TetR Family Regulator brpT Modulates Biofilm Formation in Streptococcus sanguinis

Abstract

Biofilms are a key component in bacterial communities providing protection and contributing to infectious diseases. However, mechanisms involved in S. sanguinis biofilm formation have not been clearly elucidated. Here, we report the identification of a novel S. sanguinis TetR repressor, brpT (Biofilm Regulatory Protein TetR), involved in biofilm formation. Deletion of brpT resulted in a significant increase in biofilm formation. Interestingly, the mutant accumulated more water soluble and water insoluble glucans in its biofilm compared to the wild-type and the complemented mutant. The brpT mutation led to an altered biofilm morphology and structure exhibiting a rougher appearance, uneven distribution with more filaments bound to the chains. RNA-sequencing revealed that gtfP, the only glucosyltransferase present in S. sanguinis, was significantly up-regulated. In agreement with these findings, we independently observed that deletion of gtfP in S. sanguinis led to reduced biofilm and low levels of water soluble and insoluble glucans. These results suggest that brpT is involved in the regulation of the gtfP-mediated exopolysaccharide synthesis and controls S. sanguinis biofilm formation. The deletion of brpT may have a potential therapeutic application in regulating S. sanguinis colonization in the oral cavity and the prevention of dental caries.

Fig 1. Deletion of brpT increases biofilm formation.

S. sanguinis was cultured in BM supplemented with 1% sucrose, and biofilm biomass was determined by CV staining. Data from three biological replicates were averaged and the statistical significance between the brpT mutant, ΔbrpT and the wild-type, SK36 or the complemented mutant, ΔbrpT_C was determined by Student’s t-test. **, indicates significance with P <0.01.

Fig 2. Deletion of brpT alters the biofilm structure.

(A) Wild-type S. sanguinis, SK36, the brpT mutant, ΔbrpT, and the complemented mutant, ΔbrpT_C were grown in BM as described in Materials and Methods. After 24-h growth, the biofilms were washed and stained with SYTO 9, and z-stacks of each were acquired by CLSM with a Plan-Neofluar ×10/0.3 objective lens. Representative orthogonal views from three independent experiments are displayed. (B) Quantification of biofilm thickness by CLSM for the wild-type, ΔbrpT and ΔbrpT_C. (C) Quantification of biofilm roughness for the wild-type, ΔbrpT and ΔbrpT_C. **, indicates significance with P <0.01.

Fig 3. SEM analysis further reveals altered biofilm morphology and an increase in filamentous structures.

Biofilms formed by the wild-type SK36, the brpT mutant, ΔbrpT, and the complemented mutant, ΔbrpT_C, scanned under (A) 1000x magnification and (B) 20,000x magnification revealed an altered morphology and an increase in filamentous structures for ΔbrpT compared to the wild-type and complemented mutant. White arrows indicate filamentous substances.

Fig 4. Efficiency of glucan accumulation in S. sanguinis biofilms.

S. sanguinis wild-type SK36, the brpT mutant ΔbrpT and the complemented mutant ΔbrpT_C were grown anaerobically for 24 h in BM medium containing 1% sucrose at 37°C. The amounts of (A) water soluble glucans and (B) water insoluble glucans in the biofilms were quantified using the phenol-sulfuric acid method and normalized to the concentration of genomic DNA.

Fig 5. Increased gene expression of gtfP in the brpT mutant.

qRT-PCR was used to determine the relative expression of glucosyltransferase, gtfP, in the brpT mutant using gyrA as an internal control. Data shown are mean±SD from three biological replicates. “–S” on the x-axis represents samples cultured in BHI supplemented with 1% sucrose. **, indicates significance with P <0.01.

Fig 6. Deletion of gtfP in S. sanguinis decreases biofilm attachment and glucan synthesis.

Wild-type, the gtfP mutant, ΔgtfP, and the double mutant, ΔbrpT/ΔgtfP, were cultured in BM with 1% sucrose for 24 h anaerobically and analyzed. (A) Weak attachment of the ΔgtfP and the ΔbrpT/ΔgtfP biofilm (pellicle) to the polystyrene surface and reduced biofilm biomass determined by CV staining. (B) Quantification of biofilm formation (OD₆₀₀). Quantification of (C) water soluble glucans, WSG and (D) water insoluble glucans, WIG accumulated within the biofilm. **, indicates significance with P <0.01.