SmeOP-TolCSm Efflux Pump Contributes to the Multidrug Resistance of Stenotrophomonas maltophilia.

SmeOP-TolCsm Efflux Pump Contributes to the Multidrug Resistance of Stenotrophomonas maltophilia

Cheng-Wen Lin

, Yi-Wei Huang

, Rouh-Mei Hu

, and Tsuey-Ching Yang

1

Department of Medical Laboratory Science and Biotechnology, China Medical

University, Taichung 404, Taiwan

2

Department of Biotechnology and Laboratory Science in Medicine, National Yang-

Ming University, Taipei, 112, Taiwan

3

Department of Biotechnology, Asia University, Taichung, 413, Taiwan

4

Department of Biomedical Informatics, Asia University, Taichung, 413, Taiwan

Running title: SmeOP-TolCsm of S. maltophilia

For correspondence.

E-mail: tcyang@ ym .edu.tw

Mailing address: 155 Section 2, Lie-Nong Street, Taipei, 112, Taiwan, R.O.C.

Tel: 886-2-28267289 Fax: 886-2-28264092 Abstract

A five-gene cluster, tolCsm-pcm-smeRo-smeO-smeP, of Stenotrophomonas 1

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maltophilia was characterized. The presence of smeOP and smeRo-pcm-tolCsm

operons was verified by RT-PCR. Both operons were negatively regulated by the TetR-type transcriptional regulator SmeRo, as demonstrated by quantitative-RT-PCR and promoter-fusion assay. SmeO and SmeP were associated with TolCsm for assembly of a RND-type pump. The compounds extruded by SmeOP-TolCsm mainly included nalidixic acid, doxycycline, amikacin, gentamicin, erythromycin, leucomycin, carbonyl cyanide 3-chlorophenylhydrazone, crystal violet, sodium dodecyl sulfate, and tetrachlorosalicylanilide.

Keywords: bacteria, antibiotics resistance, efflux pump

Multidrug efflux transporters capable of active extrusion of noxious compounds are classified into five families, including Resistance Nodulation cell Division (RND) family, Major Facilitator Superfamily (MFS), Small Multidrug Resistance (SMR) 22

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family, ATP Binding Cassette (ABC) family, and Multidrug and Toxic compound Extrusion (MATE) family (1). The RND systems generally form tripartite components composed of a periplasmic membrane fusion protein (MFP), an inner

membrane RND transporter, and an outer membrane protein (OMP) (2).

Stenotrophomonas maltophilia is a non fermentative Gram-negative bacillus.

Eight RND-type efflux systems, SmeABC, SmeDEF, SmeGH, SmeIJK, SmeMN, SmeOP, SmeVWX, and SmeYZ, are postulated in the S. maltophilia genome (3).

Among them, the SmeABC, SmeDEF, SmeIJK, SmeVWX, and SmeYZ have been characterized (4-7). A possible promiscuous OMP TolCsm involved in the multidrug resistance has been proposed (8). Referring from the genome sequence of S.

maltophilia, we noted that the tolCsm gene and the smeOP system are located nearby

(Fig. 1), implying that smeOP and tolCsm may be involved in a common mechanism for the antibiotics extrusion. SmeO and smeP were predicted to encode a MFP and a RND-type inner membrane transporter. A TetR-type transcription regulator (annotated as SmeRo hereafter) was located immediately upstream of the smeOP and divergently transcribed. The genes downstream of the smeRo were the pcm-tolCsm operon, which has been known to contribute to multidrug resistance (8).

SmeRo acts as a regulator for the expression of smeOP

To evaluate the regulatory role of smeRo, a smeRo deletion mutant, KJRo, was 42

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constructed. A 359-bp DNA fragment (27-bp N-terminus of pcm, intergenic region of pcm and smeRo, and 198-bp C-terminus of smeRo) and a 434-bp DNA fragment (276-

bp N-terminus of smeRo, intergenic region of smeRo and smeO, and 33-bp N terminus of smeO) were amplified with primers SmeRo3-F/SmeRo3-R and SmeRo5- F/SmeRo5-R (Fig. 1, Table S1), respectively, and sequentially cloned into pEX18Tc, yielding plasmid pRo for the construction of KJRo. The resultant in-frame deletion mutant, KJRo, was with an internal deletion in the smeRo gene from nt 273 to nt 494. The transcripts of smeO, smeP, pcm, and tolCsm in KJ and KJRo were determined by qRT-PCR. The qRT-PCR was carried out at least in triplicate (6) and the primers used are listed in Table S1. The transcripts of smeO, smeP, pcm, and tolCsm in KJRo were greater than those in KJ by a factor of 4.6  2.1, 3.8  1.9, 1.5

 0.6, and 1.4  0.5, respectively. The effect of smeRo deletion on the expression of pcm and tolCsm was notably minor. Therefore, SmeRo played a regulatory role, presumably a repressor, in the expression of smeOP.

