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Quercetin impairs Streptococcus pneumoniae biofilm formation by inhibiting sortase A activity.

ABSTRACT: Biofilm formation mediated by sortase A (srtA) is important for bacterial colonisation and resistance to antibiotics. Thus, the inhibitor of SrtA may represent a promising agent for bacterial infection. The structure of Streptococcus pneumoniae D39 srtA has been characterised by crystallisation. Site-directed mutagenesis was used for the determination of the key residues for the activity of S. pneumoniae D39 srtA. An effective srtA inhibitor, quercetin, and its mechanism was further identified using srtA activity inhibition assay and molecular modelling. In this study, the crystal structure of S. pneumoniae D39 srtA has been solved and shown to contain a unique domain B. Additionally, its transpeptidase activity was evaluated in vitro. Based on the structure, we identified Cys207 as the catalytic residue, with His141 and Arg215 serving as binding sites for the peptide substrate. We found that quercetin can specifically compete with the natural substrate, leading to a significant decrease in the catalytic activity of this enzyme. In cells co-cultured with this small molecule inhibitor, NanA cannot anchor to the cell wall effectively, and biofilm formation and biomass decrease significantly. Interestingly, when we supplemented cultures with sialic acid, a crucial signal for pneumococcal coloniation and the invasion of the host in the co-culture system, biofilm loss did not occur. This result indicates that quercetin inhibits biofilm formation by affecting sialic acid production. In conclusion, the inhibition of pneumococcal srtA by the small molecule quercetin offers a novel strategy for pneumococcal preventative therapy.

SUBMITTER: Wang J

PROVIDER: S-EPMC6237587 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

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Project description:Background: Cajuputs candy (CC), an Indonesian functional food, utilizes the bioactivity of Melaleuca cajuputi essential oil (MCEO) to maintain oral cavity health. Synergistic interaction between Candida albicans and Streptococcus mutans is a crucial step in the pathogenesis of early childhood caries. Our recent study revealed several alternative MCEOs as the main flavors in CC. The capacity of CC to interfere with the fungus-bacterium relationship remains unknown. This study aimed to evaluate CC efficacy to impair biofilm formation by these dual cariogenic microbes. Methods: The inhibition capacity of CC against mixed-biofilm comprising C. albicans and S. mutans was assessed by quantitative (crystal violet assay, tetrazolium salt [MTT] assay, colony forming unit/mL counting, biofilm-related gene expression) and qualitative analysis (light microscopy and scanning electron microscopy). Result: Both biofilm-biomass and viable cells were significantly reduced in the presence of CC. Scanning electron microscopy imaging confirmed this inhibition capacity, demonstrating morphology alteration of C. albicans, along with reduced microcolonies of S. mutans in the biofilm mass. This finding was related to the transcription level of selected biofilm-associated genes, expressed either by C. albicans or S. mutans. Based on qPCR results, CC could interfere with the transition of C. albicans yeast form to the hyphal form, while it suppressed insoluble glucan production by S. mutans. G2 derived from Mojokerto MCEO showed the greatest inhibition activity on the relationship between these cross-kingdom oral microorganisms (p < 0.05). Conclusion: In general, all CC formulas showed biofilm inhibition capacity. Candy derived from Mojokerto MCEO showed the greatest capacity to maintain the yeast form of C. albicans and to inhibit extracellular polysaccharide production by S. mutans. Therefore, the development of dual-species biofilms can be impaired effectively by the CC tested.

Project description: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.

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Quercetin impairs Streptococcus pneumoniae biofilm formation by inhibiting sortase A activity.

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