Mechanistic and biophysical characterization of polymyxin resistance response regulator PmrA in Acinetobacter baumannii

Zhenlin Ouyang; Wenbo He; Min Jiao; Qinyue Yu; Yucheng Guo; Moath Refat; Qian Qin; Jiaxin Zhang; Qindong Shi; Fang Zheng; Yurong Wen

doi:10.3389/fmicb.2024.1293990

Mechanistic and biophysical characterization of polymyxin resistance response regulator PmrA in Acinetobacter baumannii

Front Microbiol. 2024 Feb 27:15:1293990. doi: 10.3389/fmicb.2024.1293990. eCollection 2024.

Authors

Zhenlin Ouyang¹, Wenbo He¹, Min Jiao¹, Qinyue Yu¹, Yucheng Guo¹, Moath Refat², Qian Qin¹, Jiaxin Zhang¹, Qindong Shi¹, Fang Zheng², Yurong Wen¹

Affiliations

¹ Shaanxi Provincial Key Laboratory of Sepsis in Critical Care Medicine, Department of Critical Care Medicine, Center for Microbiome Research of Med-X Institute, The First Affiliated Hospital, Xi'an Jiaotong University, Xi'an, China.
² The Key Laboratory of Environment and Genes Related to Disease of Ministry of Education Health Science Center, Xi'an Jiaotong University, Xi'an, China.

Abstract

Introduction: Acinetobacter baumannii PmrAB is a crucial two-component regulatory system (TCS) that plays a vital role in conferring resistance to polymyxin. PmrA, a response regulator belonging to the OmpR/PhoB family, is composed of a C-terminal DNA-binding effector domain and an N-terminal receiver domain. The receiver domain can be phosphorylated by PmrB, a transmembrane sensor histidine kinase that interacts with PmrA. Once phosphorylated, PmrA undergoes a conformational change, resulting in the formation of a symmetric dimer in the receiver domain. This conformational change facilitates the recognition of promoter DNA by the DNA-binding domain of PmrA, leading to the activation of adaptive responses.

Methods: X-ray crystallography was carried out to solve the structure of PmrA receiver domain. Electrophoretic mobility shift assay and Isothermal titration calorimetry were recruited to validate the interaction between the recombinant PmrA protein and target DNA. Field-emission scanning electron microscopy (FE-SEM) was employed to characterize the surface morphology of A. baumannii in both the PmrA knockout and mutation strains.

Results: The receiver domain of PmrA follows the canonical α5β5 response regulator assembly, which undergoes dimerization upon phosphorylation and activation. Beryllium trifluoride is utilized as an aspartate phosphorylation mimic in this process. Mutations involved in phosphorylation and dimerization significantly affected the expression of downstream pmrC and naxD genes. This impact resulted in an enhanced cell surface smoothness with fewer modifications, ultimately contributing to a decrease in colistin (polymyxin E) and polymyxin B resistance. Additionally, a conservative direct-repeat DNA PmrA binding sequence TTTAAGNNNNNTTTAAG was identified at the promoter region of the pmrC and naxD gene. These findings provide structural insights into the PmrA receiver domain and reveal the mechanism of polymyxin resistance, suggesting that PmrA could be a potential drug target to reverse polymyxin resistance in Acinetobacter baumannii.

Keywords: Acinetobacter baumannii; PmrA; mechanism; polymyxin resistance; response regulator.

Grants and funding

The authors declare financial support was received for the research, authorship, and/or publication of this article. This work has been funded by the National Natural Science Foundation of China (Nos. 82102402, 82072237, and 32170187), Shaanxi Province Natural Science Funding (Nos. 2023-JC-JQ-63, 2021JQ-381, and 2021JM-007) and China Postdoctoral Science Foundation (Nos. 2020TQ0242 and 2020M683508), and Institutional Foundation of the First Affiliated Hospital of Xi’an Jiaotong University.