Molecular basis of florfenicol-induced increase in adherence of Staphylococcus aureus strain Newman

Maren Blickwede1, Ralph Goethe2, Christiane Wolz3, Peter Valentin-Weigand2 and Stefan Schwarz1,*

1 Institut für Tierzucht, Bundesforschungsanstalt für Landwirtschaft (FAL), Höltystrasse 10, 31535 Neustadt-Mariensee; 2 Institut für Mikrobiologie, Zentrum für Infektionsmedizin, Tierärztliche Hochschule Hannover, Bischofsholer Damm 15, 30173 Hannover; 3 Institut für Medizinische Mikrobiologie und Hygiene, Universität Tübingen, Wilhelmstrasse 31, 72074 Tübingen, Germany


* Corresponding author. Tel: +49-5034-871-241; Fax: +49-5034-871-246; E-mail: stefan.schwarz{at}fal.de

Received 8 December 2004; returned 9 May 2005; revised 19 May 2005; accepted 3 June 2005


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives: The aim of this study was to determine the molecular basis of the florfenicol-dependent increased adherence of Staphylococcus aureus strain Newman to HEp-2 cells.

Methods and results: Northern slot blot analysis showed that mRNA expression of fnbA, fnbB, coa, emp and eap, coding for adhesins, was increased in the presence of 0.5 x MIC of florfenicol. Under the same conditions expression of cap5, coding for type 5 capsular polysaccharides, was distinctly decreased. Since global regulatory systems can modulate the expression of adhesins, their role in this process was investigated by including three isogenic mutants with functionally inactive global regulator systems, agr, sar or sae. Growth in the presence of 0.5 x MIC of florfenicol significantly increased the adherence to HEp-2 cells, fibronectin and fibrinogen of the {Delta}agr and {Delta}sar mutant strains, but not that of the {Delta}sae mutant strain. In contrast to components of the agr or sar system, expression of saeRS was increased, suggesting a potential sae-directed decrease in the expression of cap5 and increase in the expression of genes coding for adhesins under the influence of florfenicol. Analysis of RNA stability revealed that the increased amount of transcripts of saeRS and adherence-associated genes was due to a stabilization of the respective mRNAs by florfenicol.

Conclusions: Our data provide evidence that an activation of the global regulator sae and a stabilization of mRNA coding for specific adhesins seem to act synergically in generating a more adherent phenotype in the presence of a high subinhibitory concentration of florfenicol.

Keywords: fibronectin , fibrinogen , capsule , RNA stability , HEp-2 cells


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Adherence to epithelial cells has been implicated as the first step in the initiation of staphylococcal infections.1 Staphylococcus aureus expresses specific surface-associated proteins, referred to as MSCRAMM (microbial surface components recognizing adhesive matrix molecules), that allow the organisms to interact specifically with extracellular matrix proteins of the host cell, such as fibronectin (Fn), and fibrinogen (Fg).2,3 Numerous in vitro and in vivo studies have underlined their role in staphylococcal infections.4 Furthermore, S. aureus can produce extracellular proteins that are able to mediate adherence to the host matrix, such as coagulase (Coa),5 the extracellular adherence protein (Eap),6 the extracellular matrix protein-binding protein (Emp)7 or the extracellular Fg-binding protein (Efb).8

Several global regulatory loci, such as agr, sar and sae, are involved in the regulation of staphylococcal adherence factors.9 The agr locus regulates the expression of cell wall-associated proteins and secreted exoproteins in response to the density of the bacterial population.10 Type 5 capsular polysaccharides (CP5), which can mask adhesins, are generally up-regulated in the post-exponential growth phase, while the production of cell surface proteins, which are mainly expressed during early growth phases, are down-regulated by agr.9,11 The agr locus specifies two transcripts, RNA II and RNA III, of which the former determines activation of agr transcription and the latter is the effector of agr-positive and -negative regulation. RNA III is thought to regulate most target genes at the level of transcription, but has also been shown to influence the translation of some genes. SarA, the major functional protein encoded by the sar locus, is believed to be required for the stimulation of transcription of agr by binding to the agr promotor(s).12 Furthermore, SarA also influences the regulation of several virulence factors independently of agr. It seems to affect directly the binding to Fn by up-regulating the transcription of the gene fnbA, coding for Fn-binding protein A (FnBPA).13 More recently, the regulatory locus sae has been described.14 It encodes a two-component regulatory system including SaeR, a response regulator, and SaeS, a histidine protein kinase. This system up-regulates the transcription of {alpha}- and ß-haemolysins, DNase, and Coa.14 Futhermore, a {Delta}sae mutant of S. aureus strain Newman showed a reduced rate of invasion of human endothelial cells, consistent with diminished transcription and expression of fnbA and increased expression of CP5.15 Thus, the complex interaction of all global regulators leads to a coordinated production of adhesins in response to changing environmental conditions.9

Several studies have demonstrated that subinhibitory concentrations of different antibiotics can affect the expression of certain staphylococcal virulence factors,1618 including those mediating the adherence to host cells.1921 We have recently shown that 0.5 x MIC of florfenicol increased the adherence of S. aureus strain Newman to epithelial cells and Fn-coated microtitre plates.22 Florfenicol, a synthetic fluorinated chloramphenicol derivative, is used exclusively in veterinary medicine.23 Its mode of action closely resembles that of chloramphenicol. Florfenicol inhibits protein synthesis by binding to the 50S ribosomal subunit of susceptible bacteria.24

