Asparagine biosynthesis as a mechanism of increased host lethality induced by Serratia marcescens in simulated microgravity environments

Abstract

While studies have shown an increase in pathogenicity in several microbes during spaceflight and after exposure to simulated microgravity, the mechanisms underlying these changes in phenotype are not understood across different pathogens, particularly in opportunistic pathogens. This study evaluates the mechanism for increased virulence of the opportunistic gram-negative bacterium, Serratia marcescens, in simulated microgravity. Low-shear modeled microgravity (LSMMG) is used in ground-based studies to simulate the effects of microgravity as experienced in spaceflight. Our previous findings showed that there was a significant increase in mortality rates of the Drosophila melanogaster host when infected with either spaceflight or LSMMG treated S. marcescens. Here, we report that LSMMG increases asparagine uptake and synthesis in S. marcescens and that the increased host lethality induced by LSMMG bacteria grown in rich media can be recapitulated in minimal media by adding only aspartate and glutamine, the substrates of asparagine biosynthesis. Interestingly, increased bacterial growth rate alone is not sufficient to contribute to maximal host lethality, since the addition of aspartate to minimal media caused an LSMMG-specific increase in bacterial growth rate that is comparable to that induced by the combination of aspartate and glutamine, but this increase in growth does not cause an equivalent rate of host mortality. However, the addition of both aspartate and glutamine cause both an increase in host mortality and an overexpression of asparagine pathway genes in a LSMMG-dependent manner. We also report that L-asparaginase-mediated breakdown of asparagine is an effective countermeasure for the increased host mortality caused by LSMMG-treated bacteria. This investigation underscores the importance of the asparagine utilization pathway by helping uncover molecular mechanisms that underlie increased mortality rates of a model host infected with microgravity-treated S. marcescens and provides a potential mitigation strategy.

Keywords: Asparagine; Drosophila melanogaster; Low shear modeled microgravity; Serratia marcescens; Virulence.

Figure 1

The asparagine pathway and increased uptake of extracellular asparagine and glutamine from nutrient-rich LB media under simulated microgravity. Measurements of amino acid consumption were performed by removing cells from media by centrifugation and utilizing spectrophotometric analysis with Megazyme™ reagent. A) The asparagine biosynthesis pathway. B) At the end of the growth period of S. marcescens in simulated microgravity (LSMMG orientation), gene expression for each sample was measured via qPCR: L-ap (L-asparaginase), L-ap1 (L-asparaginase 1), L-ap2 (L-asparaginase 2), asnB (asparagine synthetase B), iaaA (isoaspartyl peptidase/L-asparaginase), or astL (asparagine tRNA ligase). Expression values are reported as fold change relative to the RWV-Control orientation and calculated using the ΔΔ Ct method. Specific genes represented by the blue end of the color spectrum indicated gene overexpression and those towards the red end indicated inhibition of gene expression. Error bars represent one standard error. Measurements of C) extracellular asparagine and D) extracellular glutamine were performed at hours 0, 6, and 14 of growth in the rotating wall vessel, with an initial starting concentration of 1 × 10⁸ CFU.

Figure 2

Exogenously added L-asparaginase can inhibit growth of LSMMG-treated bacteria and reduce host-lethality. A) The breakdown of asparagine catalyzed by the asparaginase enzyme. B) The starting concentration of 1 × 10⁸ CFU of S. marcescens was placed in a 10 mL rotating wall vessel at 37 °C in LB media. One hundred microliters of L-asparaginase was added to the growth media, and growth of bacteria estimated by absorbance at 600 nm was measured every 3 h. L-asparaginase addition in vitro reduces the rate of growth most significantly in the LSMMG samples. Error bars represent one standard error. C and D) Bacteria were grown in RWV with LB media only. After 24 h of growth, bacteria were fixed in 20% glycerol for injections. Flies were injected with S. marcescens. One hour after infection, flies were then injected with a treatment of L-asparaginase, or sterile water (no treatment). The LSMMG bacteria kills flies at a lower rate when L-asparaginase is injected in vivo (C) and similarly in vivo injected L-asparaginase does not impact survival of the RWV-Control (D). (The statistical results for the comparisons shown in Figures 2C and 2D are included in Table 1.)

Figure 3

Growth and gene expression changes of LSMMG-treated S. marcescens in Davis minimal media supplemented with amino acids. Starting concentration of 1 × 10⁸ CFU of S. marcescens was placed in a 10 mL rotating wall vessel at 37 °C, containing A) Davis minimal media only, B) Davis minimal media supplemented with 10 mM aspartate and 10 mM glutamine, C) Davis minimal media supplemented with 10 mM glutamine, and D) Davis minimal media supplemented with 10 mM aspartate. Growth was measured every 24 h for 72 h. E) At the end of growth period, gene expression for each sample was measured via qPCR:: L-ap (L-asparaginase), L-ap1 (L-asparaginase 1), L-ap2 (L-asparaginase 2), asnB (asparagine synthetase B), iaaA (isoaspartyl peptidase/L-asparaginase), or astL (asparagine tRNA ligase). Expression values are reported as fold change in LSMMG relative to the Davis minimal media values and calculated using the ΔΔ Ct method. Even though aspartate addition increases growth in LSMMG cultures but asnB and L-ap genes are overexpressed in LSMMG compared to control only when both glutamine and aspartate are present.

Figure 4

Infection of flies with S. marcescens shows maximum host mortality with LSMMG bacteria when supplemented with both aspartate and glutamine. Sample was grown in A) Davis minimal media (DMM) either alone or with 10 mM aspartate and 10 mM glutamine, B) Davis minimal media supplemented with 10 mM aspartate, C) Davis minimal media supplemented with 10 mM glutamine. Bacteria were grown for 24 h in the RWV, then fixed in 20% glycerol for injections into D. melanogaster hosts. Both glutamine and aspartate must be present together to elicit the maximum LSMMG-induced increase in host mortality as shown in Figure 4A. (The full statistical results for all comparisons can be seen in Table 2.)