Brokaw Alyssa M, Eide Benjamin J, Muradian Michael, Boster Joshua M, Tischler Anna D
Department of Microbiology and Immunology, University of Minnesota, Minneapolis, MN, United States.
Front Microbiol. 2017 Dec 18;8:2523. doi: 10.3389/fmicb.2017.02523. eCollection 2017.
Many bacteria regulate gene expression in response to phosphate availability using a two-component signal transduction system, the activity of which is controlled by interaction with the Pst phosphate specific transporter and a cytoplasmic protein PhoU. , the causative agent of tuberculosis, requires its phosphate sensing signal transduction system for virulence and antibiotic tolerance, but the molecular mechanisms of phosphate sensing remain poorly characterized. serves as a model for studying mycobacterial pathogens including encodes two proteins with similarity to PhoU, but it was unknown if both proteins participated in signal transduction with the phosphate-responsive SenX3-RegX3 two-component system. We constructed single and double deletion mutants and tested expression of genes in the RegX3 regulon. Only the ΔΔ mutant exhibited constitutive activation of all the RegX3-regulated genes examined, suggesting that PhoU1 and PhoU2 have overlapping functions in inhibiting activity of the SenX3-RegX3 two-component system when phosphate is readily available. The ΔΔ mutant also exhibited decreased tolerance to several anti-tubercular drugs. However, a complex plasmid swapping strategy was required to generate the ΔΔ mutant, suggesting that either or is essential for growth of . Using whole-genome sequencing, we demonstrated that all five of the ΔΔ mutants we isolated had independent suppressor mutations predicted to disrupt the function of the Pst phosphate transporter, suggesting that in the absence of the PhoU proteins phosphate uptake by the Pst system is toxic. Collectively, our data demonstrate that the two PhoU orthologs have overlapping functions in both controlling SenX3-RegX3 activity in response to phosphate availability and regulating phosphate transport by the Pst system. Our results suggest that can serve as a tractable model for further characterization of the molecular mechanism of phosphate sensing in mycobacteria and to screen for compounds that would interfere with signal transduction and thereby increase the efficacy of existing anti-tubercular antibiotics.
许多细菌利用双组分信号转导系统来响应磷酸盐的可利用性调节基因表达，该系统的活性通过与Pst磷酸盐特异性转运体和细胞质蛋白PhoU相互作用来控制。结核分枝杆菌，即结核病的病原体，其毒力和抗生素耐受性需要其磷酸盐感应信号转导系统，但磷酸盐感应的分子机制仍未得到充分表征。作为研究包括结核分枝杆菌在内的分枝杆菌病原体的模型，编码两种与PhoU相似的蛋白质，但尚不清楚这两种蛋白质是否都参与了与磷酸盐响应的SenX3-RegX3双组分系统的信号转导。我们构建了单缺失和双缺失突变体，并测试了RegX3调控子中基因的表达。只有ΔΔ突变体表现出所有检测的RegX3调控基因的组成型激活，这表明当磷酸盐容易获得时，结核分枝杆菌的PhoU1和PhoU2在抑制SenX3-RegX3双组分系统的活性方面具有重叠功能。ΔΔ突变体对几种抗结核药物的耐受性也降低。然而，需要一种复杂的质粒交换策略来产生ΔΔ突变体，这表明要么是PhoU1要么是PhoU2对结核分枝杆菌的生长至关重要。通过全基因组测序，我们证明我们分离的所有五个ΔΔ突变体都有预测会破坏Pst磷酸盐转运体功能的独立抑制突变，这表明在没有PhoU蛋白的情况下，Pst系统对磷酸盐的摄取是有毒的。总体而言，我们的数据表明，这两种结核分枝杆菌PhoU直系同源物在响应磷酸盐可利用性控制SenX3-RegX3活性和调节Pst系统的磷酸盐转运方面具有重叠功能。我们的结果表明，结核分枝杆菌可作为一个易于处理的模型，用于进一步表征分枝杆菌中磷酸盐感应的分子机制，并筛选可干扰信号转导从而提高现有抗结核抗生素疗效的化合物。