The aim of this study was to gain insight into the molecular mechanisms of the florfenicol-dependent increase in staphylococcal adherence and the role of global regulatory systems in this process. Therefore, S. aureus strain Newman and three isogenic mutants with functionally inactive global regulator systems, agr, sar or sae, were treated with 0.5 x MIC of florfenicol and compared for their adherence properties and capsule expression, as well as steady-state levels of mRNA coding for specific adhesins and components of global regulator systems.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial strains and growth conditions

S. aureus strain Newman (NCTC 8178)25 and the isogenic {Delta}agr, {Delta}sar or {Delta}sae mutants Newman {Delta}agr::tet(M) (ALC 355),26 Newman {Delta}sar::Tn917LTV1 (ALC 637)13 and Newman {Delta}sae::Tn917 (AS3)27 were included in this study. MICs of florfenicol were determined by broth microdilution according to the NCCLS guideline M31-A2.28 For investigation of the adherence properties and CP5 production, all strains were cultivated for 20 h on a rotary shaker at 37°C in pure brain–heart infusion broth (BHI; Oxoid, Wesel, Germany) and in BHI supplemented with 2 mg/L florfenicol (=0.5 x MIC for S. aureus Newman). The staphylococci were washed twice and resuspended in sterile phosphate-buffered saline (PBS; Sigma, Taufkirchen, Germany) prior to their use in experiments.

For RNA isolation, cells of an overnight culture were diluted to an initial optical density at 600 nm (OD600) of 0.05 and grown under constant shaking (120 rpm) at 37°C until the bacteria reached the early-, mid- or post-exponential growth phase, corresponding to an OD600 of 0.2, 1.0 and 1.6, respectively. Then, florfenicol was added to yield a final concentration of 2 mg/L and the cultures were incubated under the same conditions for 1 h. The control cultures without florfenicol were grown until they reached the same optical densities.

Adherence to epithelial cells

Adherence assays were performed as described previously.29 Approximately 5 x 106 bacteria were added to confluent monolayers of HEp-2 cells (~3 x 105 cells per well) and incubated for 2 h at 37°C in an atmosphere containing 5% CO2. After washing with PBS, the epithelial cells were treated with 0.05% trypsin-EDTA (Invitrogen, Karlsruhe, Germany) and 0.25% Triton X-100 (Serva, Heidelberg, Germany) to release adherent bacteria. Serial dilutions of adherent bacteria were plated onto BHI agar to determine the cfu. The results were recorded as percentage of cfu of adherent bacteria compared with the total number of cfu after 2 h incubation of controls under the same conditions in the presence of epithelial cells.

For microscopic evaluation, adherence assays were performed with HEp-2 cells grown on slides containing ~75% confluent monolayers of HEp-2 cells (~2 x 105 cells per well). Washed monolayers with adherent bacteria were fixed with 0.37% formaldehyde (Sigma) overnight at 4°C, washed with PBS and covered for 1 h with blocking buffer [10% fetal calf serum (Sigma) in PBS]. This buffer was replaced by a solution containing a polyclonal antibody raised in rabbits by immunization with inactivated staphylococci (provided by Gunter Amstberg, Tierärztliche Hochschule Hannover, Germany) and incubated for 1 h at room temperature. After washing with PBS the coverslips were incubated for 1 h with a fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG antibody (Dianova, Hamburg, Germany), washed once with PBS and twice with double-distilled water. Bacteria-per-epithelial cell ratios were determined by counting the number of bacteria adherent to epithelial cells in three microscopic fields with ~50 epithelial cells per field.

Adherence to Fn- or Fg-coated microtitre plates

Wells of a 96-well microtitre plate (Roth, Karlsruhe, Germany) were coated with 1 µg bovine Fn (Invitrogen) or 5 µg bovine Fg (Sigma) for 1 h at 37°C. After the wells were washed twice with PBS, residual binding sites were blocked by adding 100 µL of bovine serum albumin (Roth) followed by incubation for 1 h at 37°C. After washing with PBS, the bacteria were resuspended in PBS containing 0.05% Tween 20 and 100 µL of the bacterial suspension (1 x 106 bacteria) was added to each well. The specificity of Fn-/Fg-binding was confirmed by adding the same bacterial suspension to wells that were not coated with Fn or Fg. The plates were incubated for 1 h at 37°C and then washed three times with PBS to remove non-adherent bacteria. Adherent bacteria were detached with trypsin-EDTA and plated onto BHI agar. The results were recorded as percentage of the difference between cfu of adherent bacteria and those of bacteria adhering to non-coated wells, compared with the total number of cfu after 1 h incubation of controls under the same conditions in the presence of Fn or Fg.

Evaluation of CP5 production

CP5 production was detected by an indirect immunofluorescence assay as previously described by Pöhlmann-Dietze et al.11 Briefly, protein A was blocked by incubation with human immunoglobulin G, and CP5 antigen was detected by using a monoclonal antibody raised against CP530 and secondary Cy3- or FITC-labelled anti-mouse antibodies. In a subsequent step, the bacteria were stained with 4',6-diamidino-2-phenylindole (DAPI) (Sigma) for 5 min at room temperature. Three microscopic fields with ~50 bacteria per field were evaluated, and the percentage of CP5-positive bacteria was determined by comparing the number of fluorescent bacteria with the total number of DAPI-stained bacteria.

