Bacitracin sensing and resistance in Staphylococcus aureus

Authors

  • Yuuma Yoshida,

    1. Department of Oral Microbiology, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan
    2. Department of Oral and Maxillofacial Surgery, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan
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  • Miki Matsuo,

    1. Department of Oral Microbiology, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan
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  • Yuichi Oogai,

    1. Department of Oral Microbiology, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan
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  • Fuminori Kato,

    1. Department of Bacteriology, Hiroshima University Graduate School of Biomedical Sciences, Hiroshima, Japan
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  • Norifumi Nakamura,

    1. Department of Oral and Maxillofacial Surgery, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan
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  • Motoyuki Sugai,

    1. Department of Bacteriology, Hiroshima University Graduate School of Biomedical Sciences, Hiroshima, Japan
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  • Hitoshi Komatsuzawa

    1. Department of Oral Microbiology, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan
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  • Editor: Roger Buxton

Correspondence: Hitoshi Komatsuzawa, Department of Oral Microbiology, Kagoshima University Graduate School of Medical and Dental Sciences, Sakuragaoka 8-35-1, Kagoshima City, Kagoshima 890-8544, Japan. Tel.: +81 99 275 6150; fax: +81 99 275 6158; e-mail: hkomatsu@dent.kagoshima-u.ac.jp

Abstract

Bacterial two-component systems (TCSs) have been demonstrated to be associated with not only the expression of virulence factors, but also the susceptibility to antibacterial agents. In Staphylococcus aureus, 16 types of TCSs have been identified. We previously found that the inactivation of one uncharacterized TCS (designated as BceRS, MW gene ID: MW2545-2544) resulted in an increase in susceptibility to bacitracin. In this study, we focused on this TCS and tried to identify the TCS-controlled factors affecting the susceptibility to bacitracin. We found that two ABC transporters were associated with the susceptibility to bacitracin. One transporter designated as BceAB (MW2543-2542) is downstream of this TCS, while another (formerly designated as VraDE: MW2620-2621) is separate from this TCS. Both transporters showed homology with several bacitracin-resistance factors in Gram-positive bacteria. Inactivation of each of these two transporters increased the susceptibility to bacitracin. Expressions of these transporters were significantly increased by the addition of bacitracin, while this induction was not observed in the TCS-inactivated mutant. These results indicate that this TCS senses bacitracin, and also positively regulates the expression of two ABC transporters.

Introduction

Staphylococcus aureus is a major pathogen to humans and often causes serious problems related to nosocomial infection. This organism, especially methicillin-resistant S. aureus (MRSA), shows multiple resistances to several chemotherapeutic agents, such as β-lactams, quinolones and aminoglycosides (Grundmann et al., 2006; Fischbach & Walsh, 2009). To date, many factors conveying resistance to antibacterial agents have been identified in S. aureus (Ito et al., 2003; Grundmann et al., 2006). Among these factors, recently, some two-component systems (TCSs) have been demonstrated to affect the susceptibility to several antibacterial agents (Sakoulas et al., 2002; Kuroda et al., 2003; Meehl et al., 2007). TCSs have mainly been characterized in terms of the expressions of virulence factors and adaptation to environmental conditions (Novick, 2003), but some TCSs have been demonstrated to affect the susceptibility to antibacterial agents. VraSR was originally identified as a factor that affects bacterial resistance to vancomycin (Kuroda et al., 2003). VraSR is a positive modulator for the regulation of cell wall biosynthesis, such as for pbpB, sgtB and murZ (Yin et al., 2006). Inhibition of VraSR leads to decreased resistance to cell wall inhibitors, including β-lactams, vancomycin, teicoplanin and fosfomycin (Kuroda et al., 2003; Gardete et al., 2006). ApsRS/GraRS has been reported to be involved in vancomycin-intermediate resistance owing to the increased expression of VraFG, an ABC transporter (Meehl et al., 2007; Howden et al., 2008). In addition, ApsRS/GraRS is involved in the susceptibility to cationic antibacterial peptides, such as defensins and LL37, by modulating the bacterial surface charge. Agr, a global regulator for virulence factors, has also been demonstrated to be associated with vancomycin and daptomycin resistance (Sakoulas et al., 2002; Tsuji et al., 2007). Loss of agr function was linked to the development of vancomycin-intermediate resistance by vancomycin exposure, although the mechanism of this linkage is unclear.

