Identification and Characterization of TriABC-OpmH, a Triclosan Efflux Pump of Pseudomonas aeruginosa Requiring Two 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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Opening the Channel: the Two Functional Interfaces of Pseudomonas aeruginosa OpmH with the Triclosan Efflux Pump TriABC

Journal of Bacteriology ◽

10.1128/jb.00535-16 ◽

2016 ◽

Vol 198 (23) ◽

pp. 3176-3185 ◽

Cited By ~ 8

Author(s):

Abigail T. Ntreh ◽

Jon W. Weeks ◽

Logan M. Nickels ◽

Helen I. Zgurskaya

Keyword(s):

Pseudomonas Aeruginosa ◽

Membrane Fusion ◽

Outer Membrane ◽

Fusion Proteins ◽

Efflux Pump ◽

Cell Envelope ◽

Functional Integration ◽

Content Type ◽

Membrane Fusion Proteins ◽

Stimulation Of

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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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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Triclosan resistance in clinical isolates of Acinetobacter baumannii

Journal of Medical Microbiology ◽

10.1099/jmm.0.008524-0 ◽

2009 ◽

Vol 58 (8) ◽

pp. 1086-1091 ◽

Cited By ~ 38

Author(s):

Yagang Chen ◽

Borui Pi ◽

Hua Zhou ◽

Yunsong Yu ◽

Lanjuan Li

Keyword(s):

Acinetobacter Baumannii ◽

Efflux Pump ◽

Resistance Mechanism ◽

Dilution Method ◽

Agar Dilution Method ◽

Active Efflux ◽

Low Level ◽

High Level ◽

Resistance Rates ◽

Triclosan Resistance

The susceptibility to triclosan of 732 clinical Acinetobacter baumannii isolates obtained from 25 hospitals in 16 cities in China from December 2004 to December 2005 was screened by using an agar dilution method. Triclosan MICs ranged between 0.015 and 16 mg l−1, and the MIC90 was 0.5 mg l−1, lower than the actual in-use concentration of triclosan. Twenty triclosan-resistant isolates (MICs ≥1 mg l−1) were characterized by antibiotic susceptibility, clonal relatedness, fabI mutation, fabI expression, and efflux pump phenotype and expression to elucidate the resistance mechanism of A. baumannii to triclosan. The resistance rates of triclosan-resistant isolates to imipenem, levofloxacin, amikacin and tetracycline were higher than those of triclosan-sensitive isolates. Triclosan resistance was artificially classified as low level (MICs 1–2 mg l−1) or high level (MICs ≥4 mg l−1). High-level triclosan resistance could be explained by a Gly95Ser mutation of FabI, whilst wild-type fabI was observed to be overexpressed in low-level resistant isolates. Active efflux did not appear to be a major reason for acquired triclosan resistance, but acquisition of resistance appeared to be dependent on a background of intrinsic triclosan efflux.

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Resistance to β-Lactam Antibiotics inPseudomonas aeruginosa Due to Interplay between the MexAB-OprM Efflux Pump and β-Lactamase

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.43.5.1301 ◽

1999 ◽

Vol 43 (5) ◽

pp. 1301-1303 ◽

Cited By ~ 49

Author(s):

Taiji Nakae ◽

Akira Nakajima ◽

Toshihisa Ono ◽

Kohjiro Saito ◽

Hiroshi Yoneyama

Keyword(s):

Pseudomonas Aeruginosa ◽

Efflux Pump ◽

Efflux System ◽

High Level

ABSTRACT We evaluated the roles of the MexAB-OprM efflux pump and β-lactamase in β-lactam resistance in Pseudomonas aeruginosa by constructing OprM-deficient, OprM basal level, and OprM fully expressed mutants from β-lactamase-negative, -inducible, and -overexpressed strains. We conclude that, with the notable exception of imipenem, the MexAB-OprM pump contributes significantly to β-lactam resistance in both β-lactamase-negative and β-lactamase-inducible strains, while the contribution of the MexAB-OprM efflux system is negligible in strains with overexpressed β-lactamase. Overexpression of the efflux pump alone contributes to the high level of β-lactam resistance in the absence of β-lactamase.

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