RNA isolation

For RNA isolation, ~109 S. aureus cells were lysed in 1 mL of Trizol reagent (Invitrogen) with 0.4 mL of zirconia-silica beads (diameter 0.1 mm; Roth) in a Mini-BeadbeaterTM (Biospec Products, Bartlesville, OK, USA). RNA was isolated as described in the instructions by the manufacturer of Trizol. Contaminating DNA was degraded by incubating 30 µg RNA samples in the presence of 1.5 mM MgCl2, 80 U of RNasin (Invitrogen) and 50 U of DNaseI (Roche, Mannheim, Germany) at 37°C for 30 min.

Slot blot hybridization

Two-fold serial dilutions of sample RNA (10 µL) were mixed with 30 µL denaturation solution [660 µL formamide, 210 µL of 37% (w/v) formaldehyde and 130 µL of 10 x MOPS, pH 7.0], denaturated for 15 min at 65°C, then mixed with 40 µL of 20 x SSC and finally transferred onto a positively charged nylon membrane (Qbiogene, Heidelberg, Germany) with a Slot-Blotter (Roth). The membranes were fixed for 2 h at 80°C, pre-incubated for 2 h at 65°C in hybridization solution [1 mM EDTA (pH 7.5), 0.5 M Na2HPO4 (pH 7.2); 7% (w/v) SDS], and then hybridized overnight with a heat-denatured 32P-labelled DNA probe and 100 mg/L yeast tRNA (Invitrogen) in hybridization solution as previously described by Church and Gilbert.31 The specific gene probes were prepared by PCR with gene-specific primers (Table 1). For this, the whole-cell DNA of S. aureus Newman served as the target DNA. All PCR products were cloned into pBlunt II TOPO (Invitrogen) and sequenced completely to verify their specificity. For use as gene probes the inserts were removed from the vector by EcoRI digestion, except for 16S rRNA, which was removed from the vector by HindIII and EcoRV double digestion, and then purified by the use of the Gel Extraction Kit (Qiagen, Hilden, Germany). Approximately 200 ng of the specific purified DNA was labelled with [{alpha}-32P]dCTP using the Nick Translation System (Invitrogen) and then used for hybridization. Finally, the blot was analysed by the use of the Bio-Rad Gel Doc 1000 Station (Bio-Rad, München, Germany) and quantified with the Multi Analyst 1.1 software (Bio-Rad). The quantitative values obtained were normalized to corresponding values for 16S rRNA, obtained from hybridization of the same membranes with a 16S rRNA-specific probe (Table 1).


View this table:
[in this window]
[in a new window]
 
Table 1. PCR primers used in this study to generate specific gene probes

 
RNA stability test

For determination of RNA stability, the bacteria, grown in the presence or absence of florfenicol for 1 h, were treated with rifampicin (150 mg/L) for 0, 1, 2.5, 5, 10, 20 and 30 min before isolating RNA. For northern blotting, 10 µg of DNase-digested RNA was denaturated in the presence of 21.6% glyoxal, 75.4% DMSO and 10 mM Na2HPO4 (pH 6.9) for 1 h at 50°C and then electrophoretically separated in 1.5% (w/v) agarose gels with an aqueous 10 mM Na2HPO4 solution (pH 6.9) as running buffer. RNA was transferred to a positively charged nylon membrane (Qbiogene) by the use of a capillary blot in the presence of 20 x SSC and then hybridized as described above. Half-lives were determined by linear regression analysis of percent RNA remaining versus time.

Statistical analysis

Each experiment was performed independently in triplicate, and within each experiment all samples were processed in duplicate. The values obtained after treatment of the bacteria with florfenicol were compared with those obtained for the untreated control by an unpaired two-sided Student's t-test. P values of ≤0.05 were considered as significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Florfenicol affects steady-state levels of mRNA coding for adhesins and CP5

We have previously shown that 0.5 x MIC of florfenicol increased the Fn-mediated adherence of S. aureus strain Newman to HEp-2 cells.22 To gain insight into the underlying mechanism(s), we first analysed the florfenicol-dependent mRNA expression of a set of genes coding for different matrix protein-binding proteins that play an important role in staphylococcal adherence, i.e. the cell wall-associated FnBPA (fnbA) and the two secreted adhesins, Eap (eap) and Emp (emp), that are able to bind Fn as well as Fg.6,7,32 Furthermore, we also included the genes coding for Coa (coa), clumping factor A (ClfA, clfA), Efb (efb), and FnBPB (fnbB), which can bind specifically either Fg or Fn,5,8,33 as well as cap5A coding for the first gene within the CP5 gene cluster,34 which is known to interfere with adherence.11 The steady-state levels of mRNA of these genes were analysed by northern slot blot analysis after S. aureus Newman was grown in the presence or absence of 0.5 x MIC of florfenicol. Because expression of these genes could be influenced by the growth phase, we analysed the florfenicol-dependent mRNA expression in the early-, mid- and post-exponential growth phases. The amount of gene-specifc transcripts of fnbA, fnbB and coa, which are expressed mainly during the early growth phase,26,35 was increased four-, two- and 23-fold, respectively, when S. aureus Newman was treated with 0.5 x MIC of florfenicol (Table 2). The amounts of transcripts of emp and eap were increased up to two- and three-fold, respectively, after treatment of S. aureus Newman with 0.5 x MIC of florfenicol during the post-exponential phase (Table 2), when the expression of these genes reached the maximum.7,36 In contrast, the expression of genes clfA and efb was not increased under the influence of florfenicol (data not shown), either during the early- or mid-exponential phase, or during the post-exponential phase. Moreover, florfenicol distinctly decreased cap5A expression to 0.1-fold compared with the untreated control during the post-exponential growth phase (Table 2), when the expression level of CP5 reaches the maximum.11 During the early- or mid-exponential growth phase no mRNA of cap5A was detectable. These data suggest that 0.5 x MIC of florfenicol increases the steady-state levels of mRNA of genes coding for several different adhesins and decreases that of mRNA of a gene coding for CP5, and thereby may potentially generate a more adherent phenotype, as seen in our previous study.22


