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Antimicrobial Agents and Chemotherapy, September 2005, p. 3955-3958, Vol. 49, No. 9
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.9.3955-3958.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Correlation between Ceftriaxone Resistance of Salmonella enterica Serovar Typhimurium and Expression of Outer Membrane Proteins OmpW and Ail/OmpX-Like Protein, Which Are Regulated by BaeR of a Two-Component System

Wensi S. Hu,* Pei-Chuan Li,{dagger} and Chao-Yin Cheng{ddagger}

Institute of Biotechnology in Medicine, School of Medical Technology and Engineering, National Yang-Ming University, Taipei, Taiwan

Received 31 December 2004/ Returned for modification 21 February 2005/ Accepted 1 July 2005


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ABSTRACT
 
Mutant 7F2 of Salmonella enterica serovar Typhimurium has a transposon inserted in the regulator gene baeR of a two-component system and showed a more-than-fourfold reduction in resistance to ceftriaxone. Complementation analysis suggested an association among the outer membrane proteins OmpW and STM3031, ceftriaxone resistance, and baeR.


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TEXT
 
The spread of multidrug-resistant Salmonella spp. in many areas of the world has become a great public health concern. Although extended-spectrum cephalosporins (ESC) and fluoroquinolones have proved effective, resistance to these agents has emerged (9, 18). In gram-negative bacteria, such as Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa, ESC resistance is always associated with beta-lactamase or cephalosporinase production, loss or reduced expression of porins, overexpression of multidrug efflux pumps, and/or alterations in penicillin-binding proteins (3, 13, 20). Most studies of the resistance mechanisms in Salmonella have concentrated on the production of extended-spectrum beta-lactamases; only a few reports have shown that outer membrane proteins may be involved in either permeability or cooperation with the transporter protein to form efflux pumps, and these changes then result in antibiotic resistance (2, 4, 5, 8, 15).

Two-component systems are a unique family of proteins that allow bacteria to modulate the expression of downstream genes in response to various environmental cues. The nature of these systems makes them exceptionally efficient at mediating adaptive changes to environmental stress, and as a result many are involved in virulence and antibiotic resistance pathways of pathogenic bacteria (7, 17). A typical two-component system consists of two types of signal transducers, a sensor kinase and its cognate response regulator. The sensor kinase monitors some environmental conditions and accordingly modulates the phosphorylation state of the response regulator. The response regulator controls gene expression and/or cell behavior. Recently, the overexpression of 15 of 32 response regulator genes has been reported to confer increased single- or multidrug resistance in E. coli (6). For instance, the overexpression of the response regulator baeR causes a novobiocin resistance phenotype by upregulating the expression of novel resistance-nodulation-cell division-type drug exporter MdtABC (1, 11); it was also found that outer membrane protein TolC is required for the resistance phenotype of MdtABC (16).

We previously established a series of ceftriaxone-resistant strains of Salmonella enterica serovar Typhimurium, derived from ceftriaxone-susceptible strain 01-4, and have established their different levels of resistance to ceftriaxone. A comparison of these adapted resistant strains with the outer membrane protein profile of susceptible strain 01-4 by two-dimensional gel electrophoresis shows the level of expression of several proteins changed (C. Y. Cheng, unpublished data). In the present study, insertion mutant 7F2, with a transposon inserted into the baeR regulator gene of the BaeSR two-component system, was generated by transposon mutagenesis and used to assess the correlation between the overexpression of the baeR regulator gene and the resistance to ceftriaxone in S. enterica serovar Typhimurium. In addition, the level of expression of two outer membrane proteins (OmpW and STM3031), whose function remains unknown, were shown to be influenced by baeR, and these proteins seem to be associated with resistance to ceftriaxone in S. enterica serovar Typhimurium.

Transposon mutagenesis and mapping. Ceftriaxone-resistant strain R200 of S. enterica serovar Typhimurium (MIC, >204.8 µg ml–1), derived from ceftriaxone-susceptible strain 01-4, was previously established by selection. Mutants of this resistant strain were generated by transposon mutagenesis using the EZ::TN <R6K rori/KAN-2> transposome system (Epicentre) in accordance with the manufacturer's instructions. Eleven hundred independent transposon insertion mutants were isolated and screened for decreased susceptibility to ceftriaxone. The susceptibility of these 1,100 insertion mutants to ceftriaxone was determined by the agar dilution method according to the guidelines of CLSI (formerly NCCLS) (12), and 10 of them showed at least a fourfold reduction (MIC, ≤51.2 µg ml–1) in resistance. Chromosomal DNA isolated from each of these 10 insertion mutants was digested with HincII and analyzed by Southern blot using a synthetic oligonucleotide probe RK primer (5'-CTCAAAATCTCTGATGTTACATTACATTGCA-3') to confirm insertion of the transposon into the S. enterica serovar Typhimurium R200 chromosome. The HincII-digested chromosomal DNA of insertion mutants was self-ligated with T4 DNA ligase and used as template DNA for PCR to obtain the insertion site and flanking region of the transposon. The primers used for amplification were KAN-2 FP-1 as forward primer (5'-ACCTACAACAAAGCTCTCATCAACC-3') and R6KAN-2 RP-1 as reverse primer (5'-CTACCCTGTGGAACACCTACATCT-3'). The PCR products were purified and sequenced by automated DNA sequencing (Applied Biosystems) to identify the insertion site and its flanking region. A BLASTP search of the translated open reading frames from each of these 10 insertion mutants revealed insertion into homologues of nine different genes of the S. enterica serovar Typhimurium LT2 strain. In one case, two insertion mutants were identified to have insertions into the same gene at different locations. Insertion mutant 7F2 was identified to have a transposon insertion positioned 42 bp away from the 3' end of the baeR regulator gene of the two-component system BaeSR.

