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. 2024 Mar 13;15(3):e0221123.
doi: 10.1128/mbio.02211-23. Epub 2024 Feb 12.

Pseudomonas aeruginosa MipA-MipB envelope proteins act as new sensors of polymyxins

Manon Janet-Maitre  1 , Viviana Job  1 , Maxime Bour  2   3 , Mylène Robert-Genthon  1 , Sabine Brugière  4 , Pauline Triponney  3 , David Cobessi  5 , Yohann Couté  4 , Katy Jeannot  2   3   6 , Ina Attrée  1
Affiliations

Pseudomonas aeruginosa MipA-MipB envelope proteins act as new sensors of polymyxins

Manon Janet-Maitre et al. mBio. .

Abstract

Due to the rising incidence of antibiotic-resistant infections, the last-line antibiotics, polymyxins, have resurged in the clinics in parallel with new bacterial strategies of escape. The Gram-negative opportunistic pathogen Pseudomonas aeruginosa develops resistance to colistin/polymyxin B by distinct molecular mechanisms, mostly through modification of the lipid A component of the LPS by proteins encoded within the arnBCDATEF-ugd (arn) operon. In this work, we characterized a polymyxin-induced operon named mipBA, present in P. aeruginosa strains devoid of the arn operon. We showed that mipBA is activated by the ParR/ParS two-component regulatory system in response to polymyxins. Structural modeling revealed that MipA folds as an outer-membrane β-barrel, harboring an internal negatively charged channel, able to host a polymyxin molecule, while the lipoprotein MipB adopts a β-lactamase fold with two additional C-terminal domains. Experimental work confirmed that MipA and MipB localize to the bacterial envelope, and they co-purify in vitro. Nano differential scanning fluorimetry showed that polymyxins stabilized MipA in a specific and dose-dependent manner. Mass spectrometry-based quantitative proteomics on P. aeruginosa membranes demonstrated that ∆mipBA synthesized fourfold less MexXY-OprA proteins in response to polymyxin B compared to the wild-type strain. The decrease was a direct consequence of impaired transcriptional activation of the mex operon operated by ParR/ParS. We propose MipA/MipB to act as membrane (co)sensors working in concert to activate ParS histidine kinase and help the bacterium to cope with polymyxin-mediated envelope stress through synthesis of the efflux pump, MexXY-OprA.IMPORTANCEDue to the emergence of multidrug-resistant isolates, antibiotic options may be limited to polymyxins to eradicate Gram-negative infections. Pseudomonas aeruginosa, a leading opportunistic pathogen, has the ability to develop resistance to these cationic lipopeptides by modifying its lipopolysaccharide through proteins encoded within the arn operon. Herein, we describe a sub-group of P. aeruginosa strains lacking the arn operon yet exhibiting adaptability to polymyxins. Exposition to sub-lethal polymyxin concentrations induced the expression and production of two envelope-associated proteins. Among those, MipA, an outer-membrane barrel, is able to specifically bind polymyxins with an affinity in the 10-µM range. Using membrane proteomics and phenotypic assays, we showed that MipA and MipB participate in the adaptive response to polymyxins via ParR/ParS regulatory signaling. We propose a new model wherein the MipA-MipB module functions as a novel polymyxin sensing mechanism.

