Transcriptional Regulation of icaADBC by both IcaR and TcaR in Staphylococcus epidermidis

Abstract

S. epidermidis is a primary cause of biofilm-mediated infections in humans due to adherence to foreign bodies. A major staphylococcal biofilm accumulation molecule is polysaccharide intracellular adhesin (PIA), which is synthesized by enzymes encoded by the icaADBC operon. Expression of PIA is highly variable among clinical isolates, suggesting that PIA expression levels are selected in certain niches of the host. However, the mechanisms that govern enhanced icaADBC transcription and PIA synthesis in these isolates are not known. We hypothesized that enhanced PIA synthesis in these isolates was due to function of IcaR and/or TcaR. Thus, two S. epidermidis isolates (1457 and CSF41498) with different icaADBC transcription and PIA expression levels were studied. Constitutive expression of both icaR and tcaR demonstrated that both repressors are functional and can completely repress icaADBC transcription in both 1457 and CSF41498. However, it was found that IcaR was the primary repressor for CSF41498 and TcaR was the primary repressor for 1457. Further analysis demonstrated that icaR transcription was repressed in 1457 in comparison to CSF41498, suggesting that TcaR functions as a repressor only in the absence of IcaR. Indeed, DNase I footprinting suggests IcaR and TcaR may bind to the same site within the icaR-icaA intergenic region. Lastly, we found mutants expressing variable amounts of PIA could rapidly be selected from both 1457 and CSF41498. Collectively, we propose that strains producing enhanced PIA synthesis are selected within certain niches of the host through several genetic mechanisms that function to repress icaR transcription, thus increasing PIA synthesis.IMPORTANCEStaphylococcus epidermidis is a commensal bacterium that resides on our skin. As a commensal, it protects humans from bacterial pathogens through a variety of mechanisms. However, it is also a significant cause of biofilm infections due to its ability to bind to plastic. Polysaccharide intercellular adhesin is a significant component of biofilm, and we propose that the expression of this polysaccharide is beneficial in certain host niches, such as providing extra strength when the bacterium is colonizing the lumen of a catheter, and detrimental in others, such as colonization of the skin surface. We show here that fine-tuning of icaADBC transcription, and thus PIA synthesis, is mediated via two transcriptional repressors, IcaR and TcaR.

Keywords: Staphylococcus epidermidis; biofilms; transcriptional regulation.

FIG 1

S. epidermidis 1457 and CSF41498 differ in icaA transcription, PIA synthesis, and biofilm formation. In comparison to 1457, less icaA transcript (A), PIA (B), and biofilm (C) was detected in CSF41498. RNA was isolated from the mid-exponential phase during microaerobic growth (5:3 flask to volume ratio; 125 rpm, 37°C). PIA was purified from the post-exponential phase. Biofilm was stained with crystal violet after 24 h of growth in TSB. RNA gel shown as a loading control. PIA dot blot and biofilm analyses were assessed using three biological replicates.

FIG 2

icaA transcription, PIA synthesis, and biofilm production in 1457 and CSF41498. (A and B) Quantitative real-time PCR was performed to evaluate icaA transcription in 1457 (A) and CSF41498 (B) ΔicaR, ΔtcaR, ΔicaR ΔtcaR, and constitutive icaR and tcaR cis complement strains. icaA (3310 and 3311) and gyrB (2301 and 2302) specific primers were used. icaA expression was calculated relative to gyrB, and all strains were compared to the wild type. RNA was isolated in the mid-exponential phase during microaerobic growth (5:3 flask/volume ratio; 125 rpm, 37°C). One-way analysis of variance (ANOVA) was performed, followed by Dunnett’s multiple-comparison test, to determine significance; all groups were compared to the wild type. **, P ≤ 0.01; ***, P ≤ 0.001. (C and D) PIA was purified from the post-exponential phase and detected using a PIA-specific antibody. The percent intensity was measured using ImageJ software. (E) Biofilm formation was determined using a Christensen biofilm assay and assessed based on the OD₅₉₅. Statistical analysis for panels C, D, and E was performed using one-way ANOVA with Tukey’s multiple-comparison test. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. PIA dot blot and biofilm analyses were assessed using three biological replicates.

FIG 3

icaR transcription in 1457 and CSF41498. Northern blot analyses of icaA and icaR transcripts in 1457 and CSF41498 and mutants were performed. Transcript was detected using a DIG-labeled icaA or icaR DNA probe. Total RNA was isolated in the mid-exponential phase after microaerobic growth (5:3 flask/volume ratio; 125 rpm, 37°C). The percent intensity was measured using ImageJ software. An RNA gel is shown as a loading control.

FIG 4

TcaR binds to multiple sites in the icaR-icaA intergenic region. Incubation with IcaR resulted in a footprint upstream of icaA. Incubation with TcaR resulted in a footprint throughout the intergenic region (three sites noted on the right). ³²P-labeled icaR-icaA intergenic DNA (amplified with the primers 2855 and 2965) was incubated with increasing concentrations of recombinant IcaR or TcaR, treated with DNase I, and electrophoresed on a 6% denaturing polyacrylamide gel. Previously proposed TcaR binding sites and experimentally derived IcaR binding site are noted on the left.

FIG 5

icaA transcription, PIA synthesis and biofilm production in CSF41498 enhanced biofilm mutants. CSF41498 was screened on Congo red agar (CRA) after selection for enhanced adherence. Strains not producing PIA, such as the 1457 ΔicaA mutant, have a smooth, round phenotype on CRA, whereas strains that produce significant amounts of PIA, such as 1457, appear crusty and rough on CRA. CSF41498 has an intermediate phenotype on CRA. (A) CSF41498 mutants selected on CRA have a colony phenotype similar to 1457. (B and C) Northern blot analyses detecting icaA and icaR transcript in CSF41498 biofilm mutants in addition to PIA synthesis (B) and biofilm formation (C) compared to CSF41498 wild type (WT). Statistical analysis was performed using one-way ANOVA with Tukey’s multiple-comparison test. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. An RNA gel is shown as a loading control. PIA dot blot and biofilm analyses were assessed from three biological replicates.

FIG 6

icaA transcript, PIA synthesis, and biofilm production in 1457 mutants that produce less biofilm. (A and B) Northern blot analyses detecting icaA and icaR transcript in 1457 biofilm mutants in addition to PIA synthesis (A) and biofilm formation (B) compared to 1457 wild type (WT). Statistical analysis was performed using one-way ANOVA with Tukey’s multiple-comparison test. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. An RNA gel is shown as a loading control. PIA dot blot and biofilm analyses were assessed from three biological replicates.