Cloning of the wild-type baeR gene and complementation studies. To obtain a wild-type baeR gene, a 730-bp DNA fragment from the S. enterica serovar Typhimurium R200 strain was amplified by PCR using forward primer NheI-baeR(RBS) (5'-GCTAGCGAGAAGTATGACTGAAATA-3') and reverse primer baeR-HindIII (5'-AAGCTTTTATACCAGGCGACACGCAT-3'). The NheI and HindIII sites are underlined. This gene fragment was then ligated into the pGEM-T Easy cloning vector (Promega) according to the manufacturer's instructions. The resulting plasmid, pGEM-baeR, was then digested with NheI and PstI restriction enzymes and then directionally inserted into pBAD18-Kan that had been cut with NheI/PstI. The resulting plasmid, pBAD-baeR, was transformed into the S. enterica serovar Typhimurium 7F2 mutant by electroporation with the Gene Pulser II system (Bio-Rad, Hercules, Calif.) according to the manufacturer's instructions. The transformants, grown on Mueller-Hinton agar containing 51.2 µg ml–1 of ceftriaxone, were selected to test whether the overexpression of the cloned baeR gene in the 7F2 mutant could restore the resistance to ceftriaxone using the agar dilution method. In the presence of arabinose induction, the MIC for the 7F2 mutant with overexpression of baeR gene showed a more-than-fourfold (≥204.8 µg ml–1 versus 51.2 µg ml–1) elevation over that of the 7F2 mutant, where there is an almost complete restoration of resistance to ceftriaxone (Table 1).


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  		TABLE 1. MICs for Salmonella enterica serovar Typhimurium strains expressing or lacking baeR

Effect of the baeR gene on drug susceptibility. To examine the effect of the baeR gene on drug susceptibility, the R200, 7F2, and 7F2/pBAD-baeR strains of S. enterica serovar Typhimurium were used to determine MICs for a number of antibiotics using the agar dilution method. As shown in Table 1, the insertion mutant of 7F2 resulted in a more-than-fourfold reduction in resistance to ceftriaxone compared to ceftriaxone-resistant strain R200. In contrast, for the same strains, there was a 2- to 32-fold elevation in resistance to tetracycline, erythromycin, novobiocin, and nalidixic acid, but no effect on ampicillin, chloramphenicol, and ciprofloxacin resistance. Although the introduction of pBAD-baeR into the insertion mutant 7F2 (7F2/pBAD-baeR) showed almost complete restoration of resistance to ceftriaxone, there was no effect on cephalothin and cefamandole resistance. In contrast, there was a >2- to 16-fold reduction in erythromycin, novobiocin, and nalidixic acid resistance. Thus, complementation of 7F2 with wild-type baeR restores resistance to erythromycin, novobiocin, nalidixic acid, and ceftriaxone in the transposon mutant strain to the same level as the original strain. However, the regulator BaeR does not have any effect on the susceptibility to ampicillin, chloramphenicol, tetracycline, and ciprofloxacin. These results suggested that the regulator BaeR has a variety of effects on antibiotic resistance. Even though ceftriaxone, cephalothin, and cefamandole belong to the same family of cephalosporins, the result showed that the baeR gene has more influence on resistance to a broad-spectrum drug (ceftriaxone) than on resistance to a narrow-spectrum drug (cephalothin) and an expanded-spectrum drug (cefamandole). The reason for the differences is not known yet and deserves further exploration.