Bacitracin is a polypeptide antibiotic produced by Bacillus subtilis and Bacillus licheniformis (Johnson et al., 1945; Azevedo et al., 1993). Bacitracin binds to undecaprenyl pyrophosphate, resulting in inhibition of cell wall biosynthesis (Stone & Strominger, 1971). We previously investigated susceptibility to various antibacterial agents using a group of S. aureus MW2 mutants that are gene inactivated in 15 TCSs, with the exception of one essential TCS (Matsuo et al., 2010). Among those mutants, one uncharacterized TCS (MW gene ID: MW2545-2544) showed association with an increased susceptibility to bacitracin, implying that this TCS may be involved in bacitracin sensing and also resistance to bacitracin. One uncharacterized ABC transporter (MW2543-2542) is located downstream of this TCS and shows homology with BceAB in B. subtilis, which is responsible for bacitracin efflux (Ohki et al., 2003) (Fig. 1). Therefore, we investigated whether this transporter, together with two other transporters (vraDE: MW2620-2621 and vraFG: MW0623-0624) showing homology with BceAB, is associated with susceptibility to bacitracin. In this study, we presented data on the characterization of the transporters related to bacitracin resistance and also the linkage between this TCS and the transporters. Based on our results, we designated the TCS (MW2545-2544) as BceRS and its downstream transporter (MW2543-42) as BceAB.

Figure 1.

 Restriction map of bceRSAB region in Staphylococcus aureus. The boxed arrows represent the ORF and the orientation of the transcripts. Two transcripts (bceRS and bceAB) represent the line above the arrows.

Materials and methods

Bacterial strains and growth conditions

The bacterial strains used in this study are listed in Table 1. Staphylococcus aureus and Escherichia coli were grown in trypticase soy broth (TSB) (Beckton Dickinson Microbiology Systems, Cockeysville, MD) and Luria–Bertani (LB) broth, respectively. Tetracycline (10 μg mL−1) or chloramphenicol (10 μg mL−1) for S. aureus was added when necessary.

Table 1. Staphylococcus aureus strains used in this study
StrainsInactivated gene ID*GenotypeReference
  • *

    Gene ID in Staphylococcus aureus MW2.

  • Two-component system.

  • Tetracycline resistance.

  • §

    § Chloramphenicol resistance.

MW2Clinical strain, methicillin resistant (mec+)Baba et al. (2002)
FK64MW0621-22 (apsRS): TCSapsR∷pCL52.1 in MW2, TCrMatsuo et al. (2010)
FK77MW2545-44 (bceRS): TCSbceR∷pCL52.1 in MW2, TCrMatsuo et al. (2010)
MM01MW0623-24 (vraFG): ABC transportervraF∷pCL52.1 in MW2, TCrThis study
MM02MW2543-42 (bceAB): ABC transporterbceA∷pCL52.1 in MW2, TCrThis study
MM03MW2620-21 (vraDE): ABC transportervraD∷pCL52.1 in MW2, TCrThis study
MM07MW2542 (bceB): ABC transporterbceB∷pCL52.1 in MW2, TCrThis study
MM08MW2544 (bceS): TCSbceS∷pCL52.1 in MW2, TCrThis study
MM09MW2544 (bceS) complementationpMM09(bceS) in MM08, TCr, CPr§This study
MM10MW2542 (bceB) complementationpMM10(bceB) in MM07, TCr, CPr§This study
MM11MW2620-21 (vraDE) complementationpMM11(vraDE) in MM03, TCr, CPr§This study
RN4220Laboratory strain, methicillin-sensitive(mec-)Kreiswirth et al. (1983)
MM04MW0623-24 (vraFG) ABC transportervraF∷pCL52.1 in RN4220, TCrThis study
MM05MW2542 (bceAB) ABC transporterbceA∷pCL52.1 in RN4220, TCrThis study
MM06MW2620-21 (vraDE) ABC transportervraD∷pCL52.1 in RN4220, TCrThis study

