ciaR impacts biofilm formation by regulating an arginine biosynthesis pathway in Streptococcus sanguinis SK36

Abstract

Streptococcus sanguinis is an early colonizer of the tooth surface and competes with oral pathogens such as Streptococcus mutans to maintain oral health. However, little is known about its mechanism of biofilm formation. Here, we show that mutation of the ciaR gene, encoding the response regulator of the CiaRH two-component system in S. sanguinis SK36, produced a fragile biofilm. Cell aggregation, gtfP gene expression and water-insoluble glucan production were all reduced, which suggested polysaccharide production was decreased in ΔciaR. RNA sequencing and qRT-PCR revealed that arginine biosynthesis genes (argR, argB, argC, argG, argH and argJ) and two arginine/histidine permease genes (SSA_1568 and SSA_1569) were upregulated in ΔciaR. In contrast to ΔciaR, most of strains constructed to contain deletions in each of these genes produced more biofilm and water-insoluble glucan than SK36. A ΔciaRΔargB double mutant was completely restored for the gtfP gene expression, glucan production and biofilm formation ability that was lost in ΔciaR, indicating that argB was essential for ciaR to regulate biofilm formation. We conclude that by promoting the expression of arginine biosynthetic genes, especially argB gene, the ciaR mutation reduced polysaccharide production, resulting in the formation of a fragile biofilm in Streptococcus sanguinis.

Figure 1

The impact of ciaR mutation on biofilm formation. (A) The biofilms of WT, ΔciaR and ΔciaR/ciaR were cultured in a 96-well plate in BM media for 24 hours. Biomass was measured by crystal violet staining. (B) Biofilms of WT and ΔciaR cultured in 4-well chambers after being washed with PBS buffer. (C) Biofilms grown in 4-well chambers were stained by SYTO9 and propidium iodide. Fluorescence (left) and differential interference microscopy images (middle) were obtained by confocal laser scanning microscopy. Fluorescence images were analyzed by COMSTAT script, and heat maps of biofilm thickness were generated, which showed the distribution of biomass in biofilms (right). (D) The fluorescence images were analyzed by COMSTAT. Biofilm biomass, average thickness, propidium iodide signal and roughness coefficient were quantified, respectively. All the data in Fig. 1D were compared with their WT control. *P ≤ 0.05, **P ≤ 0.01, Student’s t-test. Means and standard deviations from triplicate experiments are shown.

Figure 2

Polysaccharide production and cell growth of WT and ΔciaR. (A) Strains were incubated in BM at a shaking speed of 200 rpm for 24 hours. The arrow points a cellular auto-aggregate. (B) Strains were incubated in BM at a shaking speed of 200 rpm for 24 hours. Extracellular polysaccharide was stained by HHA-FITC and measured by flow cytometry. Arrow indicates a tail of increased staining in the WT strain. (C) WT and ΔciaR were cultured in BM. Every 30 minutes, cells were shaken for 3 minutes and OD₆₀₀ was read using a platereader (Top). CFU values were determined after 16 hours of incubation (bottom). (D) Biofilms were cultured in BM for 24 hours and then treated with 100 U/mL of DNase I for 1 hour. Microtiter dish biofilm assay was performed after DNase I treatment. Means and standard deviations from triplicate experiments are shown. ND: no significant difference, Student’s t-test.

Figure 3

Differentially expressed genes in ΔciaR. (A) Genes with fold change ≥ 1.5 or ≤ 0.67 and qvalue ≤ 0.01 in ΔciaR RNA sequencing data were analyzed by DAVID gene functional classification tools. The enriched pathways were shown. (B) Transcript levels of arg genes in ΔciaR and ΔciaR/ciaR. (C) qRT-PCR was performed to examine the expression of arg genes in ΔciaR. Means and standard deviations from triplicate experiments are shown.*P ≤ 0.05, **P ≤ 0.01, Student’s t-test.

Figure 4

ΔciaR impacts biofilm formation by modulating the expression of arginine biosynthesis genes. (A) Biofilm biomass was examined by microtiter dish biofilm assay and at the same time, the total protein concentration was quantified by BCA assay. The biofilm formation ability was defined as biomass (OD₅₆₀) divided by total protein concentration (μg/mL). (B) Biofilms of strains were grown in BM for 24 hours in 24-well plates. WIG levels were assayed. (C) qRT-PCR assay was performed to show the expression of gtfP gene in different strains or in WT cultured in BHI with different concentrations of L-arginine. (D) WT was cultured in BM with different concentrations of L-arginine. Biofilm formation ability was examined by microtiter dish biofilm assay. Means and standard deviations from triplicate experiments are shown. *P ≤ 0.05, **P ≤ 0.01, Student’s t-test.

Figure 5

Biofilm formation of WT and ΔciaR under flow cell conditions. Strains were marked by different fluorescent reporters and then cultured in a flow cell system. Image in Fig. 5C was obtained by CLSM. Others were recorded by the fluorescence microscopy. Biofilm biomass was quantified by COMSTAT software. (A) The biofilms biomass of WT and ΔciaR in the flow cell system at different time points. (B) WT and ΔciaR were co-cultured in the flow cell channel and biofilm biomass was measured. (C) After being co-cultured for 4 days, the structure of biofilm formed by WT – ΔciaR was examined by CLSM. For each sample in Fig. 5A and B, ten images from fluorescence microscopy were obtained to calculate the means and standard deviations. *P ≤ 0.05, **P ≤ 0.01, Student’s t-test.

Figure 6

The effect of ΔciaR on competence stimulation and the effect of CSP and competence genes on biofilm formation. The concentration of CSP was quantified by measuring the transformation frequency of a ΔcomC mutant to which was added culture supernatant (A) or cell lysate (B) of WT and ΔciaR, respectively. (C and E) biofilm formation of strains was tested by microtiter dish biofilm assay. (D) Strains were cultured in BM with different concentrations of exogenous CSP. Biofilm formation ability was tested by microtiter dish biofilm assay. Means and standard deviations from triplicate experiments are shown. ND: no significant difference, *P ≤ 0.05, **P ≤ 0.001, ND: no significant difference, Student’s t-test.