Opening the Channel: the Two Functional Interfaces of Pseudomonas aeruginosa OpmH with the Triclosan Efflux Pump TriABC

ABSTRACTTriABC-OpmH is an efflux pump fromPseudomonas aeruginosawith an unusual substrate specificity and protein composition. When overexpressed, this pump confers a high level of resistance to the biocide triclosan and the detergent SDS, which are commonly used in combinations for antimicrobial treatments. This activity requires an RND transporter (TriC), an outer membrane channel (OpmH), and two periplasmic membrane fusion proteins (TriA and TriB) with nonequivalent functions. In the active complex, TriA is responsible for the recruitment of OpmH, while TriB is responsible for stimulation of the transporter TriC. Here, we used the functional and structural differences between the two membrane fusion proteins to link their functional roles to specific interactions with OpmH. Our results provide evidence that the TriB-dependent stimulation of the TriC transporter is coupled to opening of the OpmH aperture through binding to the interprotomer groove of OpmH.IMPORTANCEMultidrug efflux transporters are important contributors to intrinsic and acquired antibiotic resistance in clinics. In Gram-negative bacteria, these transporters have a characteristic tripartite architecture spanning the entire two-membrane cell envelope. How such complexes are assembled and how the reactions separated in two different membranes are coupled to provide efficient efflux of various compounds across the cell envelope remain unclear. This study addressed these questions, and the results suggest a mechanism for functional integration of drug efflux by the inner membrane transporter and opening of the channel for transport across the outer membrane.

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Identification and Characterization of TriABC-OpmH, a Triclosan Efflux Pump of Pseudomonas aeruginosa Requiring Two Membrane Fusion Proteins

Journal of Bacteriology ◽

10.1128/jb.00850-07 ◽

2007 ◽

Vol 189 (21) ◽

pp. 7600-7609 ◽

Cited By ~ 73

Author(s):

Takehiko Mima ◽

Swati Joshi ◽

Margarita Gomez-Escalada ◽

Herbert P. Schweizer

Keyword(s):

Pseudomonas Aeruginosa ◽

Membrane Fusion ◽

Fusion Proteins ◽

Efflux Pump ◽

Divalent Cations ◽

Constitutive Expression ◽

Multiple Copies ◽

High Level ◽

Triclosan Resistance ◽

Membrane Fusion Proteins

ABSTRACT Pseudomonas aeruginosa achieves high-level (MIC > 1 mg/ml) triclosan resistance either by constitutive expression of MexAB-OprM, an efflux pump of the resistance nodulation cell division (RND) family, or expression of MexCD-OprJ, MexEF-OprN, and MexJK-OpmH in regulatory mutants. A triclosan-resistant target enzyme and perhaps other mechanisms probably act synergistically with efflux. To probe this notion, we exposed the susceptible Δ(mexAB-oprM) Δ(mexCD-oprJ) Δ(mexEF-oprN) Δ(mexJK) Δ(mexXY) strain PAO509 to increasing triclosan concentrations and derived a resistant strain, PAO509.5. This mutant overexpressed the PA0156-PA0157-PA0158 pump, which only effluxed triclosan, but not closely related compounds, antibiotics, and divalent cations, and was therefore renamed TriABC. Constitutive expression of the triABC operon was due to a single promoter-up mutation. Deletion of two adjacent genes, pcaR and PA0159, encoding transcriptional regulators had no effect on expression of this operon. TriABC is the only P. aeruginosa RND pump which contains two membrane fusion proteins, TriA and TriB, and both are required for efflux pump function. Probably owing to tight transcriptional coupling of the triABC genes, complementation of individual mutations was only partially achievable. Full complementation was only observed when a complete triABC operon was provided in trans, either in single or multiple copies. TriABC associated with OpmH, but not OprM, for assembly of a functional triclosan efflux pump. TriABC is the fifth RND pump in P. aeruginosa shown to efficiently efflux triclosan, supporting the notion that efflux is the primary mechanism responsible for this bacterium's high intrinsic and acquired triclosan resistance.

