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J Bacteriol. Nov 2007; 189(21): 7600–7609.
Published online Aug 24, 2007. doi:  10.1128/JB.00850-07
PMCID: PMC2168734

Identification and Characterization of TriABC-OpmH, a Triclosan Efflux Pump of Pseudomonas aeruginosa Requiring Two Membrane Fusion Proteins[down-pointing small open triangle]

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.

The biocide triclosan has been used for over 30 years, first in hospital settings but more recently in an increasing number of consumer products (7, 26). It was originally thought to act as a nonspecific biocide by affecting membrane structure and function, and resistance development was, therefore, thought to be highly unlikely (38, 53). However, this view changed after triclosan was shown to inhibit a specific cellular target, the enoyl-acyl carrier protein reductase (FabI or its homologs), in several bacteria (21, 23, 24, 34, 36). In the relatively short period since this discovery in 1998, it has become evident that bacteria use multiple mechanisms to develop triclosan resistance, including target mutations, increased target expression, enzymatic degradation, cellular exclusion, and active efflux from the cell. These are the same mechanisms that bacteria use to develop resistance to clinically significant antibiotics, raising the fear that, in certain instances, indiscriminate and imprudent use of this biocide may select for resistance against clinically useful drugs (30, 31, 48, 52, 54, 59, 67).

FabI target mutations have been isolated and characterized in bacteria as diverse as Escherichia coli (23, 36), Staphylococcus aureus (6, 20, 21), and Mycobacterium smegmatis (in mycobacteria, triclosan inhibits the FabI homolog InhA [21]). In Mycobacterium tuberculosis, increased InhA target expression was shown to confer triclosan resistance (61). Enzymatic degradation of triclosan has been demonstrated in two soil bacteria, Pseudomonas putida TriRY and Alcaligenes xylosoxidans subsp. denitrificans TR1, which were shown to grow on medium containing 1% triclosan (37). Triclosan degradation has also been shown in Sphingomonas sp. strain RD1, where loss of the ability to mineralize triclosan resulted in susceptibility to this biocide (28). While the authors are not aware of any bacteria that can modify triclosan, O-glucosyation and O-xylosylation of this biocide have been demonstrated in at least two fungi (25), and we should not be too surprised to find similar modification mechanisms in bacteria. Biofilm-grown Salmonella enterica serovar Typhimurium was triclosan tolerant, and it was suggested that Salmonella bacteria within biofilms could experience reduced influx (64). Small-colony variants of Staphylococcus aureus were described as a novel mode of evasion of susceptibility to antiseptics such as triclosan (3). Active efflux of triclosan from the cell has been demonstrated in many bacteria, including E. coli (35), Stenotrophomonas maltophilia (55), Campylobacter jejuni (32), Salmonella enterica (5), Bacteroides fragilis (51), P. aeruginosa (15), and others. In highly triclosan-resistant bacteria, several of these triclosan resistance mechanisms act synergistically. In P. aeruginosa, for example, high-level resistance (>1 mg/ml) can be solely achieved by efflux (15), but the presence of a triclosan-resistant enoyl-acyl carrier protein reductase (FabK) (22, 24) and perhaps other mechanisms (9) are probably also contributing factors.

Active efflux from the cell is a common mechanism for biocide and antibiotic resistance in many bacteria (5, 30, 48, 51, 55, 65), especially in organisms such as P. aeruginosa (15). We have previously demonstrated that triclosan is an excellent substrate for many of the clinically significant multidrug efflux pumps of the resistance nodulation cell division (RND) family of P. aeruginosa, including MexAB-OprM, MexCD-OprJ, MexEF-OprN, and MexJK-OpmH (15). Triclosan can be readily used as a surrogate for antibiotic substrates to select regulatory mutants overexpressing RND efflux pumps that are normally not expressed or only at very low levels, in many instances causing cross-resistance between triclosan and antibiotics (14, 17, 55). In this study, we sought to obtain triclosan-resistant mutants other than efflux pump-expressing strains by exposure of a triclosan-susceptible strain deficient in five known RND pumps. Such mutants were readily obtained, and the triclosan resistance determinant was surprisingly found to be yet another RND efflux pump, TriABC. Compared to previously described P. aeruginosa RND pumps mediating triclosan efflux, however, this pump is unique in that it is a triclosan-specific pump requiring two membrane fusion proteins for function and that pump expression in the resistant strain is caused by a promoter-up mutation.

MATERIALS AND METHODS

Bacterial growth and media.

