A Quorum-Sensing System That Regulates Streptococcus pneumoniae Biofilm Formation and Surface Polysaccharide Production

Abstract

Despite vaccines, Streptococcus pneumoniae kills more than a million people yearly. Thus, understanding how pneumococci transition from commensals to pathogens is particularly relevant. Quorum sensing regulates collective behaviors and thus represents a potential driver of commensal-to-pathogen transitions. Rgg/small hydrophobic peptide (SHP) quorum-sensing systems are widespread in streptococci, yet they remain largely uncharacterized in S. pneumoniae. Using directional transcriptome sequencing, we show that the S. pneumoniae D39 Rgg0939/SHP system induces the transcription of a single gene cluster including shp and capsule gene homologs. Capsule size measurements determined by fluorescein isothiocyanate-dextran exclusion allowed assignment of the system to the regulation of surface polysaccharide expression. We found that the SHP pheromone induced exopolysaccharide expression in R36A, an unencapsulated derivative of D39. In the encapsulated parent strain, overexpression of the Rgg system resulted in a mutant with increased capsule size. In line with previous studies showing that capsule expression is inversely associated with biofilm formation, we found that biofilm formed on lung epithelial cells was decreased in the overexpression strain and increased in an rgg deletion mutant. Although no significant differences were observed between D39 and the rgg deletion mutant in a mouse model of lung infection, in competitive assays, overexpression reduced fitness. This is the first study to reveal a quorum-sensing system in streptococci that regulates exopolysaccharide synthesis from a site distinct from the original capsule locus. IMPORTANCE Quorum sensing regulates bacterial social behaviors by production, secretion, and sensing of pheromones. In this study, we characterized a new quorum-sensing system of the Rgg/SHP class in S. pneumoniae D39. The system was found to directly induce the expression of a single gene cluster comprising the gene for the SHP pheromone and genes with putative functions in capsule synthesis. Capsule size, as measured by dextran exclusion, was increased by SHP exposure in R36A, an unencapsulated derivative of D39. In the encapsulated parent strain, overexpression of the gene cluster increased capsule size, supporting the role of Rgg/SHP in the synthesis of surface polysaccharides. Further, we found that biofilm formation on epithelial cells was reduced by overexpression of the system and increased in a mutant with an rgg deletion. Placing surface polysaccharide expression under quorum-sensing regulation may enable S. pneumoniae to tune interactions with the host and other bacteria in accordance with environmental and cell density conditions.

Keywords: Streptococcus pneumoniae; biofilms; cell signaling; mutagenesis; natural transformation systems; polysaccharides; quorum sensing; rgg; shp; transcriptional regulation.

FIG 1

Effect of the sSHP pheromone on relative shp expression in S. pneumoniae D39 and the Δrgg (SP68), and Δrgg_comp (SP101) mutants. Cultures were grown in C+Y and incubated at 37°C for 2 h at a starting OD₆₀₀ of 0.05 without treatment (control), with the addition of a nonspecific sSHP, or with the addition of the S. pneumoniae SHP (specific). The data presented are the means and standard deviation of at least three independent experiments. ****, P < 0.0001. dRn, relative mRNA expression.

FIG 2

Transcriptome analysis. (A) Scatterplot of global mRNA expression of S. pneumoniae D39 with and without addition of the S. pneumoniae sSHP (specific). Both axes show mRNA expression (number of reads) on a log₁₀ scale. The dashed red lines represent 2-fold up- and downregulation. The operon downstream of shp was highly upregulated (circle). Only annotated genes were included in this analysis; therefore, shp is not present. (B) Fold change in gene expression in the genome of S. pneumoniae D39 when the sSHP is added to the culture. The x axis shows gene names in accordance with NCBI Protein Coding Genes. Pseudogenes and tRNA were excluded from the final analyses.