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Development of New Cloning Vectors for the Production of Immunogenic Outer Membrane Fusion Proteins in Escherichia coli

Nature Biotechnology ◽

10.1038/nbt0296-203 ◽

1996 ◽

Vol 14 (2) ◽

pp. 203-208 ◽

Cited By ~ 25

Author(s):

Pierre Cornells ◽

Javier Cote Sierra ◽

Antonio Lim ◽

Achut Malur ◽

Sumalee Tungpradabkul ◽

...

Keyword(s):

Escherichia Coli ◽

Membrane Fusion ◽

Outer Membrane ◽

Fusion Proteins ◽

Cloning Vectors ◽

Membrane Fusion Proteins

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Appoptosin interacts with mitochondrial outer-membrane fusion proteins and regulates mitochondrial morphology

Journal of Cell Science ◽

10.1242/jcs.176792 ◽

2016 ◽

Vol 129 (5) ◽

pp. 994-1002 ◽

Cited By ~ 16

Author(s):

Cuilin Zhang ◽

Zhun Shi ◽

Lingzhi Zhang ◽

Zehua Zhou ◽

Xiaoyuan Zheng ◽

...

Keyword(s):

Membrane Fusion ◽

Outer Membrane ◽

Fusion Proteins ◽

Mitochondrial Outer Membrane ◽

Mitochondrial Morphology ◽

Membrane Fusion Proteins

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Localization and Regulation of the T1 Unimolecular Spanin

Journal of Virology ◽

10.1128/jvi.00380-18 ◽

2018 ◽

Vol 92 (22) ◽

Cited By ~ 5

Author(s):

Rohit Kongari ◽

Jeffrey Snowden ◽

Joel D. Berry ◽

Ry Young

Keyword(s):

Membrane Fusion ◽

Outer Membrane ◽

Fusion Proteins ◽

Transmembrane Domain ◽

Time Lapse ◽

Negative Regulator ◽

Viral Fusion ◽

Outer Membranes ◽

Two Component ◽

Membrane Fusion Proteins

ABSTRACTSpanins are bacteriophage lysis proteins responsible for disruption of the outer membrane, the final step of Gram-negative host lysis. The absence of spanins results in a terminal phenotype of fragile spherical cells. The phage T1 employs a unimolecular spanin gp11that has an N-terminal lipoylation signal and a C-terminal transmembrane domain. Upon maturation and localization, gp11ends up as an outer membrane lipoprotein with a C-terminal transmembrane domain embedded in the inner membrane, thus connecting both membranes as a covalent polypeptide chain. Unlike the two-component spanins encoded by most of the other phages, including lambda, the unimolecular spanins have not been studied extensively. In this work, we show that the gp11mutants lacking either membrane localization signal were nonfunctional and conferred a partially dominant phenotype. Translation from internal start sites within the gp11coding sequence generated a shorter product which exhibited a negative regulatory effect on gp11function. Fluorescence spectroscopy time-lapse videos of gp11-GFP expression showed gp11accumulated in distinct punctate foci, suggesting localized clusters assembled within the peptidoglycan meshwork. In addition, gp11was shown to mediate lysis in the absence of holin and endolysin function when peptidoglycan density was depleted by starvation for murein precursors. This result indicates that the peptidoglycan is a negative regulator of gp11function. This supports a model in which gp11acts by fusing the inner and outer membranes, a mode of action analogous to but mechanistically distinct from that proposed for the two-component spanin systems.IMPORTANCESpanins have been proposed to fuse the cytoplasmic and outer membranes during phage lysis. Recent work with the lambda spanins Rz-Rz1, which are similar to class I viral fusion proteins, has shed light on the functional domains and requirements for two-component spanin function. Here we report, for the first time, a genetic and biochemical approach to characterize unimolecular spanins, which are structurally and mechanistically different from two-component spanins. Considering similar predicted secondary structures within the ectodomains, unimolecular spanins can be regarded as a prokaryotic version of type II viral membrane fusion proteins. This study not only adds to our understanding of regulation of phage lysis at various levels but also provides a prokaryotic genetically tractable platform for interrogating class II-like membrane fusion proteins.