Bacterial strains used in this study are listed in Table Table1.1. LB broth Miller and LB agar Miller from EMD Chemicals (Gibbstown, NJ) were routinely used as the rich media for all bacterial strains. Unless otherwise noted, antibiotics were added at the following concentrations: 100 μg/ml ampicillin (Ap), 35 μg/ml kanamycin (Km), and 10 μg/ml tetracycline (Tc) for E. coli; 200 μg/ml carbenicillin (Cb) and 15 μg/ml gentamicin (Gm) for P. aeruginosa. Antibiotics were either purchased from Sigma-Aldrich, St. Louis, MO (ampicillin and kanamycin), EMD Biosciences, San Diego, CA (gentamicin), or Gemini Bioproducts, Sacramento, CA (carbenicillin). Triclosan was purchased from KIC Chemicals (Armonk, NY). Hexachlorophene and dichlorophene [2,2′-methylenebis(4-chlorphenol)] were purchased from Sigma-Aldrich and TCI America, Portland, OR, respectively.

TABLE 1.
Strains and plasmids used in this study

MIC determinations.

MICs for triclosan were determined using an agar incorporation method (15). Triclosan was dissolved in 75% 2-methoxyethanol and added to Mueller-Hinton II agar from Becton Dickinson (Franklin Lakes, NJ) at increasing concentrations. Cells were pregrown in LB medium until mid-log phase (optical density at 600 nm, ~0.7) and then standardized using a 0.5 McFarland standard in saline. The standardized cells were applied on the plates using a 48-pin applicator. The MICs were recorded after incubation at 37°C for 15 to 16 h. MICs for other drugs and heavy metal ions were determined in Mueller-Hinton broth from Becton Dickinson by the twofold broth microdilution method.

Isolation of triclosan-resistant mutants.

The triclosan-resistant mutant PAO509.5 was isolated in several steps. First, PAO509 was grown in LB medium containing 4 μg/ml triclosan. The resulting PAO509.1 was then grown in LB medium containing 6 μg/ml triclosan, which yielded PAO509.2. The selection steps were repeated using LB medium containing 8 μg/ml, 10 μg/ml, and 12 μg/ml, which yielded PAO509.3, PAO509.4, and PAO509.5, respectively.

Cloning and identification of the triclosan resistance determinant.

For cloning of DNA fragments carrying the triclosan resistance-conferring chromosomal region from PAO509.5, the bacteriophage mini-D3112-based in vivo cloning method of Darzins and Casadaban (18) was used with the following modifications. Strain PAO505.9 was lysogenized with D3112cts, the resulting strain was transformed with pADD948, and a lysate was prepared by heat induction (19). Cells of recipient PAO509 were grown overnight and infected with the mixed lysate as previously described (56). The samples were spread on LB plates containing 30 μg/ml of triclosan and incubated at 30°C. Colonies growing on these plates after 36 h were purified on the same medium and analyzed for the presence of recombinant plasmids. The plasmid-borne nature of Cb resistance (Cbr) and triclosan resistance (Trir) was confirmed by electroporation of PAO509. One Cbr- and Trir-conferring plasmid was retained and named pPS1680.

The Trir determinant on pPS1680 was localized by in vitro transposon mutagenesis with the EZ-Tn5 <TET-1> insertion kit from Epicenter (Madison, WI) according to the manufacturer's protocol. The mutagenized plasmid pool was isolated from E. coli Apr and Tcr transformants and an aliquot electroporated into PAO509. Cbr transformants were selected and screened for loss of Trir. Plasmids were prepared by using the QIAprep Spin Miniprep kit from QIAGEN (Valencia, CA) and the transposon insertion sites determined by nucleotide sequencing. Sequencing reactions were primed by using the TET-1 FP-1 forward primer and the TET-1 RP-1 reverse primers from the EZ-Tn5 <TET-1> insertion kit. Homologous sequences were identified by using online BLAST searches of the National Library of Medicine databases.

Subcloning of triABC.