SmeRo, pcm, and tolCsm form an operon

Based on the genetic organization, we considered the possible presence of the smeRo- pcm-tolCsm operon. To test it, an RT-PCR analysis was performed on the mRNA extracted from KJ and KJRo. Primer TolCQ-R (Table S1, Fig. 1B) was used for the 62

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obtainment of cDNA. A 670-bp amplicon I (primered by 23-F/23-R) and an 1123-bp amplicon II (primered by 123-F/23-R) were detected in KJRo, but only the amplicon I was observed in KJ (Fig. 1B). KJRo is an in-frame deletion mutant with an internal deletion in smeRo gene from nt 273 to nt 494. The primers of 123-F and 23-R targeted onto the 221-241 nts of the smeRo gene and the 48-65 nts of the tolCsm gene (Fig.

1B). Therefore, an 1123-bp amplicon II can be amplified only if smeRo-pcm-tolCsm are co-transcribed. A smeRo-pcm-tolCsm transcript was observed in KJRo, but not in KJ even though the number of PCR cycles was increased to 40 (Fig. 1B). These observations support that a smeRo-pcm-tolCsm transcript was slightly expressed in KJRo and a pcm-tolCsm transcript was expressed in KJ and KJRo.

Assays of promoter activities of smeRo-pcm-tolCsm and smeOP operons

To further study the regulatory circuits, transcriptional xylE fusions to promoters of the smeOP operon (pSmeO

), smeRo-pcm-tolCsm operon (pSmeRo

), pcm- tolCsm operon (pPCM

), and tolCsm (pTolC

) were constructed (Fig. 1A) and introduced into KJ and KJRo, respectively. The expressed C23O activities were monitored, as described previously (9). KJRo(pSmeO

) exhibited a higher C23O activity than KJ(pSmeO

) (Table 1), further confirming that SmeRo plays a repressor role in the expression of smeOP operon. Compared to KJ(pSmeRo

), 81

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KJRo(pSmeRo

) had a slightly higher C23O activity (Table 1), which signifies the slightly negative autoregulation of smeRo. KJ(pPCM

) and KJRo(pPCM

) exhibited comparable C23O activities, indicating that the promoter of the pcm-tolCsm operon is constitutively active and not subjected to the regulation of SmeRo. No significant C23O activity was observed in KJ(pTolC

) and KJRo(pTolC

), supporting that there is no promoter activity in the 243-bp upstream region of the tolCsm gene.

Substrate spectrum of the SmeOP efflux pump

A 1405-bp DNA fragment containing partial C-terminus of smeP, amplified with primers SmeP3-F and SmeP3-R, and the aforementioned 434-bp DNA fragment (primered by SmeRo5-F/SmeRo5-R) were sequentially cloned into pEX18Tc, yielding plasmid pOP (Fig. 1A, Table S1). The SmeOP in-frame deletion mutant, KJOP, was obtained using the same strategy described in Ref. 6. The 1103-bp C- terminus of smeO gene and 2802-bp N-terminus of smeP gene in mutant KJOP were deleted (Fig. 1A). The substrate spectrum of SmeOP was assessed by comparing the antimicrobials susceptibilities between KJ and KJOP as well as KJRo and KJRoOP (Table 2). Susceptibility assay was tested by the agar dilution method, as described previously (6), at least three replicates. The results concluded that SmeOP 100

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was responsible for the extrusion of nalidixic acid, doxycycline, aminoglycosides, macrolides, carbonyl cyanide 3-chlorophenylhydrazone (CCCP), crystal violet, sodium dodecyl sulfate (SDS), and tetracholorsalicylanilide (TCS). Among them, aminoglycosides, CCCP, and TCS were the most significant ones

Complementation test for smeRo mutant

To confirm whether the phenotype observed in KJRo was due to inactivation of the smeRo gene, plasmid pSmeRo (containing the full-length smeRo) was introduced into

the KJRo, yielding KJRo(pSmeRo). The transcripts of smeO, smeP, pcm, and tolCsm in KJRo(pSmeRo) were lower than those in KJRo(pRK415) by a factor of

2.6  1.1, 2.3  1.0, 1.1  0.4, and 1.1  0.5, respectively. The susceptibility of KJ(pRK415), KJRo(pRK415), and KJRo(pSmeRo) to aminoglycoside, CCCP, and TCS was tested by the agar dilution method and the disk diffusion assay. The complementation of KJRo with pSmeRo increased the resistance to aminoglycoside, CCCP, and TCS (Table S2).

