Study On The Mechanism Of Biofilm Formation And Molecular Pathogenesis Of Streptococcus Suis

Streptococcus suis (SS) is an important pathogen associated with a wide range of disease in pigs, including meningitis, septicaemia, pneumonia, endocarditis, and arthritis. Thirty-three serotypes (types 1-31,33, and 1/2) have been described based on capsular polysaccharides. Serotype 2 is the most prevalent type in association with diseases in most countries. It is also an important zoonotic agent for humans in contact with diseased pigs or their products, causing life threatening diseases.Biofilms are tightly packed communities of bacteria, encased in a self-synthesized extracellular polysaccharide matrix, growing attached to a biotic or abiotic surface. The vast majority of bacteria in most natural, industrial, and clinical environments live in biofilms and not as free-living or "planktonic" cells that are commonly studied in the laboratory. Biofilms play a key role in the pathogenesis of many bacterial infections. Many bacteria and pathogens utilize a biofilm strategy to survive inhospitable conditions and cause disease. Current research has shown that bacteria in a biofilm have increased resistance to host defenses and antimicrobial agents, making most biofilm infections difficult or impossible to be eradicated. However, the relationship between virulence and biofilm formation ability, and whether biofilms can exhibit altered virulence factors have not been investigated. Therefore, we firstly constructed the SS biofilm model in vitro and in vivo, the objective of this study was to determine whether biofilm cells can alter adherence, virulence, gene expression, and protein expression compared to planktonic cells. The LuxS protein regulating SS biofilm formation were studied by protein crystallization and gene knockout.1 Construction and structure observation of Streptococcus suis biofilm in vivo and in vitroStreptococcus suis can binds to extracellular matrix proteins and form biofilm in vitro. In this study, biofilm model in vitro was constructed by 96-well microplate. Confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM) observation showed that SS can formed a thick biofilm in vitro. The biofilm model in vivo was firstly constructed using zebrafish, the figures of light microscope and SEM revealed clusters of bacterial surrounded by what appeared to be a glycocalys in the infected zebrafish. It contributed to further study SS infection mechanism and pathogenesis.2 Comparision of biology property of Streptococcus suis cultured under biofilm and planktonic conditionsStreptococcus suis 2 (SS2) is a zoonotic pathogen that can participate in biofilm formation to survive in hostile environments. In this study, virulent SS2 strains HA9801 and ZY05719 displayed increased biofilm formation compared to SS2 avirulent strain T15. In addition, a 58% reduction in adherence to HEp-2 cells was observed for HA9801 biofilm cells, compared to HA9801 planktonic cells. The 50% lethal dose (LD50) of biofilm cells was 40-fold greater than that of planktonic cells. Quantification of expression levels of known virulence genes by real-time PCR revealed that the transcription levels of the gdh, cps2 and mrp genes in biofilm cells were downregulated, while the sly and gapdh genes were upregulated. HA9801 biofilm and planktonic vaccines provided 60% and 46% protection, respectively, when challenged with 50 times the LD50 of the HA9801 strain. These results suggest a possible connection between virulence and the ability of biofilm formation; cell adhesion, transcription levels and virulence properties are different between biofilm cells and planktonic cells. Furthermore, this work offers novel insight into bacterium infection mechanisms, which suggests that a virulent strain maybe decrease its virulence by forming a biofilm so that it can achieve persistent infection in vivo.3 Comparative proteomic analysis between Streptococcus suis biofilm and planktonic cells that identified biofilm infection-related immunogenic proteinsStreptococcus suis (SS) can bind to extracellular matrix proteins in both endothelial and epithelial cells resulting in persistent infection due to the formation of a biofilm. In this study, a comparative proteomic analysis was used to assess the differences in protein expression profiles between both planktonic and biofilm growth conditions. In addition, an immunoproteomic assay was developed to identify candidate antigens in an SS biofilm infection. The results revealed the existence of 13 proteins of varying amounts, among which six proteins were upregulated and seven proteins were downregulated in the Streptococcus biofilm compared with the planktonic controls. The convalescent serum from a specific pathogen free (SPF) mini-pig was used to perform a Western blot assay on all proteins from the biofilm that were grown in vitro and separated by two-dimensional gel electrophoresis. A total of 10 immunoreactive protein spots corresponding to nine unique proteins were identified by MALDI-TOF-MS. Of these nine proteins, five had no previously reported immune capacity in SS2 to our knowledge. The remaining four immunogenic proteins (glyceraldehyde-3-phosphate dehydrogenase, hemolysin, pyruvate dehydrogenase and molecular chaperone) were identified under both planktonic and biofilm growth conditions in previous reports. These novel immunogenic proteins may potentially be developed as vaccine candidates for SS biofilm infections, especially the four common immunogenic proteins identified in both growth conditions, as these may be used effectively to prevent both biofilm and acute infections.4 Structure and Function study of S-Ribosylhomocysteinase from Streptococcus suis serotype 2S-Ribosylhomocysteinase (LuxS) is an vital enzyme of the activated methyl cycle, which produces cell signaling molecules autoinducer (AI)-2. In this study, S-Ribosylhomocysteinase from SS2 was initially identified by bioinformatics methods. Encoding protein was obtained in vitro using recombinant gene technology and expressed in E.coli bacteria. Crystals suitable for X-ray diffraction was obtaind by screening and optimizing, The X-ray diffraction data was obtained and then processed by some related softwares. The fine three dimensional structures were solved. Inductively coupled plasma-mass spectrometry (ICP-MS) revealed the presence of Zn2+ in the purified protein. Evolution mechanism of LuxS was analysed to contrast other bacterial based on the structure, which reveals that the role of enzymes in the evolution mechanism, and provides a theoretical basis for drug design, as well as helps to find more effective anti-drug SS disease.5 Functional analysis of luxS in Streptococcus suis reveals a key role in biofilm formation and virulenceLuxS has been reported to play critical roles in both regulating various behaviors and interspecies quorum sensing in a large spectrum of bacteria. In this study, the luxS deletion mutant of SS using homologous recombination was constructed and evaluated their biofilm formation, hemolytic activity, cell adherence, virulence and expression of virulence factors. Compared to the parental strain, its biofilm formation and hemolytic activity was significantly decreased in the luxS mutant. Addition of synthetic autoinducer 2 was able to complement the deficiencies of biofilm production in the mutant strain. Furthermore, its adherence to the HEp-2 cell line was dramatically decreased by 51% compared to the parental. Expressions of the known virulence genes gdh, cps, mrp, gapdh, sly, fbps and ef in the mutant strain were decreased by 0.66,0.61,0.45,0.48,0.29,0.57 and 0.38, respectively, as quantified by real-time PCR. In a zebrafish infection model, the 50% lethal dose of the mutant strain was increased up to 10-fold. The findings demonstrated that the luxS gene deletion resulted in a significant decrease of bacterial biofilm formation, cell adhesion, hemolytic activity and transcription levels of many virulence genes in SS, and these factors may be associated with the attenuation of virulence in zebrafish. It suggested that luxS might be involved in the interruption of bacterial communication and biofilm formation that contribute to the virulence of the bacterium.