pPS1680 was digested with NotI and NruI (location of restriction sites used for subcloning and extents of DNA contained on individual plasmids are shown in Fig. Fig.1),1), and the desired DNA fragment was isolated from the agarose gel using the QIAquick gel extraction kit from QIAGEN (Valencia, CA). The isolated 5,747-bp DNA fragment was ligated into pBluescript (pBSP) II KS(−) (60), which was digested with EcoRV and NotI. Recombinant plasmids were selected and maintained in E. coli pcnB strain GBE180 (47) to reduce plasmid copy number and minimize possible toxic effects of cloned and expressed DNA segments on the host cell. Plasmid DNA was isolated from transformants and confirmed by restriction enzyme digestion, and the resulting triA+B+C+ plasmid was named pPS1824. In pPS1824 and its derivatives, the triABC operon or the individual tri genes are transcribed from the lac promoter. Next, pPS1947 (triA+) was derived from pPS1824 by digestion with SacII, followed by religation of a 5,217-bp DNA fragment. For construction of pPS1948 (triA+B+), pPS1824 was digested with BamHI and the largest 6,392-bp fragment religated. The triB-expressing pPS1950 was obtained by digesting pPS1948 DNA with SalI, which deleted most of triA, followed by religation of the largest 5,674-bp DNA fragment. The triB+C+-expressing pPS1954 was derived by ligating a 3,338-bp BamHI fragment from pPS1824 encompassing most of triC into the unique BamHI site of triB+ pPS1950. The triA+C+-expressing pPS2140 was derived in multiple steps. First, triB was deleted from pPS1948 by digestion with BbsI, followed by blunt ending with T4 DNA polymerase and self-ligation of the resulting 5,896-bp fragment, resulting in triA+ pPS2139. Second, a 3,338-bp BamHI fragment from pPS1824 encompassing most of triC was ligated into the unique BamHI site of pPS2139 to yield pPS2140. The triC+ pPS2071 was derived from pPS1824 in two steps. First, a 4,438-bp SacII fragment containing triC was isolated and ligated into the SacII site of pBSP II KS(−) to form pPS2045. Second, pPS2045 was digested with SacI and SpeI [both of these sites are from the pBSP II SK(−) polylinker], and the resulting 4,464-bp fragment was ligated between the same sites of pBSP II SK(−) (60), which yielded pPS2071.

FIG. 1.
Genetic organization of the triABC region of the P. aeruginosa chromosome. (A) The triABC operon contains genes for two membrane fusion proteins (triA and triB) and an RND transporter (triC). The operon is flanked by two genes that encode transcriptional ...

For complementation analyses, recombinant plasmids were transformed into P. aeruginosa mutant strains by electroporation using a rapid procedure (11).

Single-copy complementation was achieved by cloning DNA fragments into suitable mini-Tn7 vectors, followed by integration into the single attTn7 site on the P. aeruginosa chromosome utilizing established procedures (10, 12). Specifically, a 6,592-bp StuI [from the pBSP II SK(-) polylinker]-BamHI fragment containing triABC was subcloned into pUC18-mini-Tn7T-Gm to yield pUC18-mini-Tn7T-Gm-triA+B+C+ (or pPS2068), on which the triABC operon is expressed from its own wild-type promoter. This was achieved in two steps. First, a 3,254-bp StuI [from the pBSP II SK(−) polylinker]-BamHI fragment containing triAB plus endogenous promoter from pPS1834 was subcloned between the same sites of pUC18-mini-Tn7T-Gm to form pPS2063 (or pUC18-mini-Tn7T-Gm-triA+B+). Note that pPS1834 was derived from pPS1680 by subcloning a 6,816-bp ScaI-NotI fragment containing the triABC operon and its upstream pcaR gene between the EcoRV and NotI sites of pBSP II SK(−). Second, a 3,338-bp BamHI fragment containing most of triC was subcloned from pPS1834 into the single BamHI site of pPS2063 to form pPS2068 (or pUC18-mini-Tn7T-Gm-triA+B+C+). A mini-Tn7T-Gm-triA+B+C+ construct expressing the triABC operon from the PAO509.5 mutant promoter (pPS2155) was constructed by exchanging a 906-bp StuI [from the pBSP II SK(−) polylinker]-NruI fragment containing the respective promoter sequences. Mini-Tn7 elements expressing triA from its native promoter and triC from the tac promoter were constructed as follows. pUC18-mini-Tn7T-Gm-GW-triA+ (pPS2035) was constructed using Gateway technology. First, the triA gene was amplified using primers pcaR-156-attB2 and triA-attB1 (PCR primer sequences are available from the authors upon request), and the PCR product was recombined into pDONR221 (Invitrogen) by the BP clonase reaction to form pPS2033. Second, the LR clonase reaction was then used to transfer the triA gene from pPS2033 into pUC18-mini-Tn7T-Gm-GW to form pPS2035 (or pUC18-mini-Tn7T-Gm-GW-triA+). In this plasmid, the triA gene is transcribed from its own promoter. For construction of a mini-Tn7-triC+ element, the triC gene was cloned as a 4,464-bp SacI-SpeI fragment [both sites are from the pBSP II SK(−) polylinker] fragment from pPS2045 between the SacI and SpeI sites of pUC18-mini-Tn7T-LAC to form pUC18-mini-Tn7-LAC-triC+ (pPS2072), where triC is transcribed from the tac promoter.

Deletion of chromosomal genes.