Analysis of outer membrane protein profiles of R200, 7F2, and 7F2/pBAD-baeR strains of S. enterica serovar Typhimurium. It has been shown that outer membrane alterations are related to resistance to antimicrobial agents in bacteria, especially gram-negative strains (14). S. enterica serovar Typhimurium R200, a ceftriaxone-resistant strain, and 7F2, a strain with lower ceftriaxone resistance, provide us with the chance to examine the change(s) that occurred in the outer membrane mediated through the baeR regulator gene of the BaeSR two-component system. The outer membrane fractions were prepared from the S. enterica serovar Typhimurium R200 and 7F2 strains, respectively, by the method of Molloy et al. (10). A sample of outer membrane fraction was loaded on an 18-cm, pH 4 to 7 immobilized pH gradient gel, analyzed by two-dimensional gel electrophoresis using the Multiphor II (Amersham Pharmacia Biotech, Uppsala, Sweden) according to manufacturer's instructions, and stained with Coomassie blue. Comparison of the outer membrane protein profiles between these two strains gave three protein spots that were significantly changed, and their molecular masses were approximately 23 kDa (one spot) and 19 kDa (two spots). The level of expression of the 23-kDa protein was significantly increased, but those of the 19-kDa protein were significantly decreased, in the 7F2 strain (Fig. 1A and B). Interestingly, upon introduction of pBAD-baeR into the 7F2 background, the level of expression of 23-kDa and 19-kDa outer membrane proteins returned to that of resistant strain R200 (Fig. 1A and C), demonstrating that the response regulator BaeR influences the expression of the 23-kDa and 19-kDa proteins. These three protein spots were excised from the gel, digested in situ with trypsin, and analyzed using matrix-assisted laser desorption-ionization time of flight mass spectrometry (21). The mass spectra showed two or more peptides that were matched to a SWISS-PROT S. enterica serovar Typhimurium database homologue, thus establishing protein identity. The 23-kDa protein was determined to be outer membrane protein OmpW. The two 19-kDa proteins with different pI values were determined to be outer membrane protein STM3031, a homologue of Ail and OmpX. OmpW is the colicin S4 receptor protein in E. coli (19); however, the exact function of both OmpW and STM3031 in S. enterica serovar Typhimurium remains unclear.



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  		FIG. 1. Comparison of outer membrane protein profiles among S. enterica serovar Typhimurium strains R200 (A), 7F2 (B), and 7F2/pBAD-baeR (C) by two-dimensional polyacrylamide gel electrophoresis. The significantly changed proteins are indicated by the arrows.

The OmpW was expressed at a relatively high level and STM3031 was almost undetectable in ceftriaxone-susceptible strain 01-4. Interestingly, the OmpW expression level was dramatically decreased and STM3031 was highly induced in ceftriaxone-resistant strain R200 (data not shown). In this study, insertion mutant 7F2, with a transposon inserted in the baeR gene, showed a more-than-fourfold reduction in resistance to ceftriaxone and the levels of expression of OmpW and STM3031 are similar to those of ceftriaxone-susceptible strain 01-4. Complementation of the wild-type baeR gene in the 7F2 mutant resulted in a return to the levels of expression of OmpW and STM3031 found in resistant strain R200, and it also completely restored resistance to ceftriaxone. Taken together, these results suggested that OmpW and/or STM3031 might be associated with ceftriaxone resistance and that they are influenced by the regulator gene baeR in S. enterica serovar Typhimurium. In ceftriaxone-resistant strain R200 or 7F2/pBAD-baeR, the highly reduced expression of OmpW suggests that this protein may have a function that is similar to the porins of gram-negative bacteria (14) because it reduces ceftriaxone permeability; on the other hand, the highly increased expression of STM3031 suggests that this protein may have a function that is similar to the outer membrane protein TolC in some gram-negative bacteria (14), which is required to cooperate with a transporter protein to form the efflux pumps for drug export.

To our knowledge, this is the first reported evidence of outer membrane proteins OmpW and STM3031 (Ail/OmpX-like) associated with ceftriaxone resistance and influenced by the regulator gene baeR of a two-component system in S. enterica serovar Typhimurium. Further studies are in progress in order to explore the exact function of OmpW and STM3031 and how they are regulated by BaeR to confer ceftriaxone resistance.


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ACKNOWLEDGMENTS
 
We thank Kuang Lo Chen from CDC Department of Health Taiwan for kindly providing Salmonella enterica serovar Typhimurium strain 01-4. We also thank Yurng Chen Su for assistance with the preparation of the manuscript.

This work was in part supported by the Program for Promoting Academic Excellence of Universities from the Ministry of Education of the Republic of China.


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FOOTNOTES
 
* Corresponding author. Mailing address: Institute of Biotechnology in Medicine, School of Medical Technology and Engineering, National Yang-Ming University, 155, Li-Nong St., Sec. 2, Peitou, Taipei, Taiwan 112. Phone: 886-2-2826-7151. Fax: 886-2-2826-4092. E-mail: huws{at}ym.edu.tw. Back

{dagger} Present address: The Genomics Research Center, Academia Sinica, 128 Academia Road, Section 2, Nankang, Taipei, Taiwan 115. Back

{ddagger} Present address: Institute of Molecular Biology, Academia Sinica, 128 Academia Road, Section 2, Nankang, Taipei, Taiwan 115. Back


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Antimicrobial Agents and Chemotherapy, September 2005, p. 3955-3958, Vol. 49, No. 9
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.9.3955-3958.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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