View this table:
[in this window]
[in a new window]
 
Table 2. Effect of 0.5 x MIC of florfenicol on mRNA expression of S. aureus Newman and its isogenic {Delta}agr, {Delta}sar or {Delta}sae mutant strains

 
Florfenicol affects adherence to Fg and expression of CP5

Based on these results, we further investigated the effect of 0.5 x MIC of florfenicol on (i) the adherence of S. aureus strain Newman to microtitre plates coated with the matrix protein Fg, and (ii) the expression of CP5. When grown in the presence of 0.5 x MIC of florfenicol, S. aureus Newman showed a significant (P ≤ 0.05) five-fold increase in adherence to Fg-coated microtitre plates. In the absence of florfenicol, 5.6% (±2.4) of the bacteria adhered to Fg-coated microtitre plates, but when incubated in the presence of 0.5 x MIC of florfenicol, 27.1% (±13.0) of the bacteria adhered to Fg-coated microtitre plates. This observation was in good accordance with the results of our initial study, where the same strain showed a four-fold increase in adherence to HEp-2 cells and a five-fold increase in adherence to Fn-coated microtitre plates.22 Furthermore, indirect immunofluorescence microscopy with monoclonal antibodies against CP5 revealed that the expression of CP5 was decreased significantly (P ≤ 0.05) to 3.6% (±1.7) when the bacteria were treated with 0.5 x MIC of florfenicol as compared with 17.5% (±5.5) of the untreated control (Figure 1). These observations correlated well with the results obtained from northern slot blot analysis and suggest that 0.5 x MIC of florfenicol may reduce masking of specific adhesins by CP5 and increase binding of S. aureus Newman to the matrix proteins Fn and Fg of the host cell, with both effects resulting in a more adherent phenotype.



View larger version (41K):
[in this window]
[in a new window]
 
Figure 1. Microscopic evaluation of the effect of florfenicol on expression of CP5. S. aureus strain Newman was grown to the stationary phase in the absence (a and b) or presence (c and d) of 0.5 x MIC of florfenicol. DNA from bacterial cells were stained with DAPI (b and d), and CP5 was detected by indirect immunofluorescence (a and c). Magnification x1000. This Figure is available in colour with the online copy of this article, which can be found at http://jac.oupjournals.org.

 
Effects of florfenicol on global regulatory systems

Since it is known that global regulatory systems have an impact on the expression of adhesins and CP5, we investigated whether the agr, sar and sae systems may play a role in the observed increase in adherence. Accordingly, we compared S. aureus strain Newman and three isogenic mutants of S. aureus Newman with functionally inactive global regulator systems agr, sar and sae, respectively, for their adherence properties and expression of genes coding for the above-mentioned adhesins and CP5 after growth in the presence of 0.5 x MIC of florfenicol. All strains showed MICs of 4 mg/L florfenicol, thus, 2 mg/L represented the strain-specfic 0.5 x MIC for all strains used in this study.

Growth in the presence of 2 mg/L florfenicol increased significantly (P ≤ 0.05) the adherence to HEp-2 cells of the {Delta}agr and {Delta}sar mutant strains (Figure 2a), as has previously been shown for the wild-type strain.22 In contrast, florfenicol did not affect adherence of the {Delta}sae mutant strain to HEp-2 cells (Figure 2a). Results of microscopic evaluation of adherence to epithelial cells by immunofluorescence microscopy correlated well with those obtained by the plating technique (Figures 2a and 3). A three- and two-fold increase in the number of adherent bacteria per epithelial cell was detected in the {Delta}agr and {Delta}sar mutant strains, respectively, whereas no increase was seen in the {Delta}sae mutant strain (Figures 2a and 3). Similar results were found when comparing the adherence of the three mutant strains with Fg- or Fn-coated microtitre plates (Figure 2b and c). The Fg- and Fn-binding of the {Delta}agr or {Delta}sar mutants grown in the presence of 0.5 x MIC of florfenicol was increased two- to six-fold, whereas the Fn- and Fg-binding pattern of the {Delta}sae mutant showed no significant increase after florfenicol-treatment (Figure 2b and c). Moreover, in contrast to the {Delta}sae mutant strain that exhibited the highest CP5 expression of 47.5% (Figure 2d), the expression of CP5 was distinctly decreased in the {Delta}agr and {Delta}sar mutants from 9.5% (±5.7) to 2.1% (±1.6) and 30.5% (±12.5) to 20.2% (±9.1), respectively, when treated with 0.5 x MIC of florfenicol (Figure 2d).