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Bacterial Adaptor Membrane Fusion Proteins and the Structurally Dissimilar Outer Membrane Auxiliary Proteins Have Exchanged Central Domains inα-Proteobacteria

International Journal of Microbiology ◽

10.1155/2010/589391 ◽

2010 ◽

Vol 2010 ◽

pp. 1-5 ◽

Cited By ~ 3

Author(s):

Anthony Y. Xiao ◽

Jing Wang ◽

Milton H. Saier

Keyword(s):

Membrane Fusion ◽

Outer Membrane ◽

Fusion Proteins ◽

Transport Systems ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Transporter Protein ◽

Functional Implications ◽

Membrane Fusion Proteins ◽

Helical Region

Transport systems frequently include auxiliary proteins that perform subfunctions within the transporter protein complex. Two such proteins found in Gram-negative bacteria are the Membrane Fusion Proteins (MFPs) and the Outer Membrane Auxiliary (OMA) proteins. We here demonstrate that OMAs present inα-proteobacteria (but not in other bacterial types) contain a longα-helical region that is homologous to corresponding regions in the MFPs. The results suggest that during their evolution, OMAs, specifically fromα-proteobacteria, exchanged their ownα-helical domain for one derived from an MFP. The structural and functional implications of these findings are discussed.

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Substrate-Dependent Utilization of OprM or OpmH by the Pseudomonas aeruginosa MexJK Efflux Pump

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.49.5.2133-2136.2005 ◽

2005 ◽

Vol 49 (5) ◽

pp. 2133-2136 ◽

Cited By ~ 30

Author(s):

Rungtip Chuanchuen ◽

Takeshi Murata ◽

Naomasa Gotoh ◽

Herbert P. Schweizer

Keyword(s):

Pseudomonas Aeruginosa ◽

Cell Division ◽

Membrane Proteins ◽

Fusion Protein ◽

Membrane Fusion ◽

Outer Membrane ◽

Protein Pair ◽

Efflux Pump ◽

Outer Membrane Proteins ◽

Membrane Fusion Protein

ABSTRACT MexJK requires OprM for erythromycin efflux but not for triclosan efflux. Deletion of 15 OprM family outer membrane proteins (OMPs) revealed that only the TolC homolog OpmH functions with MexJK for triclosan efflux. This is the first report of natural utilization of multiple OMPs by a given resistance nodulation cell division transporter/membrane fusion protein pair.

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MexAB-OprM Efflux Pump Interaction with the Peptidoglycan of Escherichia coli and Pseudomonas aeruginosa

International Journal of Molecular Sciences ◽

10.3390/ijms22105328 ◽

2021 ◽

Vol 22 (10) ◽

pp. 5328

Author(s):

Miao Ma ◽

Margaux Lustig ◽

Michèle Salem ◽

Dominique Mengin-Lecreulx ◽

Gilles Phan ◽

...

Keyword(s):

Escherichia Coli ◽

Pseudomonas Aeruginosa ◽

Cell Division ◽

Membrane Proteins ◽

Outer Membrane ◽

Efflux Pump ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Active Efflux

One of the major families of membrane proteins found in prokaryote genome corresponds to the transporters. Among them, the resistance-nodulation-cell division (RND) transporters are highly studied, as being responsible for one of the most problematic mechanisms used by bacteria to resist to antibiotics, i.e., the active efflux of drugs. In Gram-negative bacteria, these proteins are inserted in the inner membrane and form a tripartite assembly with an outer membrane factor and a periplasmic linker in order to cross the two membranes to expulse molecules outside of the cell. A lot of information has been collected to understand the functional mechanism of these pumps, especially with AcrAB-TolC from Escherichia coli, but one missing piece from all the suggested models is the role of peptidoglycan in the assembly. Here, by pull-down experiments with purified peptidoglycans, we precise the MexAB-OprM interaction with the peptidoglycan from Escherichia coli and Pseudomonas aeruginosa, highlighting a role of the peptidoglycan in stabilizing the MexA-OprM complex and also differences between the two Gram-negative bacteria peptidoglycans.