DNA manipulation

Routine DNA manipulations, restriction enzyme digestion, DNA ligation and DNA sequencing were performed essentially as described previously (Sambrook et al., 1989). Restriction enzymes and shrimp alkaline phosphatase were purchased from NipponGene (Tokyo, Japan). T4 DNA ligase and PCR reagents were from Takara (Tokyo, Japan).

Construction of mutants

Inactivation of transporters in S. aureus was achieved by a method described elsewhere (Komatsuzawa et al., 2004). Since transporter consists of two orfs encoding for a permease and an ATP-binding protein, we constructed the mutants which were inactivated the both of them. Also, for the complementation experiment, we further constructed two mutants that were inactivated, the second orf in the operon of bceRS (TCS) or bceAB (ABC transporter), because we failed to construct the plasmid containing the two genes of bceRS or bceAB due to an unknown reason. Briefly, DNA fragments containing an internal region of each orf were amplified and cloned into a pCL52.1 vector, a thermosensitive vector, which could replicate at 30 °C but not at 42 °C (Subrata et al., 1997). After electroporation of the plasmid into S. aureus RN4220, the bacteria were grown at 30 °C with tetracycline (10 μg mL−1) overnight. Then, the plasmid in RN4220 was transduced into MW2 strain using phage 80α. Both strains containing the plasmid were grown overnight at 30 °C. The appropriate dilutions of the culture were poured on trypticase soy agar plates containing tetracycline (10 μg mL−1), then incubated at 42 °C overnight. Ten colonies were collected and replated on TS agar containing tetracycline. Disruption of the target gene was checked by PCR.

For the complementation experiment, the DNA fragment of bceS, bceB or vraDE amplified with specific primers was cloned into pCL15, which was an E. coli–S. aureus shuttle vector with Pspac promoter (Luong & Lee, 2006). Each plasmid was electroporated to RN4220, and then, the plasmid was transduced to the mutant by the method described above. Plasmids and primers used in this study are listed in Table 2.

Table 2.   Plasmids and primers used in this study
PlasmidInsert fragment
pCL52.1E. coli–S. aureus shuttle vector, thermo-sensitive plasmid
pMM01PCR fragment (vraF-1+vraF-2)/pCL52.1
pMM02PCR fragment (bceA-1+bceA-2)/pCL52.1
pMM03PCR fragment (vraD-1+vraD-2)/pCL52.1
pMM07PCR fragment (bceB-1+bceB-2)/pCL52.1
pMM08PCR fragment (bceS-1+bceS-2)/pCL52.1
pCL15E. coli–S. aureus shuttle vector with Pspac promoter
pMM09PCR fragment (bceS-3+bceS-4)/pCL15
pMM10PCR fragment (bceB-3+bceB-4)/pCL15
pMM11PCR fragment (vraD-3+vraE-1)/pCL15
Gene IDGene namePrimer – forwardPrimer – reverse
Quantitative PCR
 MW0006gyrA5′-aaggtgttcgcttaattcgc-3′5′-attgcatttcctggtgtttc-3′
 MW0623vraF5′-caacaggtgcactggatt-3′5′-tcgtcatccccttggtat-3′
 MW1824vraR5′-attagatgcaggtgtcgata-3′5′-ccatttctcgttctgtaagc-3′
 MW2543bceA5′-gctacgacagcacttaatca-3′5′-caccttcagttagtccatca-3′
 MW2545bceR5′-ttaaccaacatcaacctcag-3′-5′-ccccatttgtattgccat-3′
 MW2620vraD5′-cacttgccaaattccgta-3′5′-aatacctaatgctgtcgtga-3′
Construction for gene inactivation plasmid
 MW0623vraFvraF-1: 5′-cgcggatccctctggatctgggaaaac-3′vraF-2: 5′-cgcaagcttctgttggttcgtcagcaa-3′
 MW2542bceBbceB-1: 5′-tgggatccgatacttacttggtgcg -3′bceB-2: 5′-ttaagcttgtcattgtgagcgttac-3′
 MW2543bceAbceA-1: 5′-cgcggatccgaatgctacgacagcact-3′bceA-2: 5′-cgcaagcttcttttcgtttagcaccag-3′
 MW2544bceSbceS-1: 5′-gcggatccactagcacttggcgttatt-3′bceS-2: 5′-tcaagctttctcgcatacttaagtgca-3′
 MW2620vraDvraD-1: 5′-cgcggatccttcgttgcgattatgggg-3′vraD-2: 5′-cgcaagcttaaacttgctgcaaccgga-3′
Construction for complementation plasmid
 MW2542bceBbceB-3: 5′-acaagctttaacagatagcttagcac-3′bceB-4: 5′-atggatccttgtaatgtttttaggtttc-3′
 MW2544bceSbceS-3: 5′-ttaagcttctattggcgttaatgattt-3′bceS-4: 5′-agggatccaatgttactagtatgcttac-3′
 MW2620-21vraDEvraD-3: 5′-atgtcgaccaactctgtaaggttcaa-3′vraE-1: 5′-ctggatcccaaaatctcaatgaagcatc-3′