Gene disruptions by deletion were performed using a previously described method (13). Briefly, three partially overlapping DNA fragments representing flanking DNA segments and the Gmr marker from pPS856 (13) were amplified separately and then spliced together by overlap extension PCR. The resulting recombinant DNA fragments were cloned into pDONR221 (Invitrogen) and then recombined into the Gateway-compatible gene replacement vector pEX18ApGW (13). The plasmid-borne deletions were next transferred to the P. aeruginosa chromosome by homologous recombination and merodiploids resolved by sucrose counterselection. Finally, unmarked deletion mutants were obtained by Flp-mediated excision of the Gmr marker (13). Deletions were within coding sequences removing codons 15 to 330 of triA, 38 to 269 of triB, and 11 to 980 of triC, respectively, in triA, triB, and triC single mutants. The triAB deletion extended from codon 15 of triA through codon 269 of triB. Similarly, the triABC deletion extended from codon 15 of triA through codon 980 of triC. Deletions were nonpolar as judged by quantitative reverse transcription-PCR (qRT-PCR) analyses of downstream gene expression, where applicable. The opmH gene was deleted from several strains utilizing the previously described pPS1283 gene replacement plasmid (16).

Quantitative real-time PCR.

The cells were grown at 37°C in LB medium (with antibiotic addition for plasmid-containing strains) to mid-log phase (optical density at 600 nm, ~0.7). Total RNA was extracted from 1 ml of culture using the RNeasy Mini kit from QIAGEN (Valencia, CA). One μg of RNA was treated with amplification-grade DNase I from Invitrogen (Carlsbad, CA), and cDNA was synthesized using the SuperScript III First-Strand Synthesis SuperMix for qRT-PCR from Invitrogen. cDNA samples were diluted 1:100 in sterile water before analysis. Quantitative real-time RT-PCR was performed with an iQ5 real-time PCR detection system (Bio-Rad, Hercules, CA) using Invitrogen's SYBR GreenER qPCR SuperMix for iCycler. Primers were designed using the OligoPerfect primer designing tool from Invitrogen. All primer pairs were subjected to melt-curve analysis to rule out secondary products and primer-dimer formation prior to use. The standard curve method was used for the relative quantification following an instructions manual. Standard curves were constructed using a series of DNA concentrations of extracted chromosomal DNA from a PAO509 culture.

Assays of triABC promoter activity using β-galactosidase fusions.

Putative triABC promoters were predicted using the Neural Network Promoter Prediction program (http://www.fruitfly.org/seq_tools/promoter.html). DNA fragments containing the predicted promoter regions of PAO509 and PAO509.5 were amplified by PCR and cloned into pDONR221. The resulting plasmids (pPS1920 with the PAO509 promoter region and pPS1921 with the PAO509.5 promoter region) were used as the donor plasmid of the LR recombination reaction with the recipient plasmid pUC18-mini-Tn7T-Gm-lacZ-GW, resulting in pPS1930 and pPS1931, respectively. The mini-Tn7T-Gm-lacZ elements contained on these plasmids were transposed into the chromosomes of PAO509 and PAO509.5 by coelectroporation with pTNS2, insertion events were verified by PCR, and the Gmr marker was removed by Flp-mediated excision following previously described methods (10, 12). Control strains contained promoterless mini-Tn7T-Gm-lacZ integrated in the chromosome. β-Galactosidase (β-Gal) activity in LB-grown cells was measured, and activity units were calculated as described by Miller (40).

RESULTS

Isolation of a triclosan-resistant mutant.

We previously reported that P. aeruginosa mutants expressing multidrug RND efflux pumps could easily be obtained by exposure of triclosan-susceptible efflux pump deletion strains to this antimicrobial (14, 17). In an attempt to elucidate triclosan resistance mechanisms other than multidrug RND pumps, we therefore decided to isolate triclosan-resistant derivatives of PAO509, a strain defective in all triclosan-exporting efflux pumps known to date, to relatively low levels of triclosan. First, PAO509 was grown in medium containing 4 μg/ml triclosan. The resulting PAO509.1 was then grown in medium containing 6 μg/ml triclosan, which yielded PAO509.2. The selection steps were repeated using medium containing 8 μg/ml, 10 μg/ml, and 12 μg/ml, which yielded PAO509.3, PAO509.4, and PAO509.5, respectively. The MICs for triclosan for isolates obtained in the individual selection steps were 8 (PAO509.1), 64 (PAO509.2), >1,024 (PAO509.3), >1,024 (PAO509.4), and >1,024 (PAO509.5) μg/ml. These data suggested that the event(s) leading to high-level triclosan resistance occurred early in the selection process. Of the mutants thus obtained, PAO509.5 was retained for further studies.