View larger version (17K):
[in this window]
[in a new window]
 
Figure 2. Effect of 0.5 x MIC of florfenicol on adherence properties and CP5 expression of S. aureus Newman wild-type and its isogenic {Delta}agr, {Delta}sar or {Delta}sae mutant strains. Bacteria were cultivated for 20 h in BHI (white bars) or BHI supplemented with 2 mg/L florfenicol (grey bars) prior to their use in the experiments. The error bars represent the standard deviations of the mean of three independent determinations. *P ≤ 0.05.

 


View larger version (143K):
[in this window]
[in a new window]
 
Figure 3. Microscopic evaluation of the effect of florfenicol on adherence to HEp-2 cells of S. aureus Newman {Delta}agr, {Delta}sar and {Delta}sae. Magnification x500. (a) S. aureus Newman {Delta}agr grown in the absence of florfenicol and (b) grown in the presence of 0.5 x MIC of florfenicol. (c) S. aureus Newman {Delta}sar grown in the absence of florfenicol and (d) grown in the presence of 0.5 x MIC of florfenicol. (e) S. aureus Newman {Delta}sae grown in the absence of florfenicol and (f) grown in the presence of 0.5 x MIC of florfenicol. This Figure is available in colour with the online copy of this article, which can be found at http://jac.oupjournals.org.

 
The observation that increased adherence profiles and decreased capsule expression under the influence of florfenicol could not be detected in the {Delta}sae mutant strain raised the question of whether the global regulator sae, in contrast to the global regulator systems agr or sar, might be involved in this process of florfenicol-dependent increase in adherence. Therefore, we compared the wild-type and all three mutant strains for their steady-state levels of different transcripts of specific adhesins and CP5 after treatment with florfenicol by northern slot blot analysis. No distinct florfenicol-dependent differences were observed between the wild-type strain and the {Delta}agr or the {Delta}sar mutant strain (Table 2); the florfenicol-dependent expression profiles were seen in all three strains. In the {Delta}sae mutant, mRNA of the adhesin genes fnbA, fnbB, coa and emp was not detectable, while the amounts of cap5A transcripts were not affected (Table 2). In addition, we also evaluated the mRNA expression of components of the three global regulator systems. The mRNA expression of components of the global regulator system agr, RNA II and RNA III, and that of sarA of the sar system were not affected by 0.5 x MIC of florfenicol (Table 2). In contrast, the expression of saeRS was increased up to five-fold after treating the wild-type S. aureus strain Newman with 0.5 x MIC of florfenicol during post-exponential growth phase, when saeRS was maximally expressed (Table 2).15

Effects of florfenicol on mRNA stability

It has been shown that some protein biosynthesis-inhibiting antibiotics, such as chloramphenicol, erythromycin or tetracycline, may have a stabilizing effect on specific mRNAs.3739 To determine whether the florfenicol-dependent increased steady-state levels of specific transcripts were due to an increase in mRNA stability, northern blot analysis was performed with total RNA isolated from S. aureus strain Newman at various times after addition of rifampicin in the presence or absence of florfenicol (Figure 4). Rifampicin inhibits initiation of RNA synthesis by binding to the DNA-dependent RNA polymerase. The RNA stability of both components of the global regulator system agr, RNA II (Figure 4) and RNA III, was not affected by 0.5 x MIC of florfenicol. The half-life of RNA II was 28 min (Figure 4) and the half-life of RNA III was 18 min. Similar results were obtained with sarA of the sar system. The sarA-specific mRNA was not degraded during the 30 min of analysis in the presence or absence of florfenicol (Figure 4). In contrast, florfenicol stabilized the saeRS-specific RNA (Figure 4), including three different transcripts with approximate sizes of 3.0, 2.4 and 2.0 kb, respectively,15 confirming the results of northern slot blot analysis. The half-life of the saeRS transcripts was ~1–3 min in the absence of florfenicol and 20 min in the presence of florfenicol (Figure 4).



View larger version (98K):
[in this window]
[in a new window]
 
Figure 4. Effect of 0.5 x MIC of florfenicol on RNA stability of S. aureus Newman, analysed by northern blot hybridization. Before harvesting RNA, bacteria grown in the absence (control, ctr) or presence (Ff) of florfenicol for 1 h were treated with rifampicin (150 mg/L) to stop RNA synthesis. Time (min) after rifampicin addition is indicated above the different lanes.

 
The half-lives of coa and fnbA transcripts were <1 min in the absence of florfenicol and >30 min in the presence of florfenicol, and in the case of fnbB <1 min and 30 min, respectively (Figure 4). In the case of eap and emp, the half-lives were also <1 min in the absence of florfenicol, and 27 and 20 min, respectively, when the bacteria were treated with 0.5 x MIC of florfenicol (Figure 4). This observation strongly suggested that the observed increase in the amount of the RNA coding for the global regulator sae and for specific adhesins was due to mRNA stabilization leading to an accumulation of the specific transcripts after stimulation with 2 mg/L florfenicol.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
There are several examples demonstrating that subinhibitory concentrations of different antibiotics may interfere with processes of host–pathogen interactions such as adherence.18,21 Bacterial adhesion to epithelial cells represents the initial step in infectious processes and can be the result of either hydrophobic interactions between the bacteria and the host cells, binding of bacteria to specific ligands or a combination of both.40 In a previous study, we demonstrated that S. aureus Newman showed a non-significant decrease in its surface hydrophobicity, but a significant increased adherence to Fn when treated with 0.5 x MIC of florfenicol. This suggested that interactions with specific ligands, such as Fn, may play a relevant role in the observed increased adherence to epithelial cells, rather than non-specific hydrophobic interactions.22

