Growth medium-dependent antimicrobial activity of early stage MEP pathway inhibitors

Abstract

The in vivo microenvironment of bacterial pathogens is often characterized by nutrient limitation. Consequently, conventional rich in vitro culture conditions used widely to evaluate antibacterial agents are often poorly predictive of in vivo activity, especially for agents targeting metabolic pathways. In one such pathway, the methylerythritol phosphate (MEP) pathway, which is essential for production of isoprenoids in bacterial pathogens, relatively little is known about the influence of growth environment on antibacterial properties of inhibitors targeting enzymes in this pathway. The early steps of the MEP pathway are catalyzed by 1-deoxy-d-xylulose 5-phosphate (DXP) synthase and reductoisomerase (IspC). The in vitro antibacterial efficacy of the DXP synthase inhibitor butylacetylphosphonate (BAP) was recently reported to be strongly dependent upon growth medium, with high potency observed under nutrient limitation and exceedingly weak activity in nutrient-rich conditions. In contrast, the well-known IspC inhibitor fosmidomycin has potent antibacterial activity in nutrient-rich conditions, but to date, its efficacy had not been explored under more relevant nutrient-limited conditions. The goal of this work was to thoroughly characterize the effects of BAP and fosmidomycin on bacterial cells under varied growth conditions. In this work, we show that activities of both inhibitors, alone and in combination, are strongly dependent upon growth medium, with differences in cellular uptake contributing to variance in potency of both agents. Fosmidomycin is dissimilar to BAP in that it displays relatively weaker activity in nutrient-limited compared to nutrient-rich conditions. Interestingly, while it has been generally accepted that fosmidomycin activity depends upon expression of the GlpT transporter, our results indicate for the first time that fosmidomycin can enter cells by an alternative mechanism under nutrient limitation. Finally, we show that the potency and relationship of the BAP-fosmidomycin combination also depends upon the growth medium, revealing a striking loss of BAP-fosmidomycin synergy under nutrient limitation. This change in BAP-fosmidomycin relationship suggests a shift in the metabolic and/or regulatory networks surrounding DXP accompanying the change in growth medium, the understanding of which could significantly impact targeting strategies against this pathway. More generally, our findings emphasize the importance of considering physiologically relevant growth conditions for predicting the antibacterial potential MEP pathway inhibitors and for studies of their intracellular targets.

Systematic measurement of combination-drug landscapes to predict in vivo treatment outcomes for tuberculosis

“…The tendency toward antagonism depended on the growth model, with some conditions showing a balance between synergy and antagonism (intracellular and acidic) and all other conditions showing statistically significant antagonism (one-sample t test, mu = 0, p < 0.05, adjusted for multiple hypothesis testing; Figure S3B). Our findings are consistent with those from other organisms and adds to a growing body of literature that suggests synergy is a property of both drug and growth environment rather than an intrinsic property of drugs in combination (Belanger et al, 2020;Sanders et al, 2018;Dillon et al, 2019;Cokol et al, 2018).…”

Toward Understanding the Chemistry and Biology of 1-Deoxy-d-xylulose 5-Phosphate (DXP) Synthase: A Unique Antimicrobial Target at the Heart of Bacterial Metabolism

“…47 As for other inhibitors of central metabolism, assay growth medium profoundly impacts the activity of DXPS inhibitors. For example, BAP is >1,000-fold more potent against bacterial pathogens in defined, minimal medium than in rich medium, 43,48 owing to differences in cellular entry and shifts in the relative requirements for isoprenoids, ThDP, and PLP in different growth environments.…”

Copper intoxication in group BStreptococcustriggers transcriptional activation of thecopoperon that contributes to enhanced virulence during acute infection

“…To more precisely define the toxicity effects of Cu towards GBS, we performed time-kill curves using 1-5 x 10 6 CFU/mL exposed to Cu concentrations ranging between 50 μM and 1 mM. We used a minimal Chemically-Defined Medium (CDM) in the context of prior studies of antibacterial activity in minimal media (21, 22), that antibacterial activity in such assays can be strongly dependent on the culture media (23, 24), and to minimize cell-protective effects that can result from some buffering agents, such as glutathione (15). We found potent killing effects that depended on both concentration and time with ≥ 0.5 mM Cu significantly killing WT GBS after 6h of exposure (Figure 4, WT); after 24h, there was a ~230-fold reduction in CFU/mL at 0.5 mM Cu (0 mM Cu = 5.6 Log 10 CFU/mL, 0.5 mM CFU/mL = 3.2 Log 10 CFU/mL), and no viable GBS remained in cultures exposure to 1 mM Cu.…”

“…To more precisely define the toxicity effects of Cu towards GBS, we performed time-kill curves using 1-5 x 10 6 CFU/mL exposed to Cu concentrations ranging between 50 M and 1 mM. We used a minimal Chemically-Defined Medium (CDM) in the context of prior studies of antibacterial activity in minimal media (21,22), that antibacterial activity in such assays can be strongly dependent on the culture media (23,24), and to minimize cell-. CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.…”

“…We examined the effects of Cu towards survival of GBS in nutrient limited conditions by using a minimal Chemically-Defined Medium (CDM), in consideration of prior studies of antibacterial activity in minimal media (21,22), the influence of culture media (23,24), and possible cell-protective effects from buffering agents, e.g. glutathione (15).…”