Antibiotic susceptibility tests

Minimal inhibitory concentrations (MICs) of various antibiotics were determined by microdilution as described previously (Nishi et al., 2004). Oxacillin, bacitracin and vancomycin (Sigma Chemical Co. Ltd, St. Louis, MO), as well as erythromycin and ofloxacin (Wako Pure Chemical Industries Ltd., Osaka, Japan), were used. Population analysis profiles were determined by plating appropriate dilutions of an overnight culture on plates containing various concentrations of bacitracin (Nishi et al., 2004). Colonies were counted after 48 h incubation at 37 °C. All susceptibility tests were repeated at least three times to check the reproducibility of the results.

Quantitative analysis of the gene expression

A small portion of overnight culture of S. aureus was inoculated to fresh TSB. Then, S. aureus cells were grown at 37 °C with shaking. Various concentrations (0.5, 1, 8, 16 μg mL−1) of bacitracin were added to the medium when OD 660 nm reached 0.3. After 5, 15, 30 and 60 min, the cells were collected. Total RNA was extracted with a FastRNA Pro Blue kit (MP Biomedicals, Ohio) in accordance with the manufacturer's protocol. One microgram of total RNA was reverse-transcribed to cDNA using a first-strand cDNA synthesis kit (Roche, Tokyo, Japan). Using cDNA as template DNA, quantitative PCR was performed using LightCycler system (Roche). Primers for bceR, bceA, vraD, vraF and vraR were constructed and used to determine optimal conditions for analysis of their expression. The amount of gyrA was used as internal control. Primers for quantitative PCR are listed in Table 2.

Results

Antibiotic susceptibility in S. aureus mutants

All mutants used in this study were shown in Table 1. Table 3 shows the MIC results of the mutants against various antibiotics. In MW2-derived ABC transporter mutants, the MIC of bacitracin in MM02 (ΔbceAB), MM07 (ΔbceB) and MM03 (ΔvraDE), showed two- and fourfold reductions, respectively, compared with that of the wild type, while the MIC of MM01 (ΔvraFG) showed a similar level to that of the wild type. Also, the MIC of bacitracin in a TCS mutant, MM08 (ΔbceS), was reduced fourfold compared with that of the wild type, showing a similar result with that of FK77 (ΔbceRS). In addition, two RN4220-derived mutants, MM05 (ΔbceAB) and MM06 (ΔvraDE), showed increased susceptibility to bacitracin (fourfold reduction in MM05, 16-fold reduction in MM06), while another mutant MM04 (ΔvraFG) showed no change. For the complementation experiment, three complementation strains (MM09, MM10 and MM11) showed a similar susceptibility to bacitracin with that of the wild type (Table 3). MIC of oxacillin in MM02 (ΔbceAB) showed twofold reduction, while that of MM05 showed no alteration. Also, MIC of vancomycin in MM01 and MM04 (ΔvraFG) showed twofold reduction. MICs of the mutants against erythromycin and ofloxacin were similar to that of the wild type.