To elucidate the molecular mechanisms in this florfenicol-dependent increase in the staphylococcal adherence properties, we investigated at the transcriptional level the expression of genes coding for different matrix protein-binding proteins and for CP5. Northern slot blot analysis revealed that the mRNA expression of genes coding for specific adhesins, such as fnbA, fnbB, coa, eap and emp, was increased, whereas the mRNA expression of cap5A was decreased when S. aureus Newman was treated with 0.5 x MIC of florfenicol. To extend our initial observations,22 these results were confirmed phenotypically by measuring the adherence to Fg-coated microtitre plates and the expression of CP5. Compared with the untreated control, S. aureus Newman grown in the presence of florfenicol demonstrated increased binding to the matrix protein Fg, as was previously shown for Fn,22 and also showed decreased CP5 production. The resulting phenotype, which exhibited increased adherence to epithelial cells, was most evident with bacteria that were harvested during the stationary phase of growth, when the expression level of CP5 usually reaches the maximum.11 Therefore, we assume that CP5 plays a relevant role in this phenomenon. The capsule may mask adhesins that have been shown to be important virulence factors. In our study, the decreased expression of CP5 during the post-exponential growth phase may support the ability of S. aureus Newman to adhere to epithelial cells, possibly by de-masking specific Fn- or Fg-binding molecules such as eap and emp, which are maximally expressed during this growth phase.

The data presented in this study suggest that the global regulator system sae, in contrast to agr or sar, plays an additional role in the observed increased adherence. The increased adherence to epithelial cells and to the specific ligands Fn and Fg, as well as the increased mRNA expression of specific adhesins and the decreased CP5 expression after treatment with 0.5 x MIC of florfenicol, could be detected in the {Delta}agr and the {Delta}sar mutant strains as well, indicating agr- and sar-independent processes. In contrast, the {Delta}sae mutant showed no change in adherence profiles and expression of specifc adhesins and CP5. Expression of saeRS transcripts, but not that of the transcripts of components of the global regulator systems agr and sar, was increased when the strains were cultivated in the presence of 0.5 x MIC of florfenicol. This confirms that the florfenicol-induced adherence is an sae-dependent, but an agr- and sar-independent process. In a recent study, transcripts of saeRS were also found to be induced by the cell-wall synthesis inhibitors vancomycin, teicoplanin, ceftizoxime and imipenem. However, mechanism or changes in the phenotype have not been addressed.41

The contribution of sae to virulence has been demonstrated after intraperitoneal injection of bacteria into mice.42 A {Delta}sae mutant strain showed a significantly reduced rate of invasion of human endothelial cells, consistent with diminished transcription of fnbA and coa, and an activated expression of CP5.15 These data are in good accordance with the data from northern slot blot analysis presented in this study, in which a decrease in CP5 expression and an increase in mRNA expression of coa, fnbA and some other genes coding for adhesins was detectable when S. aureus Newman was treated with florfenicol. Therefore, we suggest an sae-directed decrease in the expression of cap5 and a further sae-directed increase in transcription of specific adhesins such as coa and fnbA. In the sae mutant strain, neither mRNA of fnbA and coa (as previously reported by Steinhuber et al.15) nor mRNA of emp, eap and fnbB was detectable in the present study. However, increased amounts of transcripts of all these genes were seen when the bacteria were treated with florfenicol. Therefore, we assume that the global regulator sae may play a role in the increased expression of all these specific mRNAs.

Northern blot analysis on total RNA isolated from S. aureus Newman at various times after rifampicin addition revealed that the observed increase in the amount of the gene-specific transcripts coding for the global regulator sae and also for specific adhesins in the presence of florfenicol was most likely due to a stabilization of the respective mRNAs. These results revealed an additional effect of florfenicol on gene expression in S. aureus, namely that a high subinhibitory concentration of florfenicol can result in increased stability of specific mRNA. This observation is in good accordance with data on mRNA stabilization by other protein biosynthesis inhibitors. It has already been shown by Sandler and Weisblum that in S. aureus erythromycin causes a specific increase in the half-life of transcripts of the gene erm(A) coding for an rRNA methylase involved in erythromycin resistance, whereas the half-life of cat-86 mRNA, coding for a chloramphenicol acetyltransferase, was not increased by erythromycin.38 In Bacillus subtilis, chloramphenicol proved to be able to stabilize the cat mRNA,37 and addition of subinhibitory concentrations of tetracycline resulted in stabilization of transcripts of the tetracycline resistance gene tet(L) and several other cellular mRNAs.39

In conclusion, the data from the present study indicate that 0.5 x MIC of florfenicol leads to a stabilization of the mRNA coding for the global regulator Sae, followed by a potential sae-directed decrease in the expression of cap5 and enhancement of transcription of specific adhesins. Furthermore, a stabilization of the mRNA coding for specific adhesins was observed. All effects may act synergically in generating a more adherent phenotype, and thus could alter virulence properties of this pathogen in the presence of a subinhibitory concentration of florfenicol.