Keywords: MexXY-OprA; ParR/ParS; Pseudomonas aeruginosa; antibiotic resistance; arn; nano-DSF; polymyxin; proteomics; signal transduction; two-component system.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig 1
Fig 1
Identification of P. aeruginosa strains lacking arn operon. (A) Conservation of arn locus across P. aeruginosa strains, visualized by Clinker (23). The arn-ugd region expending from algA to fruA encompasses 8.8 kb and is not present in IHMA87. (B) Neighbor-joining phylogenetic tree highlighting separation of phylogenetic group 3 into two distinct subgroups, 3A and 3B, which differ notably by the presence or absence of the arn locus.
Fig 2
Fig 2
arn operon is necessary for acquisition of stable resistance but not for adaptive resistance to PME. (A) Frequency of acquisition of stable resistance in IHMA87 and reference strains PA7, PAO1, and PA14 in the presence of 4 to 64 µg/mL PME. (B) Bactericidal activity of PME on IHMA87, PA7, PAO1, and PA14 strains over time in the presence of 8× MIC (4 µg/mL) of PME (n = 3).
Fig 3
Fig 3
MipA is synthetized in response to PMB in a ParR/ParS-dependent manner. (A) Conservation of mipBA locus across P. aeruginosa strains, visualized by Clinker (23). (B) Consensus of ParR-binding site obtained from known target promoters and ParR-binding site in PmipB (PA1797). (C) Electrophoresis mobility shift assay showing ParR binding onto the promoter of mipB/PA1797 with or without phosphorylation (P). (D)PmipBA activity in response to PMB measured by β-galactosidase assay in wild type (WT-IHMA87) and IHMA87ΔparRS mutant. ****: P < 0.0001. (E) MipA detection in response to sub-lethal concentrations of PMB and PME in a ParR/ParS-dependent manner. * indicates non-specific antibody binding used as loading control. PMB concentration: 0.25 µg/mL.
Fig 4
Fig 4
MipA and MipB are envelope proteins. (A) Schematic representation of MipA and MipB. Both proteins carry N-terminal signal peptides predicted by SignalP v.5.0 (45). MipB contains an additional “lipobox” sequence composed of “ASGC” sequence with conserved cysteine residue, which anchors proteins to the membrane. (B) MipA model generated by AlphaFold (46), without the 21 first residues containing the signal peptide. The β-strands are represented as yellow arrows, and α-helices are represented as pink ribbons. The side chains of aromatic residues that delineate the inner membrane are drawn in sticks. Figures were generated with Pymol. (C) Alignment of the catalytic sites of AmpC (E. coli) with MipB from different P. aeruginosa strains (IHMA87, PA7, PAO1, and PA14). (D) MipB model calculated using AlphaFold (46) excluding the 27 first and 8 last residues. The β-strands are represented as arrows, and α-helices are represented as ribbons. The N-terminal domain containing the β-lactamase fold is in blue and orange. The two eight-stranded anti-parallel β-barrels in the C-terminal are in red and yellow, and their short N-terminal α-helices are in green. The long loop connecting the C-terminal to the N-terminal domain is in gray. (E) Bacterial fractionation showing membrane association of MipA. XcpY, DsbA, and Ef-Tu includes controls for membranes, periplasm, and cytosolic fractions, respectively. (F) MipB3xFLAG fractionates with the membranes and, to a lesser extent, with the perisplam. OprM, DsbA, and RpoA are controls for membranes, periplasm, and cytosolic fractions, respectively. (G) Inner and outer-membrane separation by sucrose gradient showing the presence of MipA in the outer-membrane fractions. PMB concentration: 0.25 µg/mL. B, bacteria; C, cytosol; M, membrane; P, periplasm.
Fig 5
Fig 5
MipA and MipB interaction. (A) Model of the MipA-MipB complex generated using AlphaFold-Multimer (46). (Left) MipA is in cyan. The β-lactamase fold of MipB is in blue and orange. The two eight anti-parallel β-stranded barrel domains of MipB are in yellow and red with the first α-helices in green. (Right) Electrostatic surface representation showing the charges at the interface between the two proteins. The figures were generated with Pymol. (B) MipA and MipB co-purify in vitro. MipB-Strep and MipA-His6 were co-produced in E. coli. Soluble extracts were loaded onto a strep column, and proteins were eluted by the addition of a desthobiotin-containing buffer. Different fractions (Tot, Sol, W, and E) were analyzed by immunoblotting using anti-Strep-tag and anti-MipA primary antibodies. E, elution; Sol, soluble extract; Tot, total extract; W, washing.
Fig 6
Fig 6
MipA specifically binds PMB and PME. (A) PMB docking in MipA. (Left) MipA electrostatic surface. The arrow shows the negative channel entrance. (Right top) Membrane perpendicular view from the PMB bound to MipA. The best position of PMB, calculated with Autodock Vina (46), is shown in sticks: the carbon, nitrogen, and oxygen atoms are in yellow, blue, and red, respectively. (Right bottom) View from the MipA periplasmic face of the PMB bound to MipA. (B and C) MipA thermal stability increases in the presence of PMB and PME as assessed by nano-DSF. Pure MipA alone (in orange, dashed line) or incubated for 2 h at room temperature with a molar ratio from 1:2 to 1:40 of PMB (B) or PME (C) was heated from 20°C to 95°C. Protein folding/unfolding was followed by tryptophan fluorescence emitted at 330 and 350 nm. The slope of the ratio (F350:F330) was plotted at different temperatures, and its maximum corresponds to the melting temperature (Tm) of the protein. (D) Summary of calculated Tm of MipA in presence of PMB, PME and three other positively charged molecules, magainin, indolicidin, and streptomycin, used as negative controls, assessed by nano-DSF. Data from panels B and C was also included for comparison. Indolicidin was used only up to 1:4 molar ratio, which corresponds to 25 µM. Of note, indolicidin contains five tryptophans and could not be used at higher concentrations because of the high background signal.
Fig 7
Fig 7
MipA/MipB are required for P. aeruginosa response to polymyxins. (A) Normalized abundance of MipA, MipB, MexX, MexY, and OprA in IHMA87 (WT) or ΔmipBA membranes with or without sub-lethal PMB treatment (0.25 µg/mL) obtained by proteomic analysis (n = 3). Data represented by a triangle were imputed using the slsa algorithm for partially observed values in the condition and the DetQuantile algorithm for totally absent values in the condition. (B) Relative expression level of mipB, mipA, mexX, mexY, and oprA in WT and ΔparRS (left) or IHMA87 (WT) and ΔmipBA (right) with or without addition of sub-lethal concentration of PMB (0.25 µg/mL) normalized to rpoD by RT-qPCR. A Kruskal-Wallis test was applied followed by a Dunn test for each of the genes tested. Note the absence of mex induction in ΔparRS and ΔmipBA. (C) Bactericidal effect of PME/colistin used at 1 µg/mL on cultured of IHMA87 (WT), ΔmipBA, and ΔparRS over time (n = 3). *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001.
Fig 8
Fig 8
Schematic representation of the working model proposing MipA/MipB as co-sensors of PMB. In the resting state, low amounts of MipA are present in the outer membrane, and ParR/ParS TCS is inactive. PMB binding to bacterial membranes and to MipA provokes conformational changes in the MipB-MipA complex. MipA and MipB induce ParS autophosphorylation, which leads to activation of the cognate response regulator ParR. ParR binds to promoter regions of mexXY-oprA and mipBA operons, resulting in MipA, MipB, and MeXY-OprA overproduction and adaptive resistance to polymyxins.
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