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Interdependent assembly of specific regulatory lipids and membrane fusion proteins into the vertex ring domain of docked vacuoles

The Journal of Cell Biology ◽

10.1083/jcb.200409068 ◽

2004 ◽

Vol 167 (6) ◽

pp. 1087-1098 ◽

Cited By ~ 170

Author(s):

Rutilio A. Fratti ◽

Youngsoo Jun ◽

Alexey J. Merz ◽

Nathan Margolis ◽

William Wickner

Keyword(s):

Membrane Fusion ◽

Protein Interactions ◽

Fusion Proteins ◽

Membrane Microdomains ◽

Rab Gtpase ◽

Protein Protein Interactions ◽

Px Domain ◽

Protein Partitioning ◽

Membrane Fusion Proteins ◽

Rab Effector

Membrane microdomains are assembled by lipid partitioning (e.g., rafts) or by protein–protein interactions (e.g., coated vesicles). During docking, yeast vacuoles assemble “vertex” ring-shaped microdomains around the periphery of their apposed membranes. Vertices are selectively enriched in the Rab GTPase Ypt7p, the homotypic fusion and vacuole protein sorting complex (HOPS)–VpsC Rab effector complex, SNAREs, and actin. Membrane fusion initiates at vertex microdomains. We now find that the “regulatory lipids” ergosterol, diacylglycerol and 3- and 4-phosphoinositides accumulate at vertices in a mutually interdependent manner. Regulatory lipids are also required for the vertex enrichment of SNAREs, Ypt7p, and HOPS. Conversely, SNAREs and actin regulate phosphatidylinositol 3-phosphate vertex enrichment. Though the PX domain of the SNARE Vam7p has direct affinity for only 3-phosphoinositides, all the regulatory lipids which are needed for vertex assembly affect Vam7p association with vacuoles. Thus, the assembly of the vacuole vertex ring microdomain arises from interdependent lipid and protein partitioning and binding rather than either lipid partitioning or protein interactions alone.

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Target (MexB)- and Efflux-Based Mechanisms Decreasing the Effectiveness of the Efflux Pump Inhibitor D13-9001 in Pseudomonas aeruginosa PAO1: Uncovering a New Role for MexMN-OprM in Efflux of β-Lactams and a Novel Regulatory Circuit (MmnRS) Controlling MexMN Expression

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01718-18 ◽

2018 ◽

Vol 63 (2) ◽

pp. e01718-18 ◽

Cited By ~ 1

Author(s):

Srijan Ranjitkar ◽

Adriana K. Jones ◽

Mina Mostafavi ◽

Zachary Zwirko ◽

Oleg Iartchouk ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Efflux Pump ◽

Response Regulator ◽

Heavy Metal Resistance ◽

Metal Resistance ◽

Rna Seq ◽

Efflux Pump Inhibitor ◽

Content Type ◽

Regulatory Circuit ◽

Outer Membrane Channel

ABSTRACT Efflux pumps contribute to antibiotic resistance in Gram-negative pathogens. Correspondingly, efflux pump inhibitors (EPIs) may reverse this resistance. D13-9001 specifically inhibits MexAB-OprM in Pseudomonas aeruginosa. Mutants with decreased susceptibility to MexAB-OprM inhibition by D13-9001 were identified, and these fell into two categories: those with alterations in the target MexB (F628L and ΔV177) and those with an alteration in a putative sensor kinase of unknown function, PA1438 (L172P). The alterations in MexB were consistent with reported structural studies of the D13-9001 interaction with MexB. The PA1438L172P alteration mediated a >150-fold upregulation of MexMN pump gene expression and a >50-fold upregulation of PA1438 and the neighboring response regulator gene, PA1437. We propose that these be renamed mmnR and mmnS for MexMN regulator and MexMN sensor, respectively. MexMN was shown to partner with the outer membrane channel protein OprM and to pump several β-lactams, monobactams, and tazobactam. Upregulated MexMN functionally replaced MexAB-OprM to efflux these compounds but was insusceptible to inhibition by D13-9001. MmnSL172P also mediated a decrease in susceptibility to imipenem and biapenem that was independent of MexMN-OprM. Expression of oprD, encoding the uptake channel for these compounds, was downregulated, suggesting that this channel is also part of the MmnSR regulon. Transcriptome sequencing (RNA-seq) of cells encoding MmnSL172P revealed, among other things, an interrelationship between the regulation of mexMN and genes involved in heavy metal resistance.

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