Understanding the Role of the Porin Regulatory Complex in Antibiotic Resistance: Transmembrane Modulation of EnvZ by Auxiliary Protein MzrA

  • Fadhael Alrahman Hussein Hasan

Student thesis: Doctoral Thesis

Abstract

Antimicrobial resistance poses a serious threat to both public health and the global economy, leading to patient treatment failure and growing healthcare costs. In Gram-negative bacteria, the outer membrane acts as a primary defence, controlling antibiotic entry into the periplasm and facilitating molecule exchange and signal transduction. Bacteria utilise diverse strategies for nutrient uptake, including water- filled pores such as OmpF and OmpC, the major porins in Gram-negative bacteria that enable the passage of certain-sized hydrophilic compounds. Regulating the expression of the essential membrane porins OmpF and OmpC in Escherichia coli and related bacteria is controlled by two-component regulatory systems (TCSs). EnvZ/OmpR is a well-characterised TCS that plays a vital role in responding to changes in environmental osmolarity and β-lactams. Recently, a membrane protein that modulates the sensory system of EnvZ/OmpR was discovered. This protein, MzrA, is a small inner membrane protein that has been shown to interact with and modulate transcription from the EnvZ/OmpR regulon through its periplasmic domain (1,2). However, how MzrA modulates the activity of EnvZ/OmpR through the hydrophobic membrane core is unknown. Understanding MzrA action may be of importance to the discovery of inhibitors of the essential TCSs in many pathogenic bacteria and may serve as a new drug target that could lead to bacterial death in multi-drug resistant bacteria.
In this study, experiments were conducted to create a single-Cys-containing library of MzrA variants, including residue positions 2–36. The MzrA variants were expressed from Standard European Vector Architecture plasmids (pSEVA) in various E. coli K-12 strains. The library was utilised in sulfhydryl-reactivity experimentation to locate the membrane-embedded region of MzrA and to analyse the effect of EnvZ on the structure of MzrA. The data revealed that MzrA is present as a homodimer, with residue positions 2– 13, 14–24, and 25–36 located in the cytoplasmic, transmembrane (TM) and periplasmic domains of MzrA, respectively. In addition, the results showed that when EnvZ is present, MzrA becomes less dynamic and no vertical repositioning of the TM occurs. The differences between the structure of MzrA in the presence and absence of EnvZ revealed that MzrA–MzrA’ interaction in the cytoplasm and periplasm was greatly decreased or inhibited in the presence of EnvZ. However, MzrA–MzrA’ interaction in the TM core was more conserved and the presence or absence of EnvZ was found to be largely irrelevant.
The single-Cys-containing MzrA library was further used to perform a fluorescence-based assay to map the signal output from the EnvZ/OmpR circuit. The data obtained two different ways that the signals were lost, either from interrupted MzrA dimerisation, or from interfered EnvZ–MzrA interaction, or both.
However, two residues at the membrane, L18C and V21C, retained the wild-type signal output from the MzrA-activated EnvZ/OmpR circuit. In addition, mutations of the MzrA TM region suggest that two leucine zippers contribute to the stability of MzrA–MzrA’ interaction and play an important role in modulating EnvZ activity. These residues require a closer look, in term of their amino acid properties and structural changes, to obtain an overall topological picture of the TM domain.
Upon comparison of the MzrA helical formation and signal output from the MzrA-stimulated EnvZ/OmpR circuit, with structures available from the AlphaFold2 protein structure database, we could propose a helical net to better understand potential EnvZ–MzrA interactions. Our data suggest that the cytoplasmic region of MzrA, with the exception of a few highly conserved residues, is of great importance for MzrA- based activation of the EnvZ/OmpR circuit. However, the MzrA TM domain showed that two key residues, L18C and V21C, are important for EnvZ–MzrA complex formation. In addition, two leucine zippers within the TM domain are important for coiled coil interaction between two MzrA helices. Furthermore, the few residues analysed at the periplasmic domain were highly active and promoted interaction between MzrA– MzrA’ or MzrA–EnvZ or both, except for a few that composed a loop-like structure between a membrane adjacent α-helix and a subsequent β-sheet. Our results indicate that the regulatory function of the small protein, MzrA, depends on distinct contributions from multiple residues spread across the protein. Better understanding of MzrA topology and analysis of factors affecting EnvZ/OmpR structure and function will be of great importance for the discovery of new drugs targeting essential TCSs in many pathogenic bacteria.
Date of Award12 Mar 2024
Original languageEnglish
Awarding Institution
  • University of Portsmouth
SupervisorRoger Draheim (Supervisor) & Ivor Shadrack Ebenezer (Supervisor)

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