Bicarbonate enhances expression of the endocarditis and biofilm associated pilus locus, ebpR-ebpABC, in Enterococcus faecalis

Abstract

Background: We previously identified ebpR, encoding a potential member of the AtxA/Mga transcriptional regulator family, and showed that it is important for transcriptional activation of the Enterococcus faecalis endocarditis and biofilm associated pilus operon, ebpABC. Although ebpR is not absolutely essential for ebpABC expression (100-fold reduction), its deletion led to phenotypes similar to those of an ebpABC mutant such as absence of pili at the cell surface and, consequently, reduced biofilm formation. A non-piliated ebpABC mutant has been shown to be attenuated in a rat model of endocarditis and in a murine urinary tract infection model, indicating an important participation of the ebpR-ebpABC locus in virulence. However, there is no report relating to the environmental conditions that affect expression of the ebpR-ebpABC locus.

Results: In this study, we examined the effect of CO2/HCO3(-), pH, and the Fsr system on the ebpR-ebpABC locus expression. The presence of 5% CO2/0.1 M HCO3(-) increased ebpR-ebpABC expression, while the Fsr system was confirmed to be a weak repressor of this locus. The mechanism by which the Fsr system repressed the ebpR-ebpABC locus expression appears independent of the effects of CO2(-) bicarbonate. Furthermore, by using an ebpA::lacZ fusion as a reporter, we showed that addition of 0.1 M sodium bicarbonate to TSBG (buffered at pH 7.5), but not the presence of 5% CO2, induced ebpA expression in TSBG broth. In addition, using microarray analysis, we found 73 genes affected by the presence of sodium bicarbonate (abs(fold) > 2, P < 0.05), the majority of which belong to the PTS system and ABC transporter families. Finally, pilus production correlated with ebpA mRNA levels under the conditions tested.

Conclusions: This study reports that the ebp locus expression is enhanced by the presence of bicarbonate with a consequential increase in the number of cells producing pili. Although the molecular basis of the bicarbonate effect remains unclear, the pathway is independent of the Fsr system. In conclusion, E. faecalis joins the growing family of pathogens that regulates virulence gene expression in response to bicarbonate and/or CO2.

Figure 1

ebpR and ebpA expression profiles in OG1RF. A. Expression levels of ebpA and ebpR using gene promoter::lacZ fusions. OG1RF containing either P_ebpR::lacZ (black triangle) or P_ebpA::lacZ (black square) were grown in TSBG. For β-gal assays, samples were collected every hour from 3 to 8 hr, then at 10 and 24 hr after starting the culture (x axis). The left axis represents the β-gal units (OD_{420 nm}/protein concentration in mg/ml). The right axis indicates the OD_{600 nm} readings. All sets of cultures presented were analyzed concurrently. This figure is a representative of at least three independent experiments. B. qRT-PCR with RNA purified from OG1RF cultures grown aerobically in TSBG. The left axis represents the level of transcript normalized to gyrB transcript level. The right axis indicates the OD_{600 nm} readings. The dashed line shows the mean (with standard deviation) of 5 independent cultures of OG1RF grown in TSBG. The transcript levels of ebpR (black triangle) and ebpA (black square) shown represent two different data sets, each tested in duplicate that were normalized using gyrB transcript levels.

Figure 2

CO₂/NaHCO₃ induction effect on ebpA expression level. Samples were collected every hour from 3 to 8 hr, then at 10 and 24 hr after starting the culture in TSBG. The left axis represents the β-gal units (OD_{420 nm}/protein concentration in mg/ml). The right axis indicates the OD_{600 nm} readings. All sets of cultures presented were analyzed concurrently. Each figure is a representative of at least three experiments. A. Growth curves of OG1RF are shown in gray (in air) and in orange (in the presence of 5% CO₂/0.1 M NaHCO₃). OG1RF containing P_ebpR::lacZ (triangle) or P_ebpA::lacZ (square) was grown in air (closed black symbol) or in the presence of 5% CO₂/0.1 M NaHCO₃ (open orange symbol). B. The ΔebpR mutant containing P_ebpR::lacZ is represented by closed green diamond when grown in air and with open brown diamond when grown in the presence of 5% CO₂/0.1 M NaHCO₃.

