Involvement of signal peptidase I in Streptococcus sanguinis biofilm formation

Abstract

Biofilm accounts for 65-80 % of microbial infections in humans. Considerable evidence links biofilm formation by oral microbiota to oral disease and consequently systemic infections. Streptococcus sanguinis, a Gram-positive bacterium, is one of the most abundant species of the oral microbiota and it contributes to biofilm development in the oral cavity. Due to its altered biofilm formation, we investigated a biofilm mutant, ΔSSA_0351, that is deficient in type I signal peptidase (SPase) in this study. Although the growth curve of the ΔSSA_0351 mutant showed no significant difference from that of the wild-type strain SK36, biofilm assays using both microtitre plate assay and confocal laser scanning microscopy (CLSM) confirmed a sharp reduction in biofilm formation in the mutant compared to the wild-type strain and the paralogous mutant ΔSSA_0849. Scanning electron microscopy (SEM) revealed remarkable differences in the cell surface morphologies and chain length of the ΔSSA_0351 mutant compared with those of the wild-type strain. Transcriptomic and proteomic assays using RNA sequencing and mass spectrometry, respectively, were conducted on the ΔSSA_0351 mutant to evaluate the functional impact of SPase on biofilm formation. Subsequently, bioinformatics analysis revealed a number of proteins that were differentially regulated in the ΔSSA_0351 mutant, narrowing down the list of SPase substrates involved in biofilm formation to lactate dehydrogenase (SSA_1221) and a short-chain dehydrogenase (SSA_0291). With further experimentation, this list defined the link between SSA_0351-encoded SPase, cell wall biosynthesis and biofilm formation.

Fig. 1.

Characteristics of SSA_0351-encoded SPase as extracted from various databases. (a) Multiple protein sequence alignments of S. sanguinis SPases, SSA_0351 and SSA_0849, and B. subtilis SipW were conducted using MultAlin. Blue: identity across three sequences. Red: identity between two sequences. (b) Phylogenic tree showing the evolutionary relatedness of bacterial SPases (LepB from Escherichia coli str. K-12 substr. MG1655, SpsB from Staphylococcus aureus subsp. aureus NCTC 8325, SipW from Bacillus subtilis) based on protein sequence alignment with hierarchical clustering. The identity percentages of the protein sequence alignments for every SPase with respect to SSA_0351 are shown, except for SpiW, which was too low. The evolutionary distances between SPases have been calibrated to 10 PAM, where 1 per cent accepted mutation (PAM) is the time in which 1 substitution event per 100 sites is expected to have happened.

Fig. 2.

SPase-related mutants show different patterns of biofilm formation. Bacterial samples of eight replicates each were cultured anaerobically for 24 h in BM with 1 % sucrose. After crystal violet staining, biofilm formation was quantified at OD₆₀₀ and the results were compared using ANOVA and the multiple comparison method (Dunnett's test). ** indicates significance with P-value <0.01.

Fig. 3.

SPase-related mutants form different biofilm biomasses. Biofilm formation comparison of the SPase mutants and SK36 by confocal laser scanning microscopy (CLSM). CLSM imaging was conducted in triplicate and the images were analysed with Image J. (a) Representative images from wild-type and two SPase-related mutants, ΔSSA_0351 and ΔSSA_0849 are shown. (b) Biofilm thickness and (c) roughness coefficient parameters were measured using MATLAB. * indicates significance with P-value <0.05. **indicates significance with P-value <0.01.

Fig. 4.

Growth curves of wild-type (WT), ΔSSA_0849 and ΔSSA_0351. The growth rates of the wild-type strain and mutants were compared at different time intervals (hours). The growth curve assay was performed in triplicate and the average of the three experiments is shown here. Legends: empty square, WT; empty circle, ΔSSA_0351; filled circle, ΔSSA_0849.

Fig. 5.

Scanning electron microscopy (SEM) analysis of the wild-type and ΔSSA_0351 strain showed distorted cell morphology and the absence of cellular chains in the SPase-mutant ΔSSA_0351. Biofilms formed by the wild-type strain and ΔSSA_0351 were scanned under (a) 20 000× magnification and (b) 10 000× magnification.

Fig. 6.

Evaluation of the biofilm formation potential of SPase SSA_0351 targets. (a) Potential targets of SPase SSA_0351 were shown to be involved in biofilm formation, as shown by the biofilm assays of their respective mutants. Bacterial samples of eight replicates each were cultured anaerobically for 24 h in BM with 1 % sucrose. After crystal violet staining, biofilm formation was quantified at OD₆₀₀ and the results were compared using ANOVA and the multiple comparison method (Dunnett's test). Statistically significant results had a cutoff P-value <0.05. **indicates significance with P-value <0.01. (b) Evaluation of the auto-aggregation ability of potential targets of SSA_0351-encoded SPase. The auto-aggregation ability of S. sanguinis mutants with respect to the wild-type was measured using the auto-aggregation assay, where the absorbance of each bacterial culture was measured at the time points 0 and 8 h. Auto-aggregation ability was expressed as a percentage. (c) Growth curves of mutants of differentially regulated genes/proteins in ΔSSA_0351. The growth rates of the wild-type strain and mutants were compared at different time intervals (hours). The growth curve assay was performed in triplicate and the average of the three experiments is shown here. Legends: empty square, WT; filled square, ΔSSA_1948; empty circle, ΔSSA_1221; filled circle, ΔSSA_0291; empty rhombus, ΔSSA_1950; filled rhombus, ΔSSA_0371; plus sign, ΔSSA_2141; dash, ΔSSA_2141.