    Acknowledgements
 
We wish to thank Roswitha Becker for excellent technical assistance, and Jean-Michael Fournier and Gunter Amtsberg for kindly providing the monoclonal anti-capsular antibody and the staphylococcal antibody, respectively. This work was supported by the Deutsche Forschungsgemeinschaft (GK 745).


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1. von Eiff C, Becker K, Machka K et al. Nasal carriage as a source of Staphylococcus aureus bacteremia. N Engl J Med 2001; 344: 11–6.[Abstract/Free Full Text]

2. Patti JM, Allen BL, McGavin MJ et al. MSCRAMM-mediated adherence of microorganisms to host tissues. Annu Rev Microbiol 1994; 48: 585–617.[CrossRef][ISI][Medline]

3. Foster TJ, Höök M. Surface protein adhesins of Staphylococcus aureus. Trends Microbiol 1998; 6: 484–8.[CrossRef][ISI][Medline]

4. Moreillon P, Que YA, Bayer AS. Pathogenesis of streptococal and staphylococcal endocarditis. Infect Dis Clin North Am 2002; 16: 297–318.[CrossRef][ISI][Medline]

5. Dickinson RB, Nagel JA, McDevitt D et al. Quantitative comparison of clumping factor- and coagulase-mediated Staphylococcus aureus adhesion to surface-bound fibrinogen under flow. Infect Immun 1995; 63: 3143–50.[Abstract]

6. Palma M, Haggar A, Flock JI. Adherence of Staphylococcus aureus is enhanced by an endogenous secreted protein with broad binding activity. J Bacteriol 1999; 181: 2840–5.[Abstract/Free Full Text]

7. Hussain M, Becker K, von Eiff C et al. Identification and characterization of a novel 38.5-kilodalton cell surface protein of Staphylococcus aureus with extended-spectrum binding activity for extracellular matrix and plasma proteins. J Bacteriol 2001; 183: 6778–86.[Abstract/Free Full Text]

8. Palma M, Wade D, Flock M et al. Multiple binding sites in the interaction between an extracellular fibrinogen-binding protein from Staphylococcus aureus and fibrinogen. J Biol Chem 1998; 273: 13177–81.[Abstract/Free Full Text]

9. Novick RP. Autoinduction and signal transduction in the regulation of staphylococcal virulence. Mol Microbiol 2003; 48: 1429–49.[CrossRef][ISI][Medline]

10. Ji G, Beavis RC, Novick RP. Cell density control of staphylococcal virulence mediated by an octapeptide pheromone. Proc Natl Acad Sci USA 1995; 92: 12055–9.[Abstract/Free Full Text]

11. Pöhlmann-Dietze P, Ulrich M, Kiser KB et al. Adherence of Staphylococcus aureus to endothelial cells: influence of capsular polysaccharide, global regulator agr, and bacterial growth phase. Infect Immun 2000; 68: 4865–71.[Abstract/Free Full Text]

12. Chien YT, Cheung AL. Molecular interactions between two global regulators, sar and agr, in Staphylococcus aureus. J Biol Chem 1998; 273: 2645–52.[Abstract/Free Full Text]

13. Wolz C, Pöhlmann-Dietze P, Steinhuber A et al. Agr-independent regulation of fibronectin-binding protein(s) by the regulatory locus sar in Staphylococcus aureus. Mol Microbiol 2000; 36: 230–43.[CrossRef][ISI][Medline]

14. Giraudo AT, Calzolai A, Cataldi AA et al. The sae locus of Staphylococcus aureus encodes a two-component regulatory system. FEMS Microbiol Lett 1999; 177: 15–22.[CrossRef][ISI][Medline]

15. Steinhuber A, Goerke C, Bayer MG et al. Molecular architecture of the regulatory locus sae of Staphylococcus aureus and its impact on expression of virulence factors. J Bacteriol 2003; 185: 6278–86.[Abstract/Free Full Text]

16. Gemmell CG. Antibiotics and the expression of staphylococcal virulence. J Antimicrob Chemother 1995; 36: 283–91.[Abstract]

17. Gemmell CG, Ford CW. Virulence factor expression by Gram-positive cocci exposed to subinhibitory concentrations of linezolid. J Antimicrob Chemother 2002; 50: 665–72.[Abstract/Free Full Text]

18. Shibl AM. Influence of subinhibitory concentrations of antibiotics on virulence of staphylococci. Rev Infect Dis 1987; 9: 704–12.[ISI][Medline]

19. Bisognano C, Vaudaux PE, Lew DP et al. Increased expression of fibronectin-binding proteins by fluoroquinolone-resistant Staphylococcus aureus exposed to subinhibitory levels of ciprofloxacin. Antimicrob Agents Chemother 1997; 41: 906–13.[Abstract]

20. Bisognano C, Vaudaux P, Rohner P et al. Induction of fibronectin-binding proteins and increased adhesion of quinolone-resistant Staphylococcus aureus by subinhibitory levels of ciprofloxacin. Antimicrob Agents Chemother 2000; 44: 1428–37.[Abstract/Free Full Text]

21. Shibl AM. Effect of antibiotics on adherence of microorganisms to epithelial cell surfaces. Rev Infect Dis 1985; 7: 51–65.[ISI][Medline]