Compared to its parental strain PAO509, PAO509.5 resistance levels to all antibiotics (e.g., chloramphenicol, ciprofloxacin, erythromycin, gentamicin, tetracycline, and trimethoprim) and metals (e.g., Cu2+, Co2+, Ni2+, and Zn2+) tested were not significantly changed. Resistance to hexachlorophene and dichlorophene [2,2′-methylenebis(4-chlorphenol)] was only marginally increased in PAO509.5 (MICs of 32 μg/ml and 256 μg/ml, respectively) compared to PAO509 (MICs of 16 μg/ml and 128 μg/ml, respectively). These results indicated that triclosan resistance in PAO509.5 was not mediated by a multidrug efflux pump but rather by a more-or-less triclosan-specific mechanism.

Identification of the PAO509.5 triclosan resistance determinant.

In order to identify the triclosan resistance determinant(s) in PAO509.5, a plasmid conferring triclosan resistance to the parental PAO509 strain was obtained by D3112-mediated in vivo cloning. The plasmid thus isolated, pPS1680, contained a ~30-kbp chromosomal DNA insert and confirmed triclosan resistance to PAO509 upon retransformation.

The triclosan resistance determinant on pPS1680 was mapped by EZ-Tn5 <TET-1> mutagenesis. One representative plasmid, pPS1681, which lost its ability to confer triclosan resistance to PAO509 compared to pPS1680, was retained for further studies.

Sequencing using transposon-specific primers indicated that it had been inserted 842 bp downstream from the initiation codon of the PA0158 gene, which was predicted to encode an RND efflux transporter protein by the Pseudomonas Genome Project (http://www.pseudomonas.com) (63). PA0158 is located downstream of PA0156 and PA0157, both of which have been predicted to encode membrane fusion proteins (MFPs) (Fig. (Fig.1)1) and which form an integral and essential part of tripartite RND efflux pumps. These three genes are organized in an operon. The PA0156-PA0157-PA0158 operon is the only P. aeruginosa RND pump-encoding operon that encodes two MFPs. The operon does not encode an outer membrane channel protein (OMP), but it is flanked by two genes encoding the predicted transcriptional regulators pcaR and PA0159, which are located upstream and downstream of the PA0156-PA0157-PA0158 operon, respectively (Fig. (Fig.1).1). Since this operon seemed to encode a triclosan-specific efflux pump, its three comprising genes were renamed triABC.

Identification of the triclosan resistance determinant.

To confirm that the triABC genes were indeed solely responsible for the triclosan resistance phenotype conferred by pPS1680, a DNA fragment containing only these genes was subcloned into the broad-host-range vector pBSP II KS(−). The resulting pPS1824 conferred high-level triclosan resistance on PAO509, which was comparable to that observed in PAO509.5 (Table (Table2).2). These results proved that the triclosan resistance determinant on pPS1680 was identical to the triABC operon.

TABLE 2.
TriABC expression in PAO509 and its derivatives

To further confirm that expression of triABC in PAO509.5 was responsible for the high-level triclosan resistance observed in this strain, the triABC genes were deleted and the triclosan susceptibility of the resulting ΔtriABC strain PAO1095 was assessed. When compared to PAO509.5, PAO1095 completely lost its triclosan resistance phenotype and, interestingly, was also more triclosan susceptible than PAO509 (Table (Table2).2). In fact, both PAO1095 (PAO509.5 ΔtriABC) and PAO1116 (PAO509 ΔtriABC) were less triclosan resistant than PAO509. These results confirmed that the triABC operon encodes a triclosan efflux pump.

Expression of TriABC in PAO509.5 is not due to mutations in adjacent regulatory genes.

As mentioned above, the triABC operon is flanked by pcaR and PA0159. PcaR is a putative β-ketoadipate pathway transcriptional regulator, which in concert with the LysR family transcriptional regulator PcaQ is probably responsible for regulation of expression of the pcaGH operon. This operon encodes the α- and β-subunits of β-protocatechuate-3,4-dioxygenase and is sandwiched between the pcaQ and pcaR genes on the P. aeruginosa chromosome (www.pseudomonas.com). PA0159 encodes a LysR family transcriptional regulator of unknown function.

To assess possible functions, if any, of these regulators in regulation of triABC operon expression, pcaR and PA0159 were deleted from the PAO509 and PAO509.5 chromosomes and triclosan susceptibilities of the resulting strains assessed as a measure of triABC expression. Deletion of pcaR from PAO509 did not cause increased TriABC expression and also had no effect on expression of this pump in PAO509.5 (Table (Table2).2). Similarly, deletion of PA1059 from either PAO509 or PAO509.5 had no effect on TriABC expression. The results show that neither pcaR nor PA0159 plays a significant role in TriABC expression in PAO509.5. Consistent with this notion were the findings that neither the PAO509.5 pcaR nor PA0159 genes contained any mutations compared to PAO509.

Expression of TriABC in PAO509.5 is due to a promoter-up mutation.

