Assembly and regulation of the chlorhexidine-specific efflux pump AceI

Significance Acinetobacter baumannii has become challenging to treat due to its multidrug resistance mediated by active drug efflux pumps. The prototype member of the proteobacterial antimicrobial compound efflux (PACE) family, AceI of A. baumannii, is implicated in the transport of widely used antiseptic chlorhexidine, while AceR is associated with regulating the expression of the aceI gene. Here we apply native mass spectrometry to show that AceI forms dimers at high pH, and that chlorhexidine binding facilitates the functional form of the protein. Also, we demonstrate how AceR affects the interaction between RNA polymerase and promoter DNA both in the presence and in the absence of chlorhexidine. Overall, these results provide insight into the assembly and regulation of the PACE family.


Protein expression constructs:
The plasmids used for over-expression of the proteins in this study were obtained by inserting the respective gene fragment into a modified pET15b vector using an InFusion kit (Clonetech). The DNA sequences were verified by sequencing (Source Bioscience). All the proteins contain a 6×His tag at the C-terminus was overexpressed in E. coli C43(DE3) (Lucigen) cells, grown in LB medium containing ampicillin (100 µgml -1 ). When the culture reached an absorbance at 600nm of ~0.5, expression was induced with 0.5mM isopropyl β-thiogalactopyranoside (IPTG) for 3h at 37 °C.
E15Q mutant of AceI was prepared by a PCR-based method, using the plasmid that was used for expressing wild type protein. Expression and purification of this mutant is done in the same manner as for the wild type.

Protein expression and purification:
Proteins were purified in a similar manner according to the following procedure at 4 °C. Cells were pelleted by centrifugation at 5,000g and resuspended in a buffer containing 50 mM Tris-HCl (pH 8.0), 300 mM NaCl and EDTA-free protease inhibitor cocktail (Roche). The cells were then disrupted by a microfluidizer (Microfluidics). After centrifugation (20,000g for 20 min), the supernatant was filtered and loaded onto a 5 ml His Trap-HP column in case of soluble proteins while for membrane proteins, the supernatant was ultracentrifuged (200,000g), and the membrane fractions were collected. The proteins were solubilized from the membrane fraction with 20 mM Tris (pH 8.0), 150 mM NaCl, 20% glycerol, 2% DDM (Anatrace) for 2 h at 4 °C. The insoluble material was removed by ultracentrifugation. The supernatant was filtered before loading onto a 5 ml His Trap-HP column (GE Healthcare) equilibrated in 20 mM Tris (pH 8.0), 150 mM NaCl, 20 mM imidazole, 10% glycerol and 0.03% DDM. After the clarified supernatant was loaded, the column was initially washed with 50 ml of 20 mM Tris (pH 8.0), 150 mM NaCl, 20 mM imidazole, 10% glycerol and 0.03% DDM and washed again with 50 ml of 20 mM Tris (pH 8.0), 150 mM NaCl, 80 mM imidazole, 10% glycerol and 0.03% DDM. The bound protein was then eluted with 20 mM Tris (pH 8.0), 150 mM NaCl, 500 mM imidazole, 10% glycerol and 0.03% DDM. Peak fractions were pooled, dialyzed and concentrated in a buffer containing 20 mM Tris (pH 8.0) and 150 mM NaCl, 10% glycerol and 0.03% DDM. Similar buffers were used for soluble protein purification without the DDM detergent. Concentrated protein was either used immediately or flash-frozen in liquid nitrogen and stored at −80 °C. 3

Native mass spectrometry:
Prior to MS analysis, soluble proteins were buffer exchanged into 200 mM ammonium acetate pH 8.0, while membranes were buffer-exchanged into 200 mM ammonium acetate at various pHs, with 2 × CMC (critical micelle concentration) of the detergent of interest using a Biospin-6 (BioRad) column and introduced directly into the mass spectrometer using gold-coated capillary needles (prepared inhouse). Data were collected on a modified QExactive hybrid quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific) optimized for analysis of high-mass complexes, using methods previously described for membrane proteins (1). The instrument parameters were as follows: capillary voltage 1.2 kV, S-lens RF 100%, quadrupole selection from 1,000 to 15,000 m/z range, collisional activation in the HCD cell 100-200 V, argon UHV pressure 1.12 × 10 −9 mbar, temperature 60 °C, resolution of the (2), while all other data were analysed using Xcalibur 3.0 (Thermo Scientific). The relative intensities of monomers and dimers were obtained by deconvoluting the native MS data using UniDec and were converted to mole fraction to determine the monomer and dimer concentrations at equilibrium. To obtain the monomer-dimer equilibrium constants, a previously established monomer-dimer model was used (3). Similar parameters were used for data processing in UniDec when comparisons are made.
Lipids and antibiotics were diluted into a buffer containing 200 mM ammonium acetate and 0.05% (w/v) LDAO and were added in different ratios to solutions of AceI in the same buffer. All experiments were repeated three times.
Native E. coli RNAP (Creative Enzymes) was buffer exchanged into 200 mM ammonium acetate pH 8.0 before adding it to the AceR FL protein and 100 bp dsDNA. Data were collected on Q-Exactive UHMR Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo Fisher) in -ve polarity with the exception that the capillary voltage was set 1.0 kV and with the similar parameters as above operating in -ve polarity.  Table S1 6 Fig. S2. Mass spectra of AceI in different detergents: Mass spectra of AceI in DDM, OGNG, and OG look similar to the spectrum in LDAO, in all detergents AceI seem to exist in a monomer-dimer equilibrium.  Spectra for wild type protein at pH 5 and 8 are labelled with orange and green colour respectively while for E15Q the spectra are coloured in purple.   spectra of AceR FL show that this protein exists in a dimer and tetramer equilibrium, c) mass spectra of AceR CTD show that this protein is dimer in solution, d) titration of chlorhexidine binding to AceR FL , chlorhexidine adduct peaks are highlighted in orange and range from 1 to 5 molecules at higher concentration of chlorhexidine tested, e) binding affinity of chlorhexidine to AceR CTD was measured using ITC.  No interaction between RNAP and DNA in presence of AceR FL , bottom spectrum, indicative of repressive nature of AceR. The addition of chlorhexidine significantly increases the tetramerisation of AceR FL , which in turn promotes the interaction between RNAP and the promoter DNA, top spectrum.

Isothermal Titration Calorimetry
Relative ratios of AceR tetramer and RNAP-DNA complexes are shown in inserts, these clearly indicate that the increase in AceR tetramer formation is directly correlated with RNAP binding to promoter DNA.