Online_appendix – Supplemental material for Living a Life Full of Pain: Older Pain Clinic Patients’ Experience of Living With Chronic Low Back Pain

<p>Supplemental material, Online_appendix for Living a Life Full of Pain: Older Pain Clinic Patients’ Experience of Living With Chronic Low Back Pain by Meredith Stensland and Sara Sanders in Qualitative Health Research</p

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

<div><p>The <i>in vivo</i> microenvironment of bacterial pathogens is often characterized by nutrient limitation. Consequently, conventional rich <i>in vitro</i> culture conditions used widely to evaluate antibacterial agents are often poorly predictive of <i>in vivo</i> 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.</p></div

Fosmidomycin antibacterial activity in GlpT deficient <i>E</i>. <i>coli</i>.

<p>GlpT transporter-containing (■ BW25113) and deficient ( Δ<i>glpT</i> BW25113) <i>E</i>. <i>coli</i> strains treated with 2800 μM (512 μg/mL) fosmidomycin in CAMHB (a), M9-glucose (b), and M9-glycerol (c) growth medium. Cell growth was assessed at 16 h (CAMHB, M9-glucose) or 40 h (M9-glycerol) to ensure culture saturation. The large MIC shift between the parent and GlpT deficient strain in CAMHB and M9-glycerol media indicate that GlpT is a fosmidomycin transporter in these growth conditions. (n = 3, error bars represent standard error).</p

Checkerboard analysis to assess drug interaction between BAP and fosmidomycin.

<p>a) Representative heat plot showing a synergistic relationship between BAP (5400 μM, 1000 μg/mL) and fosmidomycin (64 μM, 12 μg/mL) in CAMHB growth medium (figure reproduced with permission, [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197638#pone.0197638.ref023" target="_blank">23</a>]). b) Representative heat plot showing an indifferent relationship between BAP (5 μM, 1 μg/mL) and fosmidomycin (340 μM, 62 μg/mL) in M9-glucose minimal medium with an FIC index range of 1–1.25. The most extreme FIC index value is reported above each heat plot.</p

BAP is bacteriostatic.

<p><i>E</i>. <i>coli</i> cultures at an initial inoculum density of 10⁶ CFU/mL in M9-glucose were incubated in the presence of BAP () at 4 × MIC (80 μM, or 15 μg/mL), compared to control in the absence of BAP (■) in biological triplicate. Enumeration of bacteria on agar plates over time (0, 2, 4, 6, and 20 h) indicates that BAP is bacteriostatic. (n = 3, error bars represent standard error).</p

BAP accumulation in <i>E</i>. <i>coli</i>.

<p><i>E</i>. <i>coli</i> treated with 250 μM (47 μg/mL) or 1250 μM (233 μg/mL) BAP in CAMHB (■) or M9-glucose () medium. Intracellular BAP accumulation was monitored by LC-MS (SRM method). BAP uptake is robust and dose-dependent in M9-glucose medium, and poor in CAMHB medium. (n = 3, error bars are standard error, <i>p</i>-values were calculated using an unpaired, 2-sample t-test).</p

Minimum inhibitory concentration (MIC) of fosmidomycin against pathogenic bacteria grown in CAMHB or M9-glucose minimal medium.

<p>Minimum inhibitory concentration (MIC) of fosmidomycin against pathogenic bacteria grown in CAMHB or M9-glucose minimal medium.</p

Fosmidomycin accumulation in <i>E</i>. <i>coli</i>.

<p><i>E</i>. <i>coli</i> was treated with 110 μM (20 μg/mL) or 550 μM (100 μg/mL) fosmidomycin in CAMHB (■) or M9-glucose () medium. Intracellular fosmidomycin accumulation was monitored by LC-MS (SRM method). Fosmidomycin uptake is robust and dose-dependent in CAMHB medium, and poor in M9-glucose medium. (n = 3, error bars are standard error, <i>p</i>-values above charts were calculated using an unpaired, 2-sample t-test).</p

Two early stage MEP pathway inhibitors and their targets are shown in the context of the <i>E</i>. <i>coli</i> branchpoint metabolite, DXP.

<p>Butylacetylphosphonate (BAP) is an inhibitor of DXP synthase, and fosmidomycin is an inhibitor of IspC, the first committed step in isoprenoid biosynthesis. (Pi = PO₄²⁻).</p

Challenges and Hallmarks of Establishing Alkylacetylphosphonates as Probes of Bacterial 1‑Deoxy‑d‑xylulose 5‑Phosphate Synthase

1-Deoxy-d-xylulose 5-phosphate (DXP) synthase catalyzes the thiamin diphosphate (ThDP)-dependent formation of DXP from pyruvate and d-glyceraldehyde 3-phosphate. DXP is at a metabolic branch point in bacteria, feeding into the methylerythritol phosphate pathway to indispensable isoprenoids and acting as a precursor for biosynthesis of essential cofactors in central metabolism, pyridoxal phosphate and ThDP, the latter of which is also required for DXP synthase catalysis. DXP synthase follows a unique random sequential mechanism and possesses an unusually large active site. These features have guided the design of sterically demanding alkylacetylphosphonates (alkylAPs) toward the development of selective DXP synthase inhibitors. alkylAPs studied here display selective, low μM inhibitory activity against DXP synthase. They are weak inhibitors of bacterial growth in standard nutrient rich conditions. However, bacteria are significantly sensitized to most alkylAPs in defined minimal growth medium, with minimal inhibitory concentrations (MICs) ranging from low μM to low mM and influenced by alkyl-chain length. The longest analog (C₈) displays the weakest antimicrobial activity and is a substrate for efflux via AcrAB-TolC. The dependence of inhibitor potency on growth environment emphasizes the need for antimicrobial screening conditions that are relevant to the <i>in vivo</i> microbial microenvironment during infection. DXP synthase expression and thiamin supplementation studies offer support for DXP synthase as an intracellular target for some alkylAPs and reveal both the challenges and intriguing aspects of these approaches to study target engagement