Table 3.   Susceptibility to various antibacterial agents in Staphylococcus aureus mutants
  MIC (mg L−1)
StrainRelevant genotypeOXAVANBACERYOFX
  1. OXA, oxacillin; VAN, vancomycin; BAC, bacitracin; ERY, erythromycin; OFX, ofloxacin.

MW2Wild type82320.250.5
MM01ΔvraFG in MW281320.250.5
FK77ΔbceRS in MW28280.250.5
MM08ΔbceS in MW28280.250.5
MM09bceS in MM0882320.250.5
MM02ΔbceAB in MW242160.250.5
MM07ΔbceB in MW242160.250.5
MM10bceB in MM0782320.250.5
MM03ΔvraDE in MW28280.250.5
MM11vraDE in MM0382320.250.5
RN4220Wild type0.252640.1250.5
MM04ΔvraFG in RN42200.251640.1250.5
MM05ΔbceAB in RN42200.252160.1250.5
MM06ΔvraDE in RN42200.25240.1250.5

Population analysis showed a similar tendency as the MIC results (Fig. 2). FK77 (ΔbceRS), MM02 (ΔbceAB) and MM03 (ΔvraDE) strains reduced their resistance level, revealing hetero-resistance to bacitracin. MM03 in particular showed a significant reduction compared with two other mutants, FK77 and MM02.

Figure 2.

 Population analysis of the mutants and their parental strain MW2. Staphylococcus aureus cells cultured overnight were plated on TSA containing serial dilutions of bacitracin and were incubated at 37°C for 48 h.

Bacitracin sensing in S. aureus

In wild-type MW2 strain, the expression of bceA and vraD was rapidly induced by the addition of bacitracin into the medium (Fig. 3). This induction was found to occur after 5 min of bacitracin exposure. The level of vraD transcript was more than 100-fold that of the wild type. Interestingly, a low concentration of bacitracin (0.5, 1 μg mL−1) significantly induced the expression of two genes (bceA and vraD) until 15 min, after which the expression of both decreased. In contrast, bacitracin (above 8 μg mL−1) continued to induce these two genes even after 15 min. The level of the TCS (bceR) transcript itself did not increase by addition of bacitracin after 30 min, but slightly increased after 60 min (data not shown). The level of vraF transcript was not increased by bacitracin (data not shown).

Figure 3.

 Expressions of two transporters and TCS in Staphylococcus aureus MW2 and TCS mutant. Cells of MW2, MM08 (ΔbceS) and MM09 (bceS in MM08) were collected at 5, 15, 30 and 60 min after the addition of bacitracin. After RNA isolation and cDNA synthesis, quantitative PCR was performed. Three independent experiments were performed.

The expressions of bceA and vraD in MM08 (ΔbceS) were not induced by addition of bacitracin, while the induction of their expressions by bacitracin were observed in the strain (MM09) which complemented with the gene for bceS in MM08 (Fig. 3). Also, in FK77 (ΔbceRS) strain, induction of the expression of bceA and vraD upon the addition of bacitracin was completely inhibited.