To test the possibility that mutations in the pcaR-triA intergenic region were responsible for TriABC overexpression in PAO509.5, these regions were amplified from the PAO509 and PAO509.5 chromosomes and sequenced. Sequence comparisons showed the presence of a single mutation, a C-to-T change, in the PAO509.5 intergenic region which was absent from the PAO509 sequence (Fig. (Fig.2).2). Analyses using the Neural Network Promoter Prediction program (http://www.fruitfly.org/seq_tools/promoter.html) indicated that this mutation occurred in a predicted promoter and that it resulted in a better promoter, because its calculated score (0.96) was higher than that of the one found in PAO509 (0.78). This is consistent with the finding that the C-to-T change resulted in a predicted −35 sequence that is closer to consensus (Fig. (Fig.2).2). Sequence analyses of the promoter regions in strains revealed wild-type (PAO509) promoter regions in PAO509.1 and PAO509.2 and the mutant promoter in PAO509.3, PAO509.4, and PAO509.5. These findings confirmed that the mutation leading to high-level triclosan resistance occurred very early in the multistep selection process and suggest that strains PAO509.3, PAO509.4, and PAO509.5 may in fact be the same strains.

FIG. 2.
Wild-type and mutant triABC operon promoter sequences. Predicted triABC operon promoter sequences of strains PAO509 and PAO509.5 are shown. The single base change in the putative −35 promoter region is marked with an arrow. The σ70 promoter ...

To ascertain that the single base change observed in the putative triA promoter region was responsible for the high-level triclosan resistance observed in PAO509.5, single-copy constructs expressing the triABC operon from either the PAO509 wild-type or the PAO509.5 mutant promoter were integrated into the PAO1116 (PAO509 with ΔtriABC) chromosome, and triclosan MICs were determined (Table (Table3).3). It is evident that the presence of the promoter mutation was sufficient for expression of high-level triclosan resistance. Transcription levels of triC contained on the chromosomally integrated expression constructs driven from the PAO509 or PAO509.5 promoter were approximately the same as those observed in PAO509 and PAO509.5, respectively.

TABLE 3.
A promoter-up mutation is sufficient for high-level triABC expression

Increased transcription of the triABC operon in PAO509.5 versus PAO509 was confirmed by lacZ fusion and quantitative real-time RT-PCR analyses. From the results presented in Fig. Fig.33 it is evident that only fusion constructs expressing lacZ from the PAO509.5 promoter express high-levels of β-Gal activity, irrespective of whether they are present in the PAO509 or PAO509.5 chromosome. Fusion constructs containing the PAO509 promoter express low but detectable levels of β-Gal activity, indicating the presence of a much less active promoter. The lacZ fusion results were corroborated by qRT-PCR analyses of triC expression in PAO509 and PAO509.5 (Table (Table33).

FIG. 3.
β-Galactosidase fusion analysis of triABC wild-type and mutant promoters. Wild-type PAO509 (PPAO509) or mutant PAO509.5 (PPAO509.5) promoter sequences were fused to the β-galactosidase lacZ gene and integrated into the chromosomes of strains ...

To exclude the possibility that evolved pump proteins were contributing to the observed triclosan resistance phenotype, the entire 5,747-bp pPS1824 insert was sequenced, and the triABC sequences were found to be identical to those published for PAO1.

Summarily, the results show that the cause of high-level triclosan resistance in PAO509.5 is increased expression of the triABC operon due to a single promoter-up mutation.

TriABC requires OpmH for efflux pump function.

Work by several groups over the past few years has shown that RND efflux pumps function as tripartite complexes consisting of the RND transporter, an MFP, and an OMP. Because the triABC operon is devoid of an OMP-encoding gene, experiments were initiated to determine what OMP associates with TriABC for assembly of a functional triclosan efflux pump. We previously demonstrated that both OprM and OpmH are constitutively expressed in P. aeruginosa and could be used as an OMP in the assembly of functional MexAB-OprM and MexJK-OpmH triclosan efflux pumps (15, 16, 58), and so we set out to determine possible roles of either one of these proteins in TriABC efflux pump function.

When the triABC operon was expressed from the lac promoter present on pPS1824 in OpmH+ OprM strain PAO200 or OpmH+ OprM+ strain PAO1ΔmexAB, high-level triclosan efflux was observed (MIC, >1,024 μg/ml). In contrast, expression of triABC in OprM+ OpmH strain PAO1048 or OprM OpmH strain PAO1047 did not result in significant triclosan efflux (the observed MICs were 32 and 8 μg/ml, respectively). These findings indicated that TriABC associates with and requires OpmH, but not OprM, for assembly of a functional TriABC-OpmH triclosan efflux pump. Of note is the finding that triclosan MICs in the Δ(mexAB)-oprM+ ΔopmH strain PAO1048/pPS1824 were significantly higher than those observed in Δ(mexAB-oprM) ΔopmH strain PAO1047/pPS1824 (32 versus 8 μg/ml, respectively). A similar MIC difference (>1,024 versus 64 μg/ml) was also seen between PAO1ΔmexAB and PAO200 (Δ[mexAB-oprM]) containing pPS1824. This probably indicates interaction of OprM with yet another, unknown triclosan efflux pump in P. aeruginosa.