FIG 3

Transcript map of the single 12-gene operon activated by the sSHP. The pheromone is highlighted in yellow. The first row shows the number of reads (log₂) in the forward (FW) strand (control without sSHP) for nontreated cultures, while the second shows the expression in treated cultures (specific sSHP treated). The third row shows the number of reads (log₂) in the reverse complement (RC) strand (control without sSHP) for nontreated in comparison to treated S. pneumoniae (sSHP treated). Expression values are averages of samples from two independent biological experiments.

FIG 4

Activation of shp and the downstream regulon with and without sSHP in S. pneumoniae D39 and the Δrgg (SP68) and Over-EXP (SP44) mutant strains. shp gene expression was determined by RT-PCR. Cultures were grown in C+Y and incubated at 37°C for 2 h at a starting OD₆₀₀ of 0.05 with (as indicated by a plus sign) or without 1 µM S. pneumoniae sSHP prior to RNA extraction. The y axis shows relative mRNA expression (dRn) on a log₁₀ scale. Each bar represents mean values with the standard error of the mean. Panels: A, shp; B, SPD_0940; C, SPD_0947; D, SPD_0950; E, SPD_0952.

FIG 5

Overexpression of the Rgg0939 regulon affects serotype 2 capsule thickness. Each dot represents the measurement of the FITC-dextran exclusion area for a single cell, and each bar represents the mean and the standard error of the mean of each group. (A) Representative microscopic images of FITC-dextran exclusion for D39 and the Δrgg and Over-EXP mutant strains. (B) FITC-dextran analysis of capsule thickness. The mean area (square pixels) per bacterium was greater in the overexpression strain than in D39 and the Δrgg0939 mutant. ****, P < 0.0001. (C) Light microscopy analysis of cell size (square pixels).

FIG 6

Biofilm formation of wild-type D39 and Δrgg (SP68) and Over-EXP (SP44) mutants on eukaryotic cells. Cultures were grown in CDM++ to an OD₆₀₀ of 0.1 and inoculated on top of a fixed lung cell line (A549). Every 12 h, the medium was changed to avoid bacterial lysis. At 36 h, the supernatant was removed, the biofilm was resuspended in rich medium, the wells were scraped, and the material obtained was immediately refrigerated for plating. The y axis shows the number of biofilm CFU recovered from wells as fold change compared to wild-type D39, and each value is the mean and the standard error of the mean of at least three independent biological experiments. ****, P < 0.0001.

FIG 7

Bacterial burden of wild-type D39 and Δrgg (SP68) and Over-EXP (SP44) mutant strains in a mouse early lung infection model. Each dot represents results for a single mouse, and bars represent the mean and the standard error of the mean of each group. Mice were inoculated intranasally with 5 × 10⁶ CFU in 50 µl of 1× PBS and killed after 24 h. Recovery of samples took place immediately after death, and all washes were placed at 4°C for dilution and plating. (A) Nasal cavity wash. **, P < 0.01; *, P < 0.05. (B) BALF. (C) Mixed-infection assay with D39 and Over-EXP. The first set was recovered from nasal wash. Each point represents results for a single mouse, and bars show the median CI of the group. The second and third sets show bacteria recovered from BALF and mashed lungs, respectively. The y axis shows the ratio of the bacterial counts recovered to the inoculum. A ratio of >1 indicates that the overexpression strain was predominant, while a ratio of 0 to 1 indicates that D39 was recovered in higher numbers. *, P < 0.05.

FIG 8

Production of surface polysaccharides following treatment with synthetic SHP. Each dot represents the measurement of the FITC-dextran exclusion area for a single cell, and bars represent the mean and the standard error of the mean of each group. ****, P < 0.0001.

FIG 9

Schematic representation of the Rgg/SHP system in S. pneumoniae D39. The SHP precursor is produced, processed, and exported by PptAB and Eep (16, 77). Once it reaches the quorum-sensing threshold, the mature peptide is imported by Opp (12, 15, 16), and it binds to Rgg for activation of the shp promoter. This, in turn, activates the pheromone positive-feedback loop and an operon formed by 12 genes initiated at the shp gene. High activation of this regulon upregulates polysaccharide synthesis and downregulates biofilm formation.