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

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.

细菌病原体的体内微环境通常以营养限制为核心特征。因此，广泛用于抗菌剂活性评价的常规富营养体外培养体系，往往难以准确预测其体内活性，对于靶向代谢通路的抗菌剂而言这一问题尤为突出。在这类代谢通路中，甲基赤藓糖磷酸（MEP）途径是细菌病原体合成类异戊二烯的必需通路，目前人们对生长环境如何影响靶向该通路酶类的抑制剂的抗菌活性的认知仍相对有限。MEP通路的早期步骤由1-脱氧-D-木酮糖5-磷酸（DXP）合酶与还原异构酶（IspC）催化。此前有研究报道，DXP合酶抑制剂丁酰基膦酸（BAP）的体外抗菌活性强烈依赖于培养基组成：在营养限制条件下其抑菌效力极强，而在富营养环境中活性极弱。与之相反，经典的IspC抑制剂膦霉素在富营养条件下具有强效抗菌活性，但迄今为止，尚未在更具生理相关性的营养限制条件下探究其抗菌效力。本研究的目标是全面表征BAP与膦霉素在不同生长条件下对细菌细胞的作用效果。本研究发现，两种抑制剂单独及联合使用时的活性均强烈依赖于培养基组成，细胞摄取效率的差异是二者抑菌效力存在差异的重要原因。与BAP不同，膦霉素在营养限制条件下的活性相较富营养条件下更弱。有趣的是，尽管学界普遍认为膦霉素的活性依赖于GlpT转运蛋白的表达，但我们的研究首次证实，在营养限制条件下，膦霉素可通过另一种替代机制进入细菌细胞。最后，我们还发现BAP与膦霉素联合使用的效力及其相互作用同样取决于培养基：在营养限制条件下，二者的协同效应显著消失。这种相互作用的变化表明，伴随生长环境的改变，围绕DXP的代谢及/或调控网络发生了偏移，对这一过程的理解可显著优化针对该通路的抗菌靶向策略。更广泛而言，我们的研究结果强调，在预测甲基赤藓糖磷酸通路抑制剂的抗菌潜力，以及研究其细胞内靶点时，考虑生理相关生长条件的重要性。