24 hours. Survival was then monitored post-injection. 3 replicates were performed, each with a sample size of n=20. PBS injections were used as a negative control. Growing bacteria for 24 hours appeared to cause a significant difference in survival between 1g and µg conditions with P<0.0001 in a log-rank test.

REFERENCES

Figure 4, Bacterial Load: 3 Drosophila were homogenized in 100µL PBS for each time point and plated on LB.