SmeOP requires TolCsm for efflux pump function

Inactivation of the smeOP operon decreased the MICs to some antibiotics (Table 2), indicating that the smeOP and its cognate OMP gene should be expressed constitutively in the wild-type KJ. Four possible OMP candidates, SmeC, SmeF, 119

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SmeX, and TolCsm for the RND-type efflux pumps, have been proposed (8). Of the four OMPs, the transcripts of smeF and tolCsm were observed and no significant transcripts of smeC and smeX were detected by the assessment of RT-PCR (Fig. S1).

Therefore, SmeC and SmeX were less likely to be the cognate OMP of the SmeOP efflux pump. To assess the possibility of SmeOP-SmeF, the smeDEF operon was deleted from the chromosomes of strains KJ and KJOP, generating mutants KJDEF and KJDEFOP, respectively. Compared to KJDEF, KJDEFOP obviously compromised its resistance to aminoglycoside and leucomycin (Table 2), indicating that the SmeOP pump is still functional in the case of smeF inactivation. The possibility of SmeOP-TolCsm was also evaluated. The susceptibility of KJTolC reported in our previous study (8) was also included in Table 3 for comparison. The introduction of smeOP into KJTolC did not further compromise the resistance of KJTolC to all compounds tested (Table 2, KJTolC vs. KJTolCOP), supporting that TolCsm is the cognate OMP for the SmeOP pump. Moreover, deletion of the tolCsm gene was associated with a greater decrease in MICs than those caused by

deletion of smeOP (Table 2), signifying the promiscuous role of TolCsm. TolCsm may not only participate in the function of the SmeOP pump, but also in the function

of other hitherto uncharacterized efflux systems.

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and P. aeruginosa, tolCsm of S. maltophilia locates in a pcm-tolCsm operon. The protein-L-isoaspartate O-methyltransferase (PCM) encoded by pcm is an enzyme that recognizes and catalyzes the repair of damaged L-isoaspartyl and D-aspartatyl groups in proteins.

PCM may thus involve in the repair of damaged TolCsm and keep TolCsm in a functional state. Recently, we have verified that the pcm gene is less related to the TolCsm function in the aspect of antimicrobial extrusion (8). However, in addition to antimicrobial efflux function, TolC-associated pumps are also known to play physiological roles for stresses adaption, such as envelope stress or oxidative stress (10). Therefore, it can not be immediately ruled out that PCM play an important role in the physiological function of TolCsm.

Acknowledgement

This research was supported by the National Science Council (NSC 101-2320-B-010- 053-MY3).

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efflux pump from Gram-negative bacteria. FEBS Lett. 578:5-9.

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2008. The complete genome, comparative and functional analysis of Stenotrophomonas maltophilia reveals an organism heavily shielded by drug

resistance determinants. Gen. Biol. 9:R74.

4. Li, XZ, Li Z, Poole K. 2002. SmeC, an outer membrane multidrug efflux protein

of Stenotrophomonas maltophilia. Antimicrob. Agents Chemother. 46:333-343.

5. Alonso A, Martinez JL. 2000. Cloning and characterization of SmeDEF, a novel multidrug efflux pump from Stenotrophomonas maltophilia. Antimicrob. Agents

Chemother. 44:3079–3086.

6. Chen CH, Huang CC, Chung TC, Hu RM, Huang YW, Yang TC. 2011.

Contribution of resistance-nodulation-division efflux pump operon smeU1-V-W- U2-X to multidrug resistance of Stenotrophomonas maltophilia. Antimicro. Agents Chemother. 55:5826-5833.

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7. Gould VC, Okazaki A, Avison MB. 2013. Coordinate hyperproduction of SmeZ and SmeJK efflux pumps extends drug resistance in Stenotrophomonas maltophilia.

Antimicrob. Agents Chemother. 57:655-657.

8. Huang YW, Hu RM, Yang TC. 2013. Role of the pcm-tolCsm operon in multidrug resistance of Stenotrophomonas maltophilia. J. Antimicrob. Chemother.

68:1987-1993.

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10. Zgurskaya HI, Krishnamoorthy G, Ntreh A, Lu S. 2011. Mechanism and function of the outer membrane channel TolC in multidrug resistance and physiology of Enterobacteria. Front. Microbiol. 2:189.

