Component analysis of cell-wall anchored pili of Streptococcus sanguinis

概要

Background and Objectives
Streptococcus sanguinis has garnered considerable attention in recent years due to its prevalence in the healthy oral environment and its crucial role as a pioneer colonizer of dental plaques. As previously reported, S. sanguinis exhibits a strain-specific pilus structure on the cell surface. These pili, which are composed of PiliA, PiliB, and PiliC proteins, assist bacteria in adhering to and internalizing epithelial cells, as well as forming biofilms. However, analysis of the pilus assembly mechanism has raised concerns about the possibility of an unidentified pilus component in addition to PiliA, PiliB, and PiliC. The objective of this study was to determine whether PiliX, a putative cell surface protein encoded by a gene located adjacent to piliABC, is a pilus subunit and whether it has any effect on biofilm formation.

Materials and Methods
1. Bacterial strains and culture conditions. The Streptococcus sanguinis SK36 strain used in this experiment was generously provided by Dr. M. Kilian (Institute of Medical Microbiology and Immunology, Aarhus University, Denmark). S. sanguinis SK36 wild-type and isogenic mutant strains were cultivated at 37° C in ambient air in Todd-Hewitt broth (Becton Dickinson) supplemented with 0.2% yeast extract (Becton Dickinson).

2. RT-PCR. Total RNA was isolated from the wild-type strain in the late exponential phase of growth (OD600nm = 0.9). The synthesis of cDNA from total RNA was performed using the Transcriptor High-Fidelity cDNA Synthesis Kit (Roche), whereas the contrast group was not treated with reverse transcriptase (non-RT group). RT-PCR amplifications were performed using primers designed to amplify DNA fragments that overlapped between two adjacent genes from ssal630 to ssa1636.

3. Construction of gene deletion mutants. By allelic exchange through double crossover events, piliX (ssa1635) and srtC (ssa1631) were deleted separately using the temperature-sensitive suicide vector pSET6s. In addition, a clone carrying the wild-type allele was used in this study as a revertant strain.

4. Preparation of antisera against pilin subunits. To express recombinant pilus subunit proteins in Escherichia coli cells, the coding regions of the putative pilus proteins {piHA, piliB, piliC and piliX) were cloned into the expression vector pQE30 (Qiagen) and then purified from E. coli lysates using a QIAexpress protein purification system (Qiagen). Protein eluates were dialyzed against PBS. Mouse antisera against pilus proteins were raised by immunizing BALB/c mice with purified recombinant proteins. Rabbi t antisera against pilus recombinant proteins were raised by immunizing male New Zealand white rabbits with purified recombinant proteins.

5. Immunoprecipitation. Cell wall fractions of S. sanguinis SK36 grown in exponential phase (OD600nm = 0.5) were dialyzed against PBS and incubated overnight at 4°C with anti-Pi liA (SSA1632)mouse serum or non immune mouse serum. Subsequently, the mixture was incubated with protein G-coated magnetic beads (10%, v/v) at 4°C for 5 hours. The beads were thoroughly washed five times with PBST and resuspended in a sample buffer containing SDS. Immunoblot analysis was performed on the immunoprecipitated samples using rabbit sera against pilus proteins.

6. Biofilm analysis. The ability of wild-type, piliX deletion mutant, and revertant strains to form biofilms was determined using a static assay with saliva-coated polystyrene plates 12 or 24 hours after inoculation.

Results
1. The PiliX protein is a sanguinis pilus subunit.
　a. Detection of co-transcr ipt ion of piliZwith pili ABC RT-PCR. As demonstrated by the electrophoresis results, bands were detected between piliX,piliC, piliB, pili A, and srtC (the gene encoding Sortase C), confirming the co-transcr ipt ion of piliX with piliABC and srtC as an operon. DNA contamination was ruled out using PCR analysis on samples from the previous non-RT group.
　b. Detection of PiliX in both cell wall and culture supernatant fractions. Separate immunoblots with anti-PiliX serum were performed on the cell wall and culture supernatant fractions of S. sanguinis wild-type, piliX deletion mutant, and revertant strains. The high molecular weight ladder bands were clearly recognizable in both fractions of wild-type and revertant strains, indicating the presence of a pilus structure similar to that of other Gram-positive bacteria.
　c. Detection of PiliX in the cell wall fraction of △srtCmutants. Dedicated immunoblots using anti-PiliX serum were performed on cell wall extracts from wild-type, srtC deletion mutants and revertant strains. Those ladder bands were undetectable in those from the mutant strain lacking srtC, the gene that encodes the transpeptidase Sortase C involved in the pilus subunit linkage.
　d. Effects of piliX deletion on the pilin detection pattern. Immunoblots using anti-PiliA, B, and C and cell wall extracts from piliX deletion mutants, as well as wild-type and revertant strains, revealed that deletion of piliX altered the intensity and migration pattern of ladder bands, adding to the evidence that PiliX is a pilus subunit.
　e. Detection of PiliX in cell wall fractions immunoprecipitated with anti-PiliA serum. Immunoblots were performed on the fraction immunoprecipitated with anti-PiliA using antisera against PiliB, PiliC, or PiliX. The high molecular weight ladder bands were visible in all IP groups, but not in the non-IP control groups.

2. The PiliX protein has an effect on the formation of biofilms. Furthermore, piliX deletion compromised the ability of S. sanguinis to form biofilm. PiliX deletion mutants were less capable of forming biofilms in the presence of saliva and glucose, implying that PiliX protein promoted biofilm formation.

Conclusions and Discussion
Our findings indicate that PiliX is a novel pilus subunit of the S. sanguinis pili. Additionally, PiliX has an effect on biofilm formation, particularly when saliva and glucose are present, suggesting that PiliX may potentiate the biofilm formation activity of S. sanguinis in the oral cavity.