Both membrane fusion proteins are required for TriABC efflux pump function.

The triABC operon is the only RND pump-encoding operon in P. aeruginosa which encodes two MFPs, TriA and TriB. To assess whether both MFPs are required for triclosan efflux, we constructed strains with various combinations of mutations in the PAO509.5 MFP-encoding genes and assessed efflux pump function by determining triclosan MICs (Table (Table44).

TABLE 4.
TriA and TriB MFPs are both required for TriABC efflux pump function

It is evident that deletions of either triA (PAO1092), triB (PAO1093), triAB (PAO1210), or triC (PAO1094) led to loss of triclosan resistance, just as did deletion of the entire triABC operon (PAO1095). These mutations could only be fully complemented by plasmids expressing the entire triABC operon, but not by plasmids expressing the individual genes or combinations of individual genes other than the complete operon. However, selected mutations could be partially complemented with the respective cloned genes, e.g., ΔtriC with triC-expressing pPS2071 or ΔtriAB with triAB-expressing pPS1948. qRT-PCR analyses revealed that lack of complementation was not due to polar effects on downstream gene expression in the mutants or due to lack of transcription of genes from plasmid or single-copy expression constructs. Gene dosage—multicopy versus single-copy gene expression—also had no effect on complementation efficacy.

Because at present no antibodies against any of the Tri proteins are available, we attempted to visualize individual pump components in whole-cell lysates separated and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a measure of actual protein expression. However, these attempts failed, presumably due to low expression levels bestowed by the relatively low copy number (10 to 15 copies per cell) of the expression constructs used in this study.

Although we have no experimental proof for protein expression in plasmid-containing strains, the results obtained with nonpolar deletion mutants and successful partial complementation of ΔtriAB and ΔtriC mutants with TriAB- and TriC-expressing plasmids indicated that TriABC requires both MFPs, TriA and TriB, for triclosan efflux.

DISCUSSION

While some bacteria, for example E. coli and S. aureus, are highly triclosan susceptible, P. aeruginosa exhibits high intrinsic triclosan resistance. In wild-type P. aeruginosa PAO1, the constitutively expressed MexAB-OprM RND pump alone confers high-level triclosan resistance (MIC, >1 mg/ml) (15, 57). However, despite the finding that high-level resistance can be achieved solely by efflux (15), the presence of a triclosan-resistant enoyl-ACP reductase enzyme FabK (22, 24) and other yet-unknown mechanisms are probably also contributing factors. To probe this notion, we constructed a P. aeruginosa strain defective in 5 of the 11 RND efflux pumps and selected triclosan-resistant mutant derivatives by exposure to increasing concentrations of triclosan. Mutants resistant to high levels of triclosan were readily obtained early in the selection process, and one of these, PAO509.5, was retained for further analysis.

The triclosan resistance determinant in this strain was identified as a to-date-uncharacterized RND family efflux transporter encoded by PA0156-PA0157-PA0158, which during genome annotation was denoted as a transporter of small molecules and/or divalent metal cations. However, the mutant overexpressing this efflux pump did not export any of the divalent cations tested or any antibiotics or even substances closely related to triclosan, for example, dichlorophene or hexachlorophene. Since triclosan is the only substrate for this pump known to date, it was renamed TriABC.

The components of the TriABC efflux system are most closely related to those found in the P. aeruginosa MexJK (17) RND efflux pump. The TriA and TriB MFPs share 34% and 33% identity and 51% and 49% similarity, respectively, with MexJ. The TriC RND transporter was 46% identical and 65% similar to MexK. Interestingly, both TriABC and MexJK have a narrow substrate spectrum and associate with OpmH, but not OprM, to form functional TriABC-OpmH and MexJK-OpmH triclosan efflux pumps. This may be indicative of similar OpmH interaction domains in TriC and MexK, respectively, but very little is known about domains governing OMP-RND transporter interactions, and amino acid sequence alignments did not reveal any obvious consensus sequences and/or domains that may be involved in such interactions (not shown).

Of the 11 RND pumps encoded by the P. aeruginosa chromosome, TriABC is the only RND efflux pump whose operon contains two MFP-encoding genes. These MFPs are remarkably different in that TriA has only 36% identity and 51% similarity with TriB.