Figure Legends

Fig. 1. Schematic organization of the smeOP and smeRo-pcm-tolCsm operons from S. maltophilia KJ. The smeOP operon contains genes for a membrane fusion protein (SmeO) and a RND transporter (SmeP). A TetR-type transcriptional regulator smeRo is located immediately upstream of the smeOP operon and is divergently transcribed. SmeRo, pcm, and tolCsm genes form an operon. Solid arrows represent ORFs and the direction of transcription. (A) The structure of recombinant plasmids.

The solid lines represent the PCR amplicons and the empty bars represent the deleted 216

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region for each plasmid construct. The numbers above the solid bars represent the PCR amplicon size (bps).The arrow with dashed lines indicates the xylE gene. The black bars with vertical line indicate the product of qRT-PCR. (B) The presence of smeRo-pcm-tolCsm operon. The solid bars labeled as I and II indicate the products of RT-PCR generated by primers 23-F/23-R and 123-F/23-R, respectively. The numbers in the brackets represent the PCR amplicon sizes (bps). The fine arrows indicate the positions of primers. The empty bar represents the deleted region in strain KJRo.

The agarose gels show the RT-PCR products of strains KJ and KJRo. Lane 1, strain KJ; lane 2, strain KJRo. The RT-PCR products, labeled as I and II, were generated by primers 23-F/23-R and 123-F/23-R, respectively. The subscript numbers indicate the number of PCR cycles.

Supplemental Materials

Fig. S1

Fig. S1. Agarose gel of RT-PCR products of smeC, smeF, smeX, and tolCsm. Total cellular RNA of strain KJ was extracted from exponentially growing cells using the PureLinkTM Total RNA Purification System (Invirtogen, Carlsbad, CA, USA) and treated with 1 unit of RNase-free DNaseI (Invitrogen, Carlsbad, CA, USA). The DNase-treated RNA was converted to cDNA using the MMLV Reverse Transcriptase

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1

Strand cDNA Synthesis Kit (Epicentre Biotechnologies, Taiwan). The cDNA was then used directly as a template for RT-PCR. Lane 1: transcript of partial smeC gene (primered by SmeCQ-F/SmeCQ-R); Lane 2: transcript of partial smeF gene (primered by SmeFQ-F/SmeFQ-R); Lane 3: transcript of partial smeX gene (primered by SmeXQ-F/SmeXQ-R); Lane 4: transcript of partial tolCsm gene (primered by TolCQ- F/TolCQ-R).

TABLE S1. Bacterial strains, plasmids and primers used in this study Strain,

plasmid, or primer

Genotype or properties Reference

S. maltophilia

KJ Wild type, a clinical isolate from Taiwan 1

KJOP S. maltophilia KJ double mutant of smeO and smeP genes; smeO, smeP

This study KJRo S. maltophilia KJ mutant of smeRo gene; smeRo This study KJRoOP S. maltophilia KJ mutant of smeRo, smeO, and smeP

genes; smeRo, smeO, smeP

This study KJDEF S. maltophilia KJ mutant of smeDEF operon; smeDEF This study KJDEFOP S. maltophilia KJ mutant of smeDEF and smeOP

operons; smeDEF, smeOP

This study KJTolC S. maltophilia KJ mutant of tolCsm gene; tolCsm This study KJTolCOP S. maltophilia KJ mutant of tolCsm gene and smeOP

operon; TolCsm, smeOP

This study Escherichia

coli

DH5 F- 80dlacZM15 (lacZYA-argF)U169 deoR recA1 endA1 hsdR17 (r

m

) phoA supE44 

thi-1 gyrA96

Invitrogen 266

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relA1 S17-1

Plasmids

 pir + mating strain 2

pEX18Tc sacB oriT, Tc

3

pRK415 Mobilizable broad-host-range plasmid cloning vector, RK2 origin; Tc

4 pTXylE Plasmid containing the xylE cassette; Amp

1 pRo pEX18Tc vector with a 779-bp DNA fragment of S.

maltophilia KJ, containing the smeRo gene with a internal 235-bp deletion; Tc

This study

pOP pEX18Tc vector with a 1820-bp DNA fragment of S.

maltophilia KJ, containing the partial N-terminus of smeO gene and partial C-terminus of smeP gene; Tc

This study

pDEF pEX18Tc vector with a 752-bp DNA fragment of S.

maltophilia KJ, containing the partial N-terminus of smeD gene and partial C-terminus of smeF gene; Tc

This study

pTolC pEX18Tc vector with a 1563-bp DNA fragment of S.