Discussion

In this study, we identified one uncharacterized TCS (MW2545-44) that has the ability to sense bacitracin and positively regulates two transporters responsible for bacitracin resistance. Interestingly, this TCS regulates not only a downstream gene coding for ABC transporter (MW2543-42), but also another transporter (MW2620-2621) known as vraDE, which is located separately from the genes coding for the TCS. These two ABC transporters, especially VraDE, showed a high similarity with B. subtilis ABC transporter BceAB responsible for bacitracin efflux (Ohki et al., 2003). In addition, similar transporters showing homology with BceAB have been reported in several Gram-positive bacteria, such as BcrAB of B. licheniformis (Podlesek et al., 1995), BcrAB of Enterococcus faecalis (Manson et al., 2004) and MbrAB of Streptococcus mutans (Tsuda et al., 2002). This type of ABC transporter is considered to pump out bacitracin to the exterior of the cell directly. Upstream of bceAB in B. subtilis, the gene coding for TCS, known as BceRS, is located (Ohki et al., 2003; Rietkötter et al., 2008). BceRS has been demonstrated to sense bacitracin and induce the expression of BceAB, leading to resistance to bacitracin. Like bceRSAB in B. subtilis, the results in this study showed that S. aureus has the same system, so we designated this TCS as BceRS (MW2545-2544) and the downstream transporter as BceAB (MW2543-2542). In S. aureus, another gene, MW2546, was closely located upstream of bceRS, speculating three genes were in a same operon (Fig. 1). By reverse transcriptase-PCR analysis, we found that MW2546 was cotranscribed with bceRS (data not shown), but not with bceAB, although the function of MW2546 remains unknown.

In addition, we are the first to demonstrate the regulator for vraDE together with bceAB, although vraDE has already been reported to be related to bacitracin susceptibility (Pietiäinen et al., 2009). BceRS inactivation resulted in the failure of upregulation for vraDE expression by bacitracin, indicating that BceRS regulates two genes, bceAB and vraDE. The expression of two transporters was induced rapidly from 5 min after addition of bacitracin. However, the increased expression was suppressed from 15 min after the addition of a low concentration of bacitracin, speculating that the amount of two transporters by short-time induction was sufficient to resist to low concentration of bacitracin. Also, the inactivation of bceAB, but not vraDE, reduced the oxacillin resistance slightly, suggesting that bceAB may affect the cell wall biosynthesis. Inactivation of another transporter, vraFG (MW0623-0624), showing homology with bceAB in B. subtilis, did not cause an alteration in susceptibility to bacitracin. The gene related to this transporter, vraFG, is located downstream of apsRS/graRS (MW0621-0622), one of the TCSs in S. aureus, and this TCS has been demonstrated to regulate vraFG expression (Li et al., 2007). Also, vraFG was reported to be associated with vancomycin susceptibility (Meehl et al., 2007). In this study, we also had a similar result that vraFG mutation led to increase the susceptibility to vancomycin (Table 3). We determined that apsRS inactivation did not affect the increased expression of vraDE and bceAB by bacitracin induction (data not shown), so we concluded that apsRS and vraFG were not associated with bacitracin susceptibility in S. aureus. Furthermore, it was reported that bacitracin induced the expression of vraSR (Kuroda et al., 2003), implying the possibility of the relation of BceRS with VraSR. However, we found the vraSR expression was increased by bacitracin in bceRS mutant (data not shown). Also, in vraSR mutant, the expression of bceA and vraD was significantly induced by bacitracin. These results indicate that BceRS has no effect on VraSR expression by bacitracin.

Previously, the bacA gene (MW0645) affecting bacitracin susceptibility was reported in S. aureus (Chalker et al., 2000). The gene bacA was first identified on a multicopy plasmid in E. coli, causing an increase in isoprenol kinase activity and decrease in bacitracin susceptibility. Therefore, BacA in S. aureus is considered to have an undecaprenol kinase activity related to undecaprenol pyrophosphate recycling. Inactivation of bacA resulted in an increase in the susceptibility to bacitracin, showing an MIC of 4 μg mL−1 in the bacA mutant compared with 64 μg mL−1 in the wild type (RN4220) (Chalker et al., 2000). We investigated whether bceRS regulates the expression of bacA induced by bacitracin but found no such correlation (data not shown). Therefore, S. aureus has two independent factors responsible for susceptibility to bacitracin.

In conclusion, we found that a TCS, designated BceRS, senses bacitracin and also positively regulates the expression of two ABC transporters that function in bacitracin efflux.

Acknowledgements

This work was supported by a grant-in-aid for scientific research from Health and Labor Sciences Research Grants from the Ministry of Health and Welfare of Japan.

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