Figure 3

Detection of EbpC produced by OG1RF, ΔfsrB, and ΔebpR. A. Flow cytometry analysis of OG1RF grown in air (black) or in the presence of 5% CO₂/0.1 M NaHCO₃ (green) labeled with an anti-EbpC rabbit polyclonal immune serum and detected with phycoerythrin. The cells were collected at "T4", which corresponds to the entry into stationary growth phase (4 hrs after starting the culture). The percentages between brackets indicate the percentage of positive cells (WinMDI 2.9, marker set for 500-1024). In red is represented OG1RF grown in air incubated with a pre-immune serum and detected with Phycoerythrin as negative control. B. Flow cytometry analysis was done in the same conditions as above with samples collected at "T6" which corresponds to early stationary growth phase. C. An equal amount (by BCA protein assay) of mutanolysin extract preparation was 2-fold serial diluted and spotted onto a nitrocellulose membrane. Pilus presence was detected with an anti-EbpC rabbit polyclonal immune serum.

Figure 4

fsrB expression profile in OG1RF. For β-gal assays, samples were collected every hour from 3 to 8 hr, then at 10 and 24 hr after starting the culture (x axis). All sets of cultures presented were analyzed concurrently. The figure is a representative of at least two experiments. The growth curves are represented in brown for cells grown in BHI-air and purple for cells grown in TSBG (thin line when grown in air, dense line when grown in the presence of 5% CO₂/0.1 M NaHCO₃). OG1RF containing P_fsrB::lacZ was grown in BHI air (brown closed diamond), in TSBG- air (purple closed diamond) or in TSBG-5% CO₂/0.1 M NaHCO₃ (purple open diamond). A. OD_{600 nm} readings. B. β-gal assays (β-gal units = OD_{420 nm}/protein concentration in mg/ml).

Figure 5

ebpR and ebpA expression profiles in TX5266 (ΔfsrB mutant). For β-gal assays, samples were collected every hour from 3 to 8 hr, then at 10 and 24 hr after starting the culture (x axis). The left axis represents the β-gal units (OD_420nm/protein concentration in mg/ml). The right axis indicates the OD_{600 nm} readings. All sets of cultures presented were analyzed concurrently. Each figure is a representative of at least three experiments. A. OG1RF containing either P_ebpR::lacZ (black triangle) or P_ebpA::lacZ (black square) and ΔfsrB containing either P_ebpR::lacZ (pink triangle) or P_ebpA::lacZ (pink square) were grown in TSBG aerobically. B. The ΔfsrB mutant (TX5266) containing either P_ebpR::lacZ (triangle) or P_ebpA::lacZ (square) was grown in TSBG aerobically (pink closed symbol) or in the presence of 5% CO₂/0.1 M NaHCO₃ (open blue symbol).

Figure 6

Effect of nisin induction on ebpR and ebpA expression. Cells were grown to an OD_{600 nm} of ~0.8 (3 hr, late log exponential growth phase) and at this point cells were left untreated (0) or treated with increasing concentration of nisin (from 0.005 to 10 ng/ml). Then, cells were collected and RNA extracted. After reverse transcription, ebpA and ebpR cDNA was quantified by real time PCR. The strains were OG1RF or ΔebpR (TX5514) carrying either the empty plasmid (-) or ebpR in trans under the nisin promoter (+). ebpR (gray bars) and ebpA (white bars) transcript levels were normalized with gyrB transcript levels. The data correspond to the mean of two independent experiments.

Figure 7

ebpA expression affected by NaHCO₃, and not CO₂. For β-gal assays, samples were collected every hour from 3 to 8 hr, then at 10 and 24 hr after starting the culture (x axis). Growth curves of OG1RF containing P_ebpA::lacZ are shown in air with a thin gray line, in NaHCO₃/air with thin orange line, in CO₂ with a dense gray line, and in NaHCO₃/CO₂ with a dense orange line. The β-gal assays for OG1RF containing P_ebpA::lacZ are represented with closed black square, closed orange square, open black square, and open orange square when the cells were grown in air, 5% CO₂, NaHCO₃-air, and NaHCO₃-5% CO₂, respectively. All sets of cultures presented were analyzed concurrently. This figure is a representative of at least two experiments. A. OD_{600 nm} readings. B. β-gal assays (β-gal units = OD_{420 nm}/protein concentration in mg/ml).

Figure 8

pH and NaHCO₃ effect on ebpA expression. OG1RF containing P_ebpA::lacZ was used in these experiments. Growth curves are represented in thin gray line for pH 7 aerobically, thin orange line for pH 7-Air/NaHCO₃, dense gray line for pH 8 aerobically, and dense orange line for pH 8-Air/NaHCO₃. All sets of cultures presented were analyzed concurrently. This figure is a representative of at least two experiments. A. OD_{600 nm} readings. B. β-gal assays (β-gal units = OD_{420 nm}/protein concentration in mg/ml).