maltophilia KJ, containing the tolC gene with a internal 100-bp deletion; Tc

5

pSmeRo

pRK415 with a 117-bp DNA fragment upstream from the smeRo start codon and a P

::xylE

transcriptional fusion

This study

pSmeO

pRK415 with a 117-bp DNA fragment upstream from the smeO start codon and a P

::xylE

transcriptional fusion

This study

pPCM

pRK415 with a 359-bp DNA fragment upstream from the pcm start codon and a P

::xylE

transcriptional fusion

This study

pTolC

pRK415 with a 243-bp DNA fragment upstream from the tolCsm start codon and a P

::xylE transcriptional fusion

This study

Primers SmeRo3-F:

SmeRo3-R SmeRo5-F SmeRo5-R

5’-CGGCCTGCAGGAAATCACCGAC-3’

5’-GCGAGCTCGGGCGTAATCAATC-3’

5’-CCCAAAGCTTACCGCCCATCCAG-3’

5’-CCACTGCAGGGCATCGGGCAG-3’

This study

This study

This study

This study

SmeP3-F SmeP3-R SmeD5-F SmeD5-R SmeF3-F SmeF3-R PCMQ-F PCMQ-R TolCQ-F TolCQ-R SmeOQ-F SmeOQ-R SmePQ-F SmePQ-R SmeCQ-F SmeCQ-R SmeFQ-F SmeFQ-R SmeXQ-F SmeXQ-R rDNA-F rDNA-R OPIG-F OPIG-R 23-F 23-R 123-F

5’-GACAAGCTTCCTGCTGCTGTTCCG-3’

5’-AGCCCGCAAGCTTGACTTAACC-3’

5’-CCAGATCTTCATCGAGCTGTCC-3’

5’-GAGGTACCATACCACGTTGTCC-3’

5’-AGACTCTAGATGTCAACGAACAGTTCAC-3’

5’-TAGCAAGCTTCGTCCAGGCTGACATTCAAC-3’

5’-CGATTGATTACGCCCACGCC-3’

5’-TCCAGCACTTCGTCACCCG-3’

5’-CCTGACCCTGAACGTGCC -3’

5’-CTCTGTGCCGAGACCACC-3’

5’-GTACTGGTGGTTCACCCC-3’

5’-GTCAGCCACGTCCAGTTC-3’

5’- GTCAGCCAGTTCCTGTCC -3’

5’- TACTCCATCGTCGCCACC -3’

5’-GCGATGCCAACAGCGAGACC-3’

5’-GTCGCCACTTCAGCCACCAG-3’

5’-CCCGAGCATCTCGCTGAC-3’

5’-AAGCCCACCTGGATCGAC-3’

5’-TACGACCGCCGCAAGCAACC-3’

5’-CAGCTCGAAGTAGTTGCGTGCC-3’

5’-GACCTTGCGCGATTGAATG-3’

5’-CGGATCGTCGCCTTGGT-3’

5’-TGAACGCGGGTGACTGGG-3’

5’-CGCACCACCATCGCCTTG-3’

5’-GGACGTGCTGGACATCAAGG-3’

5’-GCGGCATGGGCAGACAAC-3’

5’-GTTCTCCGAGGCCGTGCAGT-3’

This study This study This study This study This study This study

5 5 5 5 This study This study

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TABLE S2. Antimicrobial susceptibilities of strains KJ(pRK415), KJRo(pRK415), and KJRo(pSmeRo)

Strains

MIC (g ml

)

Diameter of inhibition zone (mm)

AMK GEN KAN CCCP TCS

KJ(pRK415) KJRo(pRK415) KJRo(pSmeRo)

1024

>1024 1024

512 1024

256

256 512 256

14  1 10  1 15  0.5

15  1 10  0.5

14  1 AMK, amikacin; GEN, gentamicin; KAN, kanamycin; CCCP, carbonyl cyanide 3-

chlorophenylhydrazone; TCS, tetrachlorosalicylanilide

a

The Mueller-Hinton agar contains 30 g/ml tetracycline in addition to the antibiotic

indicated.

b

The bacterial cultures of 10

cfu/ml were plated on the Mueller-Hinton agar 283

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containing 30 g/ml tetracycline. Paper disk (6 mm in diameter; BBL) impregnated with CCCP (2048 g/ml) or TCS (8192 g/ml) was placed on the top. The diameters of growth inhibition zone were observed after 24-h incubation at 37C.

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1993.

6. Chen CH, Huang CC, Chung TC, Hu RM, Huang YW, Yang TC. 2011.

Contribution of resistance-nodulation-division efflux pump operon smeU1-V-W- U2-X to multidrug resistance of Stenotrophomonas maltophilia. Antimicro. Agents Chemother. 55:5826-5833.

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