Structure analyses of AcrB (42) and TolC (29) revealed that they both form homotrimers, but despite structure information for AcrA (39) and MexA (1) the association states of these MFPs remain unknown. While soluble AcrA and P. aeruginosa MexA are both monomeric in vitro (1, 68), in vivo cross-linking studies with AcrA suggested a trimeric form (69). If MFPs interacted with the RND transporter and the OM channel protein in a 3:3:3 complex, it would be difficult to envision how the two quite distinct TriA and TriB MFPs interact individually with TriC and OpmH in the formation of the efflux channel. Since both MFPs are required for pump function, one might either envision interaction of the TriAB heterotrimer or perhaps TriA or TriB homotrimer complexes with TriC and OpmH. Heteromultimeric RND pumps have previously been described. For example, the MdtABC system of E. coli consists of the transmembrane MdtBC heteromultimeric RND transporters and the MdtA MFP (2, 43). While the MdtBCA-TolC complex effluxes bile salts and novobiocin, the MdtAC-TolC complex only confers bile salt resistance. It was speculated that the evolution from a MdtC homomultimer to a MdtBC heteromultimer contributed to extend the substrate spectrum. Though some promiscuity exists in assembly of efflux pumps from components not encoded by the respective operons, functional heteromultimers are not too common. For example, though AcrAB of E. coli can assemble with P. aeruginosa OprM into a AcrAB-OprM complex, it is not functional unless the OprM hairpin domain is replaced with the corresponding domain from AcrA (62). Similarly, even though P. aeruginosa MexJK can associate with both OprM and OpmH, the assembly of functional MexJK-OprM erythromycin or MexJK-OpmH triclosan efflux systems is determined by the presence of the respective substrates (16). One could therefore speculate that the assembly of functional TriABC-OpmH homomultimeric or heteromultimeric pumps is dependent on triclosan or other yet-unidentified substrates, with triclosan favoring assembly of a TriABC-OpmH heteromultimeric pump. Surprisingly, mutations in the individual triABC operon genes could not be efficiently complemented with the respective individual genes or gene combinations other than the intact triABC operon. This was not due to gene dosage imbalances, since both single-copy and multicopy constructs gave similar results. This complementation failure by individual genes provided in trans may reflect the necessity for tight transcriptional coupling of the three overlapping genes encoded by the triABC operon.

Regulation of efflux pump expression in laboratory-derived mutants and clinical isolates is achieved by mechanisms that fall broadly into four groups: (i) mutations in local repressor or, more rarely, activator genes; (ii) mutations in global regulatory genes; (iii) mutations in the regulatory regions of the efflux pump-encoding gene(s); and (iv) insertion elements upstream of the efflux pump-encoding gene(s) (reviewed in reference 46). Most of these mechanisms have been implicated in regulation of P. aeruginosa RND efflux pump expression (reviewed in reference 46). For example, mutations in the local repressor genes mexR, nfxB, and mexL cause overexpression of MexAB-OprM, MexCD-OprJ, and MexJK, respectively (17, 49, 50). NalC and NalD are encoded by distantly located genes and negatively regulate expression of mexAB-oprM (8, 41). Additional evidence for the presence of global efflux pump regulatory genes in P. aeruginosa was obtained by analysis of mutants that simultaneously expressed MexAB-OprM and MexXY (33) and by the discovery of a SoxR homolog which among other genes regulates expression of an efflux pump implicated in quorum-sensing signal homeostasis (45). MexAB-OprM expression in a clinical P. aeruginosa isolate was attributed to insertion of an insertion sequence element in mexR (4). This is to our knowledge the first report demonstrating high-level efflux pump expression in P. aeruginosa caused solely by a promoter-up mutation. Although there are numerous reports of S. aureus NorA expression caused by mutations in the norA regulatory region (27, 44), such mutations seem to play minor roles in P. aeruginosa RND efflux pump gene expression and, perhaps more broadly, in efflux pump gene expression in gram-negative bacteria. While mutations in local regulatory genes had no effects on triABC expression, the possible involvement of global regulators and/or other mechanisms in regulation of triABC operon expression cannot be ruled out at the present time. In fact, although transfer of the promoter-up mutation into a virgin strain clearly demonstrated that it is the determinant for the observed high-level triABC expression in PAO509.5, and therefore also PAO509.3 and PAO509.4, other unknown contributing factors may explain the low-level MIC (64 μg/ml) observed in PAO509.2.

Besides MexAB-OprM, MexCD-OprJ, MexEF-OprN, and MexJK-OpmH, TriABC is the fifth RND pump in P. aeruginosa shown to efficiently efflux triclosan, further supporting the notion that efflux is the primary mechanism responsible for this bacterium's high intrinsic and acquired triclosan resistance.

Acknowledgments

This work was supported by NIH grant AI051588.

Footnotes

[down-pointing small open triangle]Published ahead of print on 24 August 2007.

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