22. Blickwede M, Valentin-Weigand P, Schwarz S. Subinhibitory concentrations of florfenicol enhance the adherence of florfenicol-susceptible and florfenicol-resistant Staphylococcus aureus. J Antimicrob Chemother 2004; 54: 286–8.[Free Full Text]

23. Schwarz S, Chaslus-Dancla E. Use of antimicrobials in veterinary medicine and mechanisms of resistance. Vet Res 2001; 32: 201–25.[CrossRef][ISI][Medline]

24. Schwarz S, Kehrenberg C, Doublet B et al. Molecular basis of bacterial resistance to chloramphenicol and florfenicol. FEMS Microbiol Rev 2004; 28: 519–42.[CrossRef][ISI][Medline]

25. Duthie ES, Lorenz LL. Staphylococcal coagulase: mode of action and antigenicity. J Gen Microbiol 1952; 6: 95–107.[ISI][Medline]

26. Wolz C, McDevitt D, Foster TJ et al. Influence of agr on fibrinogen binding in Staphylococcus aureus Newman. Infect Immun 1996; 64: 3142–7.[Abstract]

27. Goerke C, Fluckiger U, Steinhuber A et al. Impact of the regulatory loci agr, sarA and sae of Staphylococcus aureus on the induction of {alpha}-toxin during device-related infection resolved by direct quantitative transcript analysis. Mol Microbiol 2001; 40: 1439–47.[CrossRef][ISI][Medline]

28. National Committee for Clinical Laboratory Standards. Performance Standards for Antimicrobial Disc and Dilution Susceptibility Tests for Bacteria isolated from Animals—Second Edition: Approved Standard M31-A2. NCCLS, Wayne, PA, USA, 2002.

29. Dziewanowska K, Patti JM, Deobald CF et al. Fibronectin binding protein and host cell tyrosine kinase are required for internalization of Staphylococcus aureus by epithelial cells. Infect Immun 1999; 67: 4673–8.[Abstract/Free Full Text]

30. Hoeger PH, Lenz W, Boutonnier A et al. Staphylococcal skin colonization in children with atopic dermatitis: prevalence, persistence, and transmission of toxigenic and nontoxigenic strains. J Infect Dis 1992; 165: 1064–8.[ISI][Medline]

31. Church GM, Gilbert W. Genomic sequencing. Proc Natl Acad Sci USA 1984; 81: 1991–5.[Abstract/Free Full Text]

32. Wann ER, Gurusiddappa S, Höök M. The fibronectin-binding MSCRAMM FnbpA of Staphylococcus aureus is a bifunctional protein that also binds to fibrinogen. J Biol Chem 2000; 275: 13863–71.[Abstract/Free Full Text]

33. Jönsson K, Signäs C, Müller HP et al. Two different genes encode fibronectin binding proteins in Staphylococcus aureus. Eur J Biochem 1991; 202: 1041–8.[Abstract]

34. O'Riordan K, Lee JC. Staphylococcus aureus capsular polysaccharides. Clin Microbiol Rev 2004; 17: 218–34.[Abstract/Free Full Text]

35. Saravia-Otten P, Müller HP, Arvidson S. Transcription of Staphylococcus aureus fibronectin binding protein is negatively regulated by agr and an agr-independent mechanism. J Bacteriol 1997; 179: 5259–63.[Abstract/Free Full Text]

36. Haggar A, Hussain M, Lönnies H et al. Extracellular adherence protein from Staphylococcus aureus enhances internalization into eukaryotic cells. Infect Immun 2003; 71: 2310–7.[Abstract/Free Full Text]

37. Dreher J, Matzura H. Chloramphenicol-induced stabilization of cat messenger RNA in Bacillus subtilis. Mol Microbiol 1991; 5: 3025–34.[ISI][Medline]

38. Sandler P, Weisblum B. Erythromycin-induced stabilization of ermA messenger RNA in Staphylococcus aureus and Bacillus subtilis. J Mol Biol 1988; 203: 905–15.[CrossRef][ISI][Medline]

39. Wei Y, Bachhofer DH. Tetracycline induces stabilization of mRNA in Bacillus subtilis. J Bacteriol 2002; 184: 889–94.[Abstract/Free Full Text]

40. Ofek I, Hasty DL, Doyle RJ. Basic concepts in bacterial adhesion. In: Ofek I, Hasty DL, Doyle RJ, eds. Bacterial Adhesion to Animal Cells and Tissues. Washington, DC: ASM Press, 2003.

41. Kuroda M, Kuroda H, Oshima T et al. Two-component system VraSR positively modulates the regulation of cell-wall biosynthesis pathway in Staphylococcus aureus. Mol Microbiol 2003; 49: 807–21.[CrossRef][ISI][Medline]

42. Giraudo, AT, Rampone H, Calzolai A et. al. Phenotypic characterization and virulence of sae agr mutant of Staphylococcus aureus. Can J Microbiol 1996; 42: 120–3.[ISI][Medline]





This Article
Abstract
Full Text (PDF)
Supplementary Data
All Versions of this Article:
56/2/315    most recent
dki233v1
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Search for citing articles in:
ISI Web of Science (1)
Disclaimer
Request Permissions
Google Scholar
Articles by Blickwede, M.
Articles by Schwarz, S.
PubMed
PubMed Citation
Articles by Blickwede, M